ARF1·GTP, Tyrosine-based Signals, and Phosphatidylinositol 4,5-Bisphosphate Constitute a Minimal Machinery to Recruit the AP-1 Clathrin Adaptor to Membranes

PubMed Central

Crottet, Pascal; Meyer, Daniel M.; Rohrer, Jack; Spiess, Martin

2002-01-01

At the trans-Golgi network, clathrin coats containing AP-1 adaptor complexes are formed in an ARF1-dependent manner, generating vesicles transporting cargo proteins to endosomes. The mechanism of site-specific targeting of AP-1 and the role of cargo are poorly understood. We have developed an in vitro assay to study the recruitment of purified AP-1 adaptors to chemically defined liposomes presenting peptides corresponding to tyrosine-based sorting motifs. AP-1 recruitment was found to be dependent on myristoylated ARF1, GTP or nonhydrolyzable GTP-analogs, tyrosine signals, and small amounts of phosphoinositides, most prominently phosphatidylinositol 4,5-bisphosphate, in the absence of any additional cytosolic or membrane bound proteins. AP-1 from cytosol could be recruited to a tyrosine signal independently of the lipid composition, but the rate of recruitment was increased by phosphatidylinositol 4,5-bisphosphate. The results thus indicate that cargo proteins are involved in coat recruitment and that the local lipid composition contributes to specifying the site of vesicle formation. PMID:12388765

AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.

PubMed

Setta-Kaffetzi, Niovi; Simpson, Michael A; Navarini, Alexander A; Patel, Varsha M; Lu, Hui-Chun; Allen, Michael H; Duckworth, Michael; Bachelez, Hervé; Burden, A David; Choon, Siew-Eng; Griffiths, Christopher E M; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M B; Prins, Christa; Smahi, Asma; Trembath, Richard C; Fraternali, Franca; Smith, Catherine H; Barker, Jonathan N; Capon, Francesca

2014-05-01

Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval

PubMed Central

Hirst, Jennifer; Itzhak, Daniel N.; Antrobus, Robin; Borner, Georg H. H.

2018-01-01

The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5–associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders. PMID:29381698

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 contribute to neurologic pathologies. Functional screening of genes regulated during growth of noninjured axons revealed CD2AP as a positive regulator of axon outgrowth. A novel association of CD2AP with TrkA and p85 suggests a distinct intracellular signaling pathway regulating growth of noninjured axons. This may also represent a novel mechanism of generating specificity in multifunctional NGF signaling. Divergent regulation of CD2AP in different axon growth conditions suggests that separate mechanisms exist for different modes of axon growth. CD2AP is the first signaling molecule associated with adult sensory axonal collateral sprouting, and this association may offer new insights for NGF/TrkA-related Alzheimer's disease mechanisms. PMID:27076424

Adaptor protein complex 2-mediated, clathrin-dependent endocytosis, and related gene activities, are a prominent feature during maturation stage amelogenesis.

PubMed

Lacruz, Rodrigo S; Brookes, Steven J; Wen, Xin; Jimenez, Jaime M; Vikman, Susanna; Hu, Ping; White, Shane N; Lyngstadaas, S Petter; Okamoto, Curtis T; Smith, Charles E; Paine, Michael L

2013-03-01

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real-time PCR, we show that the expression of clathrin and adaptor protein subunits are upregulated in maturation stage rodent enamel organ cells. AP complex 2 (AP-2) is the most upregulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts, with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin-dependent endocytosis, thus implying the likelihood of specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also upregulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1); cluster of differentiation 63 and 68 (Cd63 and Cd68); ATPase, H(+) transporting, lysosomal V0 subunit D2 (Atp6v0d2); ATPase, H(+) transporting, lysosomal V1 subunit B2 (Atp6v1b2); chloride channel, voltage-sensitive 7 (Clcn7); and cathepsin K (Ctsk). Immunohistologic data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain showed upregulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor-regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation. Copyright © 2013 American Society for Bone and Mineral Research.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga

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{supmore » -}) 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 trafficking of kidney {alpha}-intercalated cells.« less

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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 Central

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

2016-01-01

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

VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3

PubMed Central

Li, Haiyan; Santos, Magda S.; Park, Chihyung K.; Dobry, Yuriy; Voglmaier, Susan M.

2017-01-01

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling. PMID:29123471

VGLUT2 Trafficking Is Differentially Regulated by Adaptor Proteins AP-1 and AP-3.

PubMed

Li, Haiyan; Santos, Magda S; Park, Chihyung K; Dobry, Yuriy; Voglmaier, Susan M

2017-01-01

Release of the major excitatory neurotransmitter glutamate by synaptic vesicle exocytosis depends on glutamate loading into synaptic vesicles by vesicular glutamate transporters (VGLUTs). The two principal isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in adult brain that broadly distinguishes cortical (VGLUT1) and subcortical (VGLUT2) systems, and correlates with distinct physiological properties in synapses expressing these isoforms. Differential trafficking of VGLUT1 and 2 has been suggested to underlie their functional diversity. Increasing evidence suggests individual synaptic vesicle proteins use specific sorting signals to engage specialized biochemical mechanisms to regulate their recycling. We observed that VGLUT2 recycles differently in response to high frequency stimulation than VGLUT1. Here we further explore the trafficking of VGLUT2 using a pHluorin-based reporter, VGLUT2-pH. VGLUT2-pH exhibits slower rates of both exocytosis and endocytosis than VGLUT1-pH. VGLUT2-pH recycling is slower than VGLUT1-pH in both hippocampal neurons, which endogenously express mostly VGLUT1, and thalamic neurons, which endogenously express mostly VGLUT2, indicating that protein identity, not synaptic vesicle membrane or neuronal cell type, controls sorting. We characterize sorting signals in the C-terminal dileucine-like motif, which plays a crucial role in VGLUT2 trafficking. Disruption of this motif abolishes synaptic targeting of VGLUT2 and essentially eliminates endocytosis of the transporter. Mutational and biochemical analysis demonstrates that clathrin adaptor proteins (APs) interact with VGLUT2 at the dileucine-like motif. VGLUT2 interacts with AP-2, a well-studied adaptor protein for clathrin mediated endocytosis. In addition, VGLUT2 also interacts with the alternate adaptors, AP-1 and AP-3. VGLUT2 relies on distinct recycling mechanisms from VGLUT1. Abrogation of these differences by pharmacological and molecular inhibition reveals that these mechanisms are dependent on the adaptor proteins AP-1 and AP-3. Further, shRNA-mediated knockdown reveals differential roles for AP-1 and AP-3 in VGLUT2 recycling.

Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

PubMed Central

LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

2012-01-01

Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together define an endocytotic pathway likely used by ameloblasts to remove the enamel matrix during enamel maturation. PMID:23044750

The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

PubMed Central

Nakatsu, Fubito; Hase, Koji; Ohno, Hiroshi

2014-01-01

The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP)-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis). Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells. PMID:25387275

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

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes

PubMed Central

Valencia, Julio C.; Watabe, Hidenori; Chi, An; Rouzaud, Francois; Chen, Kevin G.; Vieira, Wilfred D.; Takahashi, Kaoruko; Yamaguchi, Yuji; Berens, Werner; Nagashima, Kunio; Shabanowitz, Jeffrey; Hunt, Donald F.; Appella, Ettore; Hearing, Vincent J.

2015-01-01

Summary Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms μ1A and μ1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of μ1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (μ1A or μ1B) showed two distinct distribution patterns that involved Pmel17, and only μ1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking μ1B expression. Finally, we established that expression of μ1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature. PMID:16492709

Adaptor protein complex 2-mediated endocytosis is crucial for male reproductive organ development in Arabidopsis.

PubMed

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

2013-08-01

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.

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

THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS1

PubMed Central

Purdy, Amanda K.; Alvarez-Arias, Diana A.; Oshinsky, Jennifer; James, Ashley M.; Serebriiskii, Ilya; Campbell, Kerry S.

2014-01-01

Stable surface expression of human inhibitory killer cell immunoglobulin-like receptors (KIR) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC-I+ cells. Our recent experiments, however, have found that antibody-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon antibody engagement with the receptor, as compared to untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIR though their ITIM domains. Based upon our results, we propose a model in which non-engaged KIR are internalized by this mechanism, whereas engagement with MHC-I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors. PMID:25238755

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. Copyright © 2015 Elsevier GmbH. All rights reserved.

Serine Phosphorylation of HIV-1 Vpu and Its Binding to Tetherin Regulates Interaction with Clathrin Adaptors

PubMed Central

Sumner, Jonathan C.; Pickering, Suzanne; Neil, Stuart J. D.

2015-01-01

HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu’s mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism. PMID:26317613

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. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Artemisinin resistance in rodent malaria - mutation in the AP2 adaptor μ-chain suggests involvement of endocytosis and membrane protein trafficking

PubMed Central

2013-01-01

Background The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. Methods Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared. Results An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the μ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene. Conclusion An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum. PMID:23561245

Enigma interacts with adaptor protein with PH and SH2 domains to control insulin-induced actin cytoskeleton remodeling and glucose transporter 4 translocation.

PubMed

Barrès, Romain; Grémeaux, Thierry; Gual, Philippe; Gonzalez, Teresa; Gugenheim, Jean; Tran, Albert; Le Marchand-Brustel, Yannick; Tanti, Jean-François

2006-11-01

APS (adaptor protein with PH and SH2 domains) initiates a phosphatidylinositol 3-kinase-independent pathway involved in insulin-stimulated glucose transport. We recently identified Enigma, a PDZ and LIM domain-containing protein, as a partner of APS and showed that APS-Enigma complex plays a critical role in actin cytoskeleton organization in fibroblastic cells. Because actin rearrangement is important for insulin-induced glucose transporter 4 (Glut 4) translocation, we studied the potential involvement of Enigma in insulin-induced glucose transport in 3T3-L1 adipocytes. Enigma mRNA was expressed in differentiated adipocytes and APS and Enigma were colocalized with cortical actin. Expression of an APS mutant unable to bind Enigma increased the insulin-induced Glut 4 translocation to the plasma membrane. By contrast, overexpression of Enigma inhibited insulin-stimulated glucose transport and Glut 4 translocation without alterations in proximal insulin signaling. This inhibitory effect was prevented with the deletion of the LIM domains of Enigma. Using time-lapse fluorescent microscopy of green fluorescent protein-actin, we demonstrated that the overexpression of Enigma altered insulin-induced actin rearrangements, whereas the expression of Enigma without its LIM domains was without effect. A physiological link between increased expression of Enigma and an alteration in insulin-induced glucose uptake was suggested by the increase in Enigma mRNA expression in adipose tissue of diabetic obese patients. Taken together, these data strongly suggest that the interaction between APS and Enigma is involved in insulin-induced Glut 4 translocation by regulating cortical actin remodeling and raise the possibility that modification of APS/Enigma ratio could participate in the alteration of insulin-induced glucose uptake in adipose tissue.

AP2 adaptor complex mediates bile salt export pump internalization and modulates its hepatocanalicular expression and transport function.

PubMed

Hayashi, Hisamitsu; Inamura, Kaori; Aida, Kensuke; Naoi, Sotaro; Horikawa, Reiko; Nagasaka, Hironori; Takatani, Tomozumi; Fukushima, Tamio; Hattori, Asami; Yabuki, Takashi; Horii, Ikuo; Sugiyama, Yuichi

2012-06-01

The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and μ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis. Copyright © 2012 American Association for the Study of Liver Diseases.

BLOC-2, AP-3, and AP-1 Proteins Function in Concert with Rab38 and Rab32 Proteins to Mediate Protein Trafficking to Lysosome-related Organelles*

PubMed Central

Bultema, Jarred J.; Ambrosio, Andrea L.; Burek, Carolyn L.; Di Pietro, Santiago M.

2012-01-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis. PMID:22511774

BLOC-2, AP-3, and AP-1 proteins function in concert with Rab38 and Rab32 proteins to mediate protein trafficking to lysosome-related organelles.

PubMed

Bultema, Jarred J; Ambrosio, Andrea L; Burek, Carolyn L; Di Pietro, Santiago M

2012-06-01

Lysosome-related organelles (LROs) are synthesized in specialized cell types where they largely coexist with conventional lysosomes. Most of the known cellular transport machinery involved in biogenesis are ubiquitously expressed and shared between lysosomes and LROs. Examples of common components are the adaptor protein complex-3 (AP-3) and biogenesis of lysosome-related organelle complex (BLOC)-2. These protein complexes control sorting and transport of newly synthesized integral membrane proteins from early endosomes to both lysosomes and LROs such as the melanosome. However, it is unknown what factors cooperate with the ubiquitous transport machinery to mediate transport to LROs in specialized cells. Focusing on the melanosome, we show that the ubiquitous machinery interacts with cell type-specific Rab proteins, Rab38 and Rab32, to facilitate transport to the maturing organelle. BLOC-2, AP-3, and AP-1 coimmunoprecipitated with Rab38 and Rab32 from MNT-1 melanocytic cell extracts. BLOC-2, AP-3, AP-1, and clathrin partially colocalized with Rab38 and Rab32 by confocal immunofluorescence microscopy in MNT-1 cells. Rab38- and Rab32-deficient MNT-1 cells displayed abnormal trafficking and steady state levels of known cargoes of the BLOC-2, AP-3, and AP-1 pathways, the melanin-synthesizing enzymes tyrosinase and tyrosinase-related protein-1. These observations support the idea that Rab38 and Rab32 are the specific factors that direct the ubiquitous machinery to mediate transport from early endosomes to maturing LROs. Additionally, analysis of tyrosinase-related protein-2 and total melanin production indicates that Rab32 has unique functions that cannot be carried out by Rab38 in melanosome biogenesis.

Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity

PubMed Central

Casson, Cierra N.; Lefkovith, Ariel J.

2017-01-01

Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 –which optimizes toll-like receptor signaling from phagosomes–sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to Salmonella Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1β and IL-18 in response to particulate stimuli in vitro, but caspase-1 and IL-1β levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1β, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients. PMID:29253868

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 autophagy and small GTPase regulation.

Distinct Ubiquitin Binding Modes Exhibited by SH3 Domains: Molecular Determinants and Functional Implications

PubMed Central

Ortega Roldan, Jose L.; Casares, Salvador; Ringkjøbing Jensen, Malene; Cárdenes, Nayra; Bravo, Jerónimo; Blackledge, Martin; Azuaga, Ana I.; van Nuland, Nico A. J.

2013-01-01

SH3 domains constitute a new type of ubiquitin-binding domains. We previously showed that the third SH3 domain (SH3-C) of CD2AP binds ubiquitin in an alternative orientation. We have determined the structure of the complex between first CD2AP SH3 domain and ubiquitin and performed a structural and mutational analysis to decipher the determinants of the SH3-C binding mode to ubiquitin. We found that the Phe-to-Tyr mutation in CD2AP and in the homologous CIN85 SH3-C domain does not abrogate ubiquitin binding, in contrast to previous hypothesis and our findings for the first two CD2AP SH3 domains. The similar alternative binding mode of the SH3-C domains of these related adaptor proteins is characterised by a higher affinity to C-terminal extended ubiquitin molecules. We conclude that CD2AP/CIN85 SH3-C domain interaction with ubiquitin constitutes a new ubiquitin-binding mode involved in a different cellular function and thus changes the previously established mechanism of EGF-dependent CD2AP/CIN85 mono-ubiquitination. PMID:24039852

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

Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex.

PubMed

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

2013-03-29

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.

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

Interaction with epsin 1 regulates the constitutive clathrin-dependent internalization of ErbB3.

PubMed

Szymanska, Monika; Fosdahl, Anne Marthe; Raiborg, Camilla; Dietrich, Markus; Liestøl, Knut; Stang, Espen; Bertelsen, Vibeke

2016-06-01

In contrast to other members of the EGF receptor family, ErbB3 is constitutively internalized in a clathrin-dependent manner. Previous studies have shown that ErbB3 does not interact with the coated pit localized adaptor complex 2 (AP-2), and that ErbB3 lacks two AP-2 interacting internalization signals identified in the EGF receptor. Several other clathrin-associated sorting proteins which may recruit cargo into coated pits have, however, been identified, and the study was performed to identify adaptors needed for constitutive internalization of ErbB3. A high-throughput siRNA screen was used to identify adaptor proteins needed for internalization of ErbB3. Upon knock-down of candidate proteins internalization of ErbB3 was identified using an antibody-based internalization assay combined with automatic fluorescence microscopy. Among 29 candidates only knock-down of epsin 1 turned out to inhibit ErbB3. Epsin 1 has ubiquitin interacting motifs (UIMs) and we show that ErbB3 interacts with an epsin 1 deletion mutant containing these UIMs. In support of an ErbB3-epsin 1 UIM dependent interaction, we show that ErbB3 is constitutively ubiquitinated, but that both ubiquitination and the ErbB3-epsin 1 interaction increase upon ligand binding. Altogether the results are consistent with a model whereby both constitutive and ligand-induced internalization of ErbB3 are regulated through interaction with epsin 1. Internalization is an important regulator of growth factor receptor mediated signaling and the current study identify mechanisms regulating plasma membrane turnover of ErbB3. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

HIV-1 Vpu Antagonizes CD317/Tetherin by Adaptor Protein-1-Mediated Exclusion from Virus Assembly Sites

PubMed Central

Pujol, François M.; Laketa, Vibor; Schmidt, Florian; Mukenhirn, Markus; Müller, Barbara; Boulant, Steeve; Grimm, Dirk; Keppler, Oliver T.

2016-01-01

ABSTRACT The host cell restriction factor CD317/tetherin traps virions at the surface of producer cells to prevent their release. The HIV-1 accessory protein Vpu antagonizes this restriction. Vpu reduces the cell surface density of the restriction factor and targets it for degradation; however, these activities are dispensable for enhancing particle release. Instead, Vpu has been suggested to antagonize CD317/tetherin by preventing recycling of internalized CD317/tetherin to the cell surface, blocking anterograde transport of newly synthesized CD317/tetherin, and/or displacing the restriction factor from virus assembly sites at the plasma membrane. At the molecular level, antagonism relies on the physical interaction of Vpu with CD317/tetherin. Recent findings suggested that phosphorylation of a diserine motif enables Vpu to bind to adaptor protein 1 (AP-1) trafficking complexes via two independent interaction motifs and to couple CD317/tetherin to the endocytic machinery. Here, we used a panel of Vpu proteins with specific mutations in individual interaction motifs to define which interactions are required for antagonism of CD317/tetherin. Impairing recycling or anterograde transport of CD317/tetherin to the plasma membrane was insufficient for antagonism. In contrast, excluding CD317/tetherin from HIV-1 assembly sites depended on Vpu motifs for interaction with AP-1 and CD317/tetherin and correlated with antagonism of the particle release restriction. Consistently, interference with AP-1 function or its expression blocked these Vpu activities. Our results define displacement from HIV-1 assembly sites as active principle of CD317/tetherin antagonism by Vpu and support a role of tripartite complexes between Vpu, AP-1, and CD317/tetherin in this process. IMPORTANCE CD317/tetherin poses an intrinsic barrier to human immunodeficiency virus type 1 (HIV-1) replication in human cells by trapping virus particles at the surface of producer cells and thereby preventing their release. The viral protein Vpu antagonizes this restriction, and molecular interactions with the restriction factor and adaptor protein complex 1 (AP-1) were suggested to mediate this activity. Vpu modulates intracellular trafficking of CD317/tetherin and excludes the restriction factor from HIV-1 assembly sites at the plasma membrane, but the relative contribution of these effects to antagonism remain elusive. Using a panel of Vpu mutants, as well as interference with AP-1 function and expression, we show here that Vpu antagonizes CD317/tetherin by blocking its recruitment to viral assembly sites in an AP-1-dependent manner. These results refine our understanding of the molecular mechanisms of CD317/tetherin antagonism and suggest complexes of Vpu with the restriction factor and AP-1 as targets for potential therapeutic intervention. PMID:27170757

Recessive loss-of-function mutations in AP4S1 cause mild fever-sensitive seizures, developmental delay and spastic paraplegia through loss of AP-4 complex assembly

PubMed Central

Hardies, Katia; May, Patrick; Djémié, Tania; Tarta-Arsene, Oana; Deconinck, Tine; Craiu, Dana; Helbig, Ingo; Suls, Arvid; Balling, Rudy; Weckhuysen, Sarah; De Jonghe, Peter; Hirst, Jennifer; Afawi, Zaid; Barisic, Nina; Baulac, Stéphanie; Caglayan, Hande; Depienne, Christel; De Kovel, Carolien G.F.; Dimova, Petia; Guerrero-López, Rosa; Guerrini, Renzo; Hjalgrim, Helle; Hoffman-Zacharska, Dorota; Jahn, Johanna; Klein, Karl Martin; Koeleman, Bobby P.C.; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes; Lerche, Holger; Marini, Carla; Muhle, Hiltrud; Rosenow, Felix; Serratosa, Jose M.; Møller, Rikke S.; Stephani, Ulrich; Striano, Pasquale; Talvik, Tiina; Von Spiczak, Sarah; Weber, Yvonne; Zara, Federico

2015-01-01

We report two siblings with infantile onset seizures, severe developmental delay and spastic paraplegia, in whom whole-genome sequencing revealed compound heterozygous mutations in the AP4S1 gene, encoding the σ subunit of the adaptor protein complex 4 (AP-4). The effect of the predicted loss-of-function variants (p.Gln46Profs*9 and p.Arg97*) was further investigated in a patient's fibroblast cell line. We show that the premature stop mutations in AP4S1 result in a reduction of all AP-4 subunits and loss of AP-4 complex assembly. Recruitment of the AP-4 accessory protein tepsin, to the membrane was also abolished. In retrospect, the clinical phenotype in the family is consistent with previous reports of the AP-4 deficiency syndrome. Our study reports the second family with mutations in AP4S1 and describes the first two patients with loss of AP4S1 and seizures. We further discuss seizure phenotypes in reported patients, highlighting that seizures are part of the clinical manifestation of the AP-4 deficiency syndrome. We also hypothesize that endosomal trafficking is a common theme between heritable spastic paraplegia and some inherited epilepsies. PMID:25552650

In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.

PubMed

Pagano, Adriana; Crottet, Pascal; Prescianotto-Baschong, Cristina; Spiess, Martin

2004-11-01

The involvement of clathrin and associated adaptor proteins in receptor recycling from endosomes back to the plasma membrane is controversial. We have used an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. Cells expressing the asialoglycoprotein receptor H1, a typical recycling receptor, were surface biotinylated and then allowed to endocytose for 10 min. After stripping away surface-biotin, the cells were permeabilized and the cytosol washed away. In a temperature-, cytosol-, and nucleotide-dependent manner, the formation of sealed vesicles containing biotinylated H1 could be reconstituted. Vesicle formation was strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated and addition of the purified protein strongly inhibited vesicle production. The results indicate that recycling is mediated by AP-1/clathrin-coated vesicles and regulated by rab4 and rabaptin-5/rabex-5.

Molecular Basis for Association of PIPKIγ-p90 with Clathrin Adaptor AP-2*

PubMed Central

Kahlfeldt, Nina; Vahedi-Faridi, Ardeschir; Koo, Seong Joo; Schäfer, Johannes G.; Krainer, Georg; Keller, Sandro; Saenger, Wolfram; Krauss, Michael; Haucke, Volker

2010-01-01

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the Iγ-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P2 metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKIγ-p90 associates with both the μ and β2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKIγ-p90 tail binds to a cognate recognition site on the sandwich subdomain of the β2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2μ, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKIγ-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKIγ tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2β and AP-2μ. Our data also suggest that interactions between AP-2 and the tail domain of PIPKIγ-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKIγ-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P2 synthesis during clathrin-mediated SV endocytosis. PMID:19903820

Several adaptor proteins promote intracellular localisation of the transporter MRP4/ABCC4 in platelets and haematopoietic cells.

PubMed

Schaletzki, Yvonne; Kromrey, Marie-Luise; Bröderdorf, Susanne; Hammer, Elke; Grube, Markus; Hagen, Paul; Sucic, Sonja; Freissmuth, Michael; Völker, Uwe; Greinacher, Andreas; Rauch, Bernhard H; Kroemer, Heyo K; Jedlitschky, Gabriele

2017-01-05

The multidrug resistance protein 4 (MRP4/ABCC4) has been identified as an important transporter for signalling molecules including cyclic nucleotides and several lipid mediators in platelets and may thus represent a novel target to interfere with platelet function. Besides its localisation in the plasma membrane, MRP4 has been also detected in the membrane of dense granules in resting platelets. In polarised cells it is localised at the basolateral or apical plasma membrane. To date, the mechanism of MRP4 trafficking has not been elucidated; protein interactions may regulate both the localisation and function of this transporter. We approached this issue by searching for interacting proteins by in vitro binding assays, followed by immunoblotting and mass spectrometry, and by visualising their co-localisation in platelets and haematopoietic cells. We identified the PDZ domain containing scaffold proteins ezrin-binding protein 50 (EBP50/NHERF1), postsynaptic density protein 95 (PSD95), and sorting nexin 27 (SNX27), but also the adaptor protein complex 3 subunit β3A (AP3B1) and the heat shock protein HSP90 as putative interaction partners of MRP4. The knock-down of SNX27, PSD95, and AP3B1 by siRNA in megakaryoblastic leukaemia cells led to a redistribution of MRP4 from intracellular structures to the plasma membrane. Inhibition of HSP90 led to a diminished expression and retention of MRP4 in the endoplasmic reticulum. These results indicate that MRP4 localisation and function are regulated by multiple protein interactions. Changes in the adaptor proteins can hence lead to altered localisation and function of the transporter.

Two patients with Hermansky Pudlak syndrome type 2 and novel mutations in AP3B1

PubMed Central

Wenham, Matt; Grieve, Samantha; Cummins, Michelle; Jones, Matthew L.; Booth, Sarah; Kilner, Rachel; Ancliff, Philip J.; Griffiths, Gillian M.; Mumford, Andrew D.

2010-01-01

Hermansky Pudlak syndrome type 2 (HPS2) is a rare disorder associated with mutations in the Adaptor Protein 3 (AP-3) complex, which is involved in sorting transmembrane proteins to lysosomes and related organelles. We now report 2 unrelated subjects with HPS2 who show a characteristic clinical phenotype of oculocutaneous albinism, platelet and T-lymphocyte dysfunction and neutropenia. The subjects were homozygous for different deletions within AP3B1 (g.del180242-180866, c.del153-156), which encodes the AP-3β3A subunit, resulting in frame shifts and introduction of nonsense substitutions (p.E693fsX13, p.E52fsX11). In the subject with p.E693fsX13, this resulted in expression of a truncated variant β3A protein. Cytotoxic T-lymphocyte (CTL) clones from both study subjects showed increased cell-surface expression of CD63 and reduced cytotoxicity. Platelets showed impaired aggregation and reduced uptake of 3H-serotonin. These findings are consistent with CTL granule and platelet dense granule defects, respectively. This report extends the clinical and laboratory description of HPS2. PMID:19679886

AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis

PubMed Central

Gu, Mingyu; Liu, Qiang; Watanabe, Shigeki; Sun, Lin; Hollopeter, Gunther; Grant, Barth D; Jorgensen, Erik M

2013-01-01

The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes. DOI: http://dx.doi.org/10.7554/eLife.00190.001 PMID:23482940

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

PubMed

Yoneyama, Yosuke; Lanzerstorfer, Peter; Niwa, Hideaki; Umehara, Takashi; Shibano, Takashi; Yokoyama, Shigeyuki; Chida, Kazuhiro; Weghuber, Julian; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2018-04-11

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling. © 2018, Yoneyama et al.

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling

PubMed Central

Yoneyama, Yosuke; Lanzerstorfer, Peter; Niwa, Hideaki; Umehara, Takashi; Shibano, Takashi; Yokoyama, Shigeyuki; Chida, Kazuhiro; Weghuber, Julian

2018-01-01

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)−1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling. PMID:29661273

Defective pigment granule biogenesis and aberrant behavior caused by mutations in the Drosophila AP-3beta adaptin gene ruby.

PubMed Central

Kretzschmar, D; Poeck, B; Roth, H; Ernst, R; Keller, A; Porsch, M; Strauss, R; Pflugfelder, G O

2000-01-01

Lysosomal protein trafficking is a fundamental process conserved from yeast to humans. This conservation extends to lysosome-like organelles such as mammalian melanosomes and insect eye pigment granules. Recently, eye and coat color mutations in mouse (mocha and pearl) and Drosophila (garnet and carmine) were shown to affect subunits of the heterotetrameric adaptor protein complex AP-3 involved in vesicle trafficking. Here we demonstrate that the Drosophila eye color mutant ruby is defective in the AP-3beta subunit gene. ruby expression was found in retinal pigment and photoreceptor cells and in the developing central nervous system. ruby mutations lead to a decreased number and altered size of pigment granules in various cell types in and adjacent to the retina. Humans with lesions in the related AP-3betaA gene suffer from Hermansky-Pudlak syndrome, which is caused by defects in a number of lysosome-related organelles. Hermansky-Pudlak patients have a reduced skin pigmentation and suffer from internal bleeding, pulmonary fibrosis, and visual system malfunction. The Drosophila AP-3beta adaptin also appears to be involved in processes other than eye pigment granule biogenesis because all ruby allele combinations tested exhibited defective behavior in a visual fixation paradigm. PMID:10790396

CD2-associated Protein (CD2AP) Enhances Casitas B Lineage Lymphoma-3/c (Cbl-3/c)-mediated Ret Isoform-specific Ubiquitination and Degradation via Its Amino-terminal Src Homology 3 Domains*

PubMed Central

Calco, Gina N.; Stephens, Olivia R.; Donahue, Laura M.; Tsui, Cynthia C.; Pierchala, Brian A.

2014-01-01

Ret is the receptor tyrosine kinase for the glial cell line-derived neurotrophic factor (GDNF) family of neuronal growth factors. Upon activation by GDNF, Ret is rapidly polyubiquitinated and degraded. This degradation process is isoform-selective, with the longer Ret51 isoform exhibiting different degradation kinetics than the shorter isoform, Ret9. In sympathetic neurons, Ret degradation is induced, at least in part, by a complex consisting of the adaptor protein CD2AP and the E3-ligase Cbl-3/c. Knockdown of Cbl-3/c using siRNA reduced the GDNF-induced ubiquitination and degradation of Ret51 in neurons and podocytes, suggesting that Cbl-3/c was a predominant E3 ligase for Ret. Coexpression of CD2AP with Cbl-3/c augmented the ubiquitination of Ret51 as compared with the expression of Cbl-3/c alone. Ret51 ubiquitination by the CD2AP·Cbl-3/c complex required a functional ring finger and TKB domain in Cbl-3/c. The SH3 domains of CD2AP were sufficient to drive the Cbl-3/c-dependent ubiquitination of Ret51, whereas the carboxyl-terminal coiled-coil domain of CD2AP was dispensable. Interestingly, activated Ret induced the degradation of CD2AP, but not Cbl-3/c, suggesting a potential inhibitory feedback mechanism. There were only two major ubiquitination sites in Ret51, Lys1060 and Lys1107, and the combined mutation of these lysines almost completely eliminated both the ubiquitination and degradation of Ret51. Ret9 was not ubiquitinated by the CD2AP·Cbl-3/c complex, suggesting that Ret9 was down-regulated by a fundamentally different mechanism. Taken together, these results suggest that only the SH3 domains of CD2AP were necessary to enhance the E3 ligase activity of Cbl-3/c toward Ret51. PMID:24425877

HIV-1 Nef disrupts MHC-I trafficking by recruiting AP-1 to the MHC-I cytoplasmic tail

PubMed Central

Roeth, Jeremiah F.; Williams, Maya; Kasper, Matthew R.; Filzen, Tracey M.; Collins, Kathleen L.

2004-01-01

To avoid immune recognition by cytotoxic T lymphocytes (CTLs), human immunodeficiency virus (HIV)-1 Nef disrupts the transport of major histocompatibility complex class I molecules (MHC-I) to the cell surface in HIV-infected T cells. However, the mechanism by which Nef does this is unknown. We report that Nef disrupts MHC-I trafficking by rerouting newly synthesized MHC-I from the trans-Golgi network (TGN) to lysosomal compartments for degradation. The ability of Nef to target MHC-I from the TGN to lysosomes is dependent on expression of the μ1 subunit of adaptor protein (AP) AP-1A, a cellular protein complex implicated in TGN to endolysosomal pathways. We demonstrate that in HIV-infected primary T cells, Nef promotes a physical interaction between endogenous AP-1 and MHC-I. Moreover, we present data that this interaction uses a novel AP-1 binding site that requires amino acids in the MHC-I cytoplasmic tail. In sum, our evidence suggests that binding of AP-1 to the Nef–MHC-I complex is an important step required for inhibition of antigen presentation by HIV. PMID:15569716

Mutational Analysis of the Adaptor Protein 2 Sigma Subunit (AP2S1) Gene: Search for Autosomal Dominant Hypocalcemia Type 3 (ADH3)

PubMed Central

Rogers, Angela; Nesbit, M. Andrew; Hannan, Fadil M.; Howles, Sarah A.; Gorvin, Caroline M.; Cranston, Treena; Allgrove, Jeremy; Bevan, John S.; Bano, Gul; Brain, Caroline; Datta, Vipan; Grossman, Ashley B.; Hodgson, Shirley V.; Izatt, Louise; Millar-Jones, Lynne; Pearce, Simon H.; Robertson, Lisa; Selby, Peter L.; Shine, Brian; Snape, Katie; Warner, Justin

2014-01-01

Context: Autosomal dominant hypocalcemia (ADH) types 1 and 2 are due to calcium-sensing receptor (CASR) and G-protein subunit-α11 (GNA11) gain-of-function mutations, respectively, whereas CASR and GNA11 loss-of-function mutations result in familial hypocalciuric hypercalcemia (FHH) types 1 and 2, respectively. Loss-of-function mutations of adaptor protein-2 sigma subunit (AP2σ 2), encoded by AP2S1, cause FHH3, and we therefore sought for gain-of-function AP2S1 mutations that may cause an additional form of ADH, which we designated ADH3. Objective: The objective of the study was to investigate the hypothesis that gain-of-function AP2S1 mutations may cause ADH3. Design: The sample size required for the detection of at least one mutation with a greater than 95% likelihood was determined by binomial probability analysis. Nineteen patients (including six familial cases) with hypocalcemia in association with low or normal serum PTH concentrations, consistent with ADH, but who did not have CASR or GNA11 mutations, were ascertained. Leukocyte DNA was used for sequence and copy number variation analysis of AP2S1. Results: Binomial probability analysis, using the assumption that AP2S1 mutations would occur in hypocalcemic patients at a prevalence of 20%, which is observed in FHH patients without CASR or GNA11 mutations, indicated that the likelihood of detecting at least one AP2S1 mutation was greater than 95% and greater than 98% in sample sizes of 14 and 19 hypocalcemic patients, respectively. AP2S1 mutations and copy number variations were not detected in the 19 hypocalcemic patients. Conclusion: The absence of AP2S1 abnormalities in hypocalcemic patients, suggests that ADH3 may not occur or otherwise represents a rare hypocalcemic disorder. PMID:24708097

Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

PubMed

Roskoski, Robert

2005-11-11

Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of interaction with several enzymes and adaptor proteins.

Arabidopsis BPM proteins function as substrate adaptors to a cullin3-based E3 ligase to affect fatty acid metabolism in plants.

PubMed

Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R; Hellmann, Hanjo

2013-06-01

Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that cullin3-based E3 ligases have the potential to interact with a broad range of ethylene response factor (ERF)/APETALA2 (AP2) transcription factors, mediated by Math-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the wrinkled1 ERF/AP2 protein. Furthermore, loss of Math-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members.

Structural and functional characterization of cargo-binding sites on the μ4-subunit of adaptor protein complex 4.

PubMed

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-canonical site of μ4.

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-canonical site of μ4. PMID:24498434

An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

PubMed Central

Skorobogata, Olga; Escobar-Restrepo, Juan M.; Rocheleau, Christian E.

2014-01-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling. PMID:25329472

An AGEF-1/Arf GTPase/AP-1 ensemble antagonizes LET-23 EGFR basolateral localization and signaling during C. elegans vulva induction.

PubMed

Skorobogata, Olga; Escobar-Restrepo, Juan M; Rocheleau, Christian E

2014-10-01

LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling.

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

PubMed Central

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

2016-01-01

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

Adaptor protein 1 B mu subunit does not contribute to the recycling of kAE1 protein in polarized renal epithelial cells.

PubMed

Almomani, Ensaf Y; Touret, Nicolas; Cordat, Emmanuelle

2018-04-13

Mutations in the gene encoding the kidney anion exchanger 1 (kAE1) can lead to distal renal tubular acidosis (dRTA). dRTA mutations reported within the carboxyl (C)-terminal tail of kAE1 result in apical mis-targeting of the exchanger in polarized renal epithelial cells. As kAE1 physically interacts with the μ subunit of epithelial adaptor protein 1 B (AP-1B), we investigated the role of heterologously expressed μ1B subunit of the AP-1B complex for kAE1 retention to the basolateral membrane in polarized porcine LLC-PK1 renal epithelial cells that are devoid of endogenous AP-1B. We confirmed the interaction and close proximity between kAE1 and μ1B using immunoprecipitation and proximity ligation assay, respectively. Expressing the human μ1B subunit in these cells decreased significantly the amount of cell surface kAE1 at the steady state, but had no significant effect on kAE1 recycling and endocytosis. We show that (i) heterologous expression of μ1B displaces the physical interaction of endogenous GAPDH with kAE1 WT supporting that both AP-1B and GAPDH proteins bind to an overlapping site on kAE1 and (ii) phosphorylation of tyrosine 904 within the potential YDEV interaction motif does not alter the kAE1/AP-1B interaction. We conclude that μ1B subunit is not involved in recycling of kAE1.

Partially overlapping distribution of epsin1 and HIP1 at the synapse: analysis by immunoelectron microscopy.

PubMed

Yao, Pamela J; Bushlin, Ittai; Petralia, Ronald S

2006-01-10

Synapses of neurons use clathrin-mediated endocytic pathways for recycling of synaptic vesicles and trafficking of neurotransmitter receptors. Epsin 1 and huntingtin-interacting protein 1 (HIP1) are endocytic accessory proteins. Both proteins interact with clathrin and the AP2 adaptor complex and also bind to the phosphoinositide-containing plasma membrane via an epsin/AP180 N-terminal homology (ENTH/ANTH) domain. Epsin1 and HIP1 are found in neurons; however, their precise roles in synapses remain largely unknown. Using immunogold electron microscopy, we examine and compare the synaptic distribution of epsin1 and HIP1 in rat CA1 hippocampal synapse. We find that epsin1 is located across both sides of the synapse, whereas HIP1 displays a preference for the postsynaptic compartment. Within the synaptic compartments, espin1 is distributed similarly throughout, whereas postsynaptic HIP1 is concentrated near the plasma membrane. Our results suggest a dual role for epsin1 and HIP1 in the synapse: as broadly required factors for promoting clathrin assembly and as adaptors for specific endocytic pathways.

Arabidopsis BPM Proteins Function as Substrate Adaptors to a CULLIN3-Based E3 Ligase to Affect Fatty Acid Metabolism in Plants[W

PubMed Central

Chen, Liyuan; Lee, Joo Hyun; Weber, Henriette; Tohge, Takayuki; Witt, Sandra; Roje, Sanja; Fernie, Alisdair R.; Hellmann, Hanjo

2013-01-01

Regulation of transcriptional processes is a critical mechanism that enables efficient coordination of the synthesis of required proteins in response to environmental and cellular changes. Transcription factors require accurate activity regulation because they play a critical role as key mediators assuring specific expression of target genes. In this work, we show that CULLIN3-based E3 ligases have the potential to interact with a broad range of ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 (AP2) transcription factors, mediated by MATH-BTB/POZ (for Meprin and TRAF [tumor necrosis factor receptor associated factor] homolog)-Broad complex, Tramtrack, Bric-a-brac/Pox virus and Zinc finger) proteins. The assembly with an E3 ligase causes degradation of their substrates via the 26S proteasome, as demonstrated for the WRINKLED1 ERF/AP2 protein. Furthermore, loss of MATH-BTB/POZ proteins widely affects plant development and causes altered fatty acid contents in mutant seeds. Overall, this work demonstrates a link between fatty acid metabolism and E3 ligase activities in plants and establishes CUL3-based E3 ligases as key regulators in transcriptional processes that involve ERF/AP2 family members. PMID:23792371

Arabidopsis adaptor protein 1G is critical for pollen development.

PubMed

Feng, Chong; Wang, Jia-Gang; Liu, Hai-Hong; Li, Sha; Zhang, Yan

2017-09-01

Pollen development is a pre-requisite for sexual reproduction of angiosperms, during which various cellular activities are involved. Pollen development accompanies dynamic remodeling of vacuoles through fission and fusion, disruption of which often compromises pollen viability. We previously reported that the Y subunit of adaptor protein 1 (AP1G) mediates synergid degeneration during pollen tube reception. Here, we demonstrate that AP1G is essential for pollen development. AP1G loss-of-function resulted in male gametophytic lethality due to defective pollen development. By ultrastructural analysis and fluorescence labeling, we demonstrate that AP1G loss-of-function compromised dynamic vacuolar remodeling during pollen development and impaired vacuolar acidification of pollen. Results presented here support a key role of vacuoles in gametophytic pollen development. © 2017 Institute of Botany, Chinese Academy of Sciences.

Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system

PubMed Central

Yang, Xi; Arines, Felichi Mae; Zhang, Weichao

2018-01-01

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors. PMID:29355480

HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

PubMed

Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui; Lee, Il-Hyung; Hurley, James H

2015-10-23

The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat. Copyright © 2015, American Association for the Advancement of Science.

Clathrin- and AP-2-binding sites in HIP1 uncover a general assembly role for endocytic accessory proteins.

PubMed

Mishra, S K; Agostinelli, N R; Brett, T J; Mizukami, I; Ross, T S; Traub, L M

2001-12-07

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.

Loss of AP-5 results in accumulation of aberrant endolysosomes: defining a new type of lysosomal storage disease

PubMed Central

Hirst, Jennifer; Edgar, James R.; Esteves, Typhaine; Darios, Frédéric; Madeo, Marianna; Chang, Jaerak; Roda, Ricardo H.; Dürr, Alexandra; Anheim, Mathieu; Gellera, Cinzia; Li, Jun; Züchner, Stephan; Mariotti, Caterina; Stevanin, Giovanni; Blackstone, Craig; Kruer, Michael C.; Robinson, Margaret S.

2015-01-01

Adaptor proteins (AP 1–5) are heterotetrameric complexes that facilitate specialized cargo sorting in vesicular-mediated trafficking. Mutations in AP5Z1, encoding a subunit of the AP-5 complex, have been reported to cause hereditary spastic paraplegia (HSP), although their impact at the cellular level has not been assessed. Here we characterize three independent fibroblast lines derived from skin biopsies of patients harbouring nonsense mutations in AP5Z1 and presenting with spastic paraplegia accompanied by neuropathy, parkinsonism and/or cognitive impairment. In all three patient-derived lines, we show that there is complete loss of AP-5 ζ protein and a reduction in the associated AP-5 µ5 protein. Using ultrastructural analysis, we show that these patient-derived lines consistently exhibit abundant multilamellar structures that are positive for markers of endolysosomes and are filled with aberrant storage material organized as exaggerated multilamellar whorls, striated belts and ‘fingerprint bodies’. This phenotype can be replicated in a HeLa cell culture model by siRNA knockdown of AP-5 ζ. The cellular phenotype bears striking resemblance to features described in a number of lysosomal storage diseases (LSDs). Collectively, these findings reveal an emerging picture of the role of AP-5 in endosomal and lysosomal homeostasis, illuminates a potential pathomechanism that is relevant to the role of AP-5 in neurons and expands the understanding of recessive HSPs. Moreover, the resulting accumulation of storage material in endolysosomes leads us to propose that AP-5 deficiency represents a new type of LSDs. PMID:26085577

Integrative Cardiac Health Project

DTIC Science & Technology

2014-10-01

Cathelicidin antimicrobial peptide CAMP –1.27 Defense response 200615_s_at Adaptor-related protein complex 2, β1 AP2B1 –1.26 Protein transport, defense response...from self-reported 72-hour dietary recall questionnaires. Food Processor v8.4.0 (ESHA Research) was used to determine daily caloric intake and...JR, Reitman M, et al. The effect of food intake on gene expression in human peripheral blood. Hum Mol Genet. 2010;19:159–169. 47. Chon H, Gaillard

Protein kinase A-induced internalization of Slack channels from the neuronal membrane occurs by adaptor protein-2/clathrin-mediated endocytosis.

PubMed

Gururaj, Sushmitha; Evely, Katherine M; Pryce, Kerri D; Li, Jun; Qu, Jun; Bhattacharjee, Arin

2017-11-24

The sodium-activated potassium (K Na ) channel Kcnt1 (Slack) is abundantly expressed in nociceptor (pain-sensing) neurons of the dorsal root ganglion (DRG), where they transmit the large outward conductance I KNa and arbitrate membrane excitability. Slack channel expression at the DRG membrane is necessary for their characteristic firing accommodation during maintained stimulation, and reduced membrane channel density causes hyperexcitability. We have previously shown that in a pro-inflammatory state, a decrease in membrane channel expression leading to reduced Slack-mediated I KNa expression underlies DRG neuronal sensitization. An important component of the inflammatory milieu, PKA internalizes Slack channels from the DRG membrane, reduces I KNa , and produces DRG neuronal hyperexcitability when activated in cultured primary DRG neurons. Here, we show that this PKA-induced retrograde trafficking of Slack channels also occurs in intact spinal cord slices and that it is carried out by adaptor protein-2 (AP-2) via clathrin-mediated endocytosis. We provide mass spectrometric and biochemical evidence of an association of native neuronal AP-2 adaptor proteins with Slack channels, facilitated by a dileucine motif housed in the cytoplasmic Slack C terminus that binds AP-2. By creating a competitive peptide blocker of AP-2-Slack binding, we demonstrated that this interaction is essential for clathrin recruitment to the DRG membrane, Slack channel endocytosis, and DRG neuronal hyperexcitability after PKA activation. Together, these findings uncover AP-2 and clathrin as players in Slack channel regulation. Given the significant role of Slack in nociceptive neuronal excitability, the AP-2 clathrin-mediated endocytosis trafficking mechanism may enable targeting of peripheral and possibly, central neuronal sensitization. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Drosophila pigmentation gene pink (p) encodes a homologue of human Hermansky-Pudlak syndrome 5 (HPS5).

PubMed

Falcón-Pérez, Juan M; Romero-Calderón, Rafael; Brooks, Elizabeth S; Krantz, David E; Dell'Angelica, Esteban C

2007-02-01

Lysosome-related organelles comprise a group of specialized intracellular compartments that include melanosomes and platelet dense granules (in mammals) and eye pigment granules (in insects). In humans, the biogenesis of these organelles is defective in genetic disorders collectively known as Hermansky-Pudlak syndrome (HPS). Patients with HPS-2, and two murine HPS models, carry mutations in genes encoding subunits of adaptor protein (AP)-3. Other genes mutated in rodent models include those encoding VPS33A and Rab38. Orthologs of all of these genes in Drosophila melanogaster belong to the 'granule group' of eye pigmentation genes. Other genes associated with HPS encode subunits of three complexes of unknown function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2 and -3, for which the Drosophila counterparts had not been characterized. Here, we report that the gene encoding the Drosophila ortholog of the HPS5 subunit of BLOC-2 is identical to the granule group gene pink (p), which was first studied in 1910 but had not been identified at the molecular level. The phenotype of pink mutants was exacerbated by mutations in AP-3 subunits or in the orthologs of VPS33A and Rab38. These results validate D. melanogaster as a genetic model to study the function of the BLOCs.

Contrasting roles of DAP10 and KARAP/DAP12 signaling adaptors in activation of the RBL-2H3 leukemic mast cell line.

PubMed

Anfossi, Nicolas; Lucas, Mathias; Diefenbach, Andreas; Bühring, Hans-Jörg; Raulet, David; Tomasello, Elena; Vivier, Eric

2003-12-01

A common feature of hematopoietic activating immunoreceptors resides in their association at the cell surface with transmembrane signaling adaptors. Several adaptors, such as the CD3 molecules, FcRgamma and KARAP/DAP12, harbor intracytoplasmic immunoreceptor tyrosine-based activation motifs (ITAM) that activate Syk-family protein tyrosine kinases. In contrast, another transmembrane adaptor, DAP10, bears a YxxM motif that delivers signals by activation of lipid kinase pathways. We show here that the human signal-regulatory protein SIRPbeta1 can associate with both DAP10 and KARAP/DAP12 in a model of RBL-2H3 cell transfectants. In association with KARAP/DAP12, SIRPbeta1 complexes are capable of inducing serotonin release and tumor necrosis factor (TNF) secretion. By contrast,in the absence of KARAP/DAP12, engagement of SIRPbeta1:DAP10 complexes does not lead to detectable serotonin release or TNF secretion by RBL-2H3 transfectants. However, triggering of SIRPbeta1:DAP10 complexes co-stimulates RBL-2H3 effector function induced by sub-optimal stimulation of the endogenous FcepsilonRI complex. Therefore, we report here a cellular model in which the association of a cell surface receptor with various signaling adaptors dictates the co-stimulatory or the direct stimulatory properties of the complex.

Functional Analysis of AP-2 α and μ2 Subunits

PubMed Central

Motley, Alison M.; Berg, Nicola; Taylor, Marcus J.; Sahlender, Daniela A.; Hirst, Jennifer; Owen, David J.

2006-01-01

The AP-2 adaptor complex plays a key role in cargo recognition and clathrin-coated vesicle formation at the plasma membrane. To investigate the functions of individual binding sites and domains of the AP-2 complex in vivo, we have stably transfected HeLa cells with wild-type and mutant small interfering RNA–resistant α and μ2 subunits and then used siRNA knockdowns to deplete the endogenous proteins. Mutating the PtdIns(4,5)P2 binding site of α, the phosphorylation site of μ2, or the YXXΦ binding site of μ2 impairs AP-2 function, as assayed by transferrin uptake. In contrast, removing the C-terminal appendage domain of α, or mutating the PtdIns(4,5)P2 binding site of μ2, has no apparent effect. However, adding a C-terminal GFP tag to α renders it completely nonfunctional. These findings demonstrate that there is some functional redundancy in the binding sites of the various AP-2 subunits, because no single mutation totally abolishes function. They also help to explain why GFP-tagged AP-2 never appears to leave the plasma membrane in some live cell imaging studies. Finally, they establish a new model system that can be used both for additional structure-function analyses, and as a way of testing tagged constructs for function in vivo. PMID:17035630

Early stages of clathrin aggregation at a membrane in coarse-grained simulations

NASA Astrophysics Data System (ADS)

Giani, M.; den Otter, W. K.; Briels, W. J.

2017-04-01

The self-assembly process of clathrin coated pits during endocytosis has been simulated by combining and extending coarse grained models of the clathrin triskelion, the adaptor protein AP2, and a flexible network membrane. The AP2's core, upon binding to membrane and cargo, releases a motif that can bind clathrin. In conditions where the core-membrane-cargo binding is weak, the binding of this motif to clathrin can result in a stable complex. We characterize the conditions and mechanisms resulting in the formation of clathrin lattices that curve the membrane, i.e., clathrin coated pits. The mechanical properties of the AP2 β linker appear crucial to the orientation of the curved clathrin lattice relative to the membrane, with wild-type short linkers giving rise to the inward curving buds enabling endocytosis while long linkers produce upside-down cages and outward curving bulges.

Mapping the interactome of HPV E6 and E7 oncoproteins with the ubiquitin-proteasome system.

PubMed

Poirson, Juline; Biquand, Elise; Straub, Marie-Laure; Cassonnet, Patricia; Nominé, Yves; Jones, Louis; van der Werf, Sylvie; Travé, Gilles; Zanier, Katia; Jacob, Yves; Demeret, Caroline; Masson, Murielle

2017-10-01

Protein ubiquitination and its reverse reaction, deubiquitination, regulate protein stability, protein binding activity, and their subcellular localization. These reactions are catalyzed by the enzymes E1, E2, and E3 ubiquitin (Ub) ligases and deubiquitinases (DUBs). The Ub-proteasome system (UPS) is targeted by viruses for the sake of their replication and to escape host immune response. To identify novel partners of human papillomavirus 16 (HPV16) E6 and E7 proteins, we assembled and screened a library of 590 cDNAs related to the UPS by using the Gaussia princeps luciferase protein complementation assay. HPV16 E6 was found to bind to the homology to E6AP C terminus-type Ub ligase (E6AP), three really interesting new gene (RING)-type Ub ligases (MGRN1, LNX3, LNX4), and the DUB Ub-specific protease 15 (USP15). Except for E6AP, the binding of UPS factors did not require the LxxLL-binding pocket of HPV16 E6. LNX3 bound preferentially to all high-risk mucosal HPV E6 tested, whereas LNX4 bound specifically to HPV16 E6. HPV16 E7 was found to bind to several broad-complex tramtrack and bric-a-brac domain-containing proteins (such as TNFAIP1/KCTD13) that are potential substrate adaptors of Cullin 3-RING Ub ligases, to RING-type Ub ligases implicated in innate immunity (RNF135, TRIM32, TRAF2, TRAF5), to the substrate adaptor DCAF15 of Cullin 4-RING Ub ligase and to some DUBs (USP29, USP33). The binding to UPS factors did not require the LxCxE motif but rather the C-terminal region of HPV16 E7 protein. The identified UPS factors interacted with most of E7 proteins across different HPV types. This study establishes a strategy for the rapid identification of interactions between host or pathogen proteins and the human ubiquitination system. © 2017 Federation of European Biochemical Societies.

Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex

PubMed Central

Elbediwy, Ahmed; Zihni, Ceniz; Terry, Stephen J.; Clark, Peter

2012-01-01

Epithelial cell–cell adhesion and morphogenesis require dynamic control of actin-driven membrane remodeling. The Rho guanosine triphosphatase (GTPase) Cdc42 regulates sequential molecular processes during cell–cell junction formation; hence, mechanisms must exist that inactivate Cdc42 in a temporally and spatially controlled manner. In this paper, we identify SH3BP1, a GTPase-activating protein for Cdc42 and Rac, as a regulator of junction assembly and epithelial morphogenesis using a functional small interfering ribonucleic acid screen. Depletion of SH3BP1 resulted in loss of spatial control of Cdc42 activity, stalled membrane remodeling, and enhanced growth of filopodia. SH3BP1 formed a complex with JACOP/paracingulin, a junctional adaptor, and CD2AP, a scaffolding protein; both were required for normal Cdc42 signaling and junction formation. The filamentous actin–capping protein CapZ also associated with the SH3BP1 complex and was required for control of actin remodeling. Epithelial junction formation and morphogenesis thus require a dual activity complex, containing SH3BP1 and CapZ, that is recruited to sites of active membrane remodeling to guide Cdc42 signaling and cytoskeletal dynamics. PMID:22891260

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

PubMed Central

Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

2001-01-01

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

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

Specificity in Toll-like receptor signalling through distinct effector functions of TRAF3 and TRAF6.

PubMed

Häcker, Hans; Redecke, Vanessa; Blagoev, Blagoy; Kratchmarova, Irina; Hsu, Li-Chung; Wang, Gang G; Kamps, Mark P; Raz, Eyal; Wagner, Hermann; Häcker, Georg; Mann, Matthias; Karin, Michael

2006-01-12

Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains, but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-beta) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

NASA Astrophysics Data System (ADS)

Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

2017-04-01

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

PubMed

Beautrait, Alexandre; Paradis, Justine S; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M; Khoury, Etienne; Audet, Martin; Roux, Philippe P; Veprintsev, Dmitry B; Laporte, Stéphane A; Bouvier, Michel

2017-04-18

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

PubMed Central

Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

2017-01-01

In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways. PMID:28416805

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2

PubMed Central

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-01-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of possible 18 α-chains and one of possible 8 β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalised by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalisation by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with AP2 C-µ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions. PMID:26779610

Selective integrin endocytosis is driven by interactions between the integrin α-chain and AP2.

PubMed

De Franceschi, Nicola; Arjonen, Antti; Elkhatib, Nadia; Denessiouk, Konstantin; Wrobel, Antoni G; Wilson, Thomas A; Pouwels, Jeroen; Montagnac, Guillaume; Owen, David J; Ivaska, Johanna

2016-02-01

Integrins are heterodimeric cell-surface adhesion molecules comprising one of 18 possible α-chains and one of eight possible β-chains. They control a range of cell functions in a matrix- and ligand-specific manner. Integrins can be internalized by clathrin-mediated endocytosis (CME) through β subunit-based motifs found in all integrin heterodimers. However, whether specific integrin heterodimers can be selectively endocytosed was unknown. Here, we found that a subset of α subunits contain an evolutionarily conserved and functional YxxΦ motif directing integrins to selective internalization by the most abundant endocytic clathrin adaptor, AP2. We determined the structure of the human integrin α4-tail motif in complex with the AP2 C-μ2 subunit and confirmed the interaction by isothermal titration calorimetry. Mutagenesis of the motif impaired selective heterodimer endocytosis and attenuated integrin-mediated cell migration. We propose that integrins evolved to enable selective integrin-receptor turnover in response to changing matrix conditions.

It takes two to tango to the melanosome

PubMed Central

Lakkaraju, Aparna; Carvajal-Gonzalez, Jose Maria

2009-01-01

The role of clathrin adaptor proteins in sorting cargo in the biosynthetic and recycling routes is an area of intense research. In this issue, Delevoye et al. (2009. J. Cell Biol. doi:10.1083/jcb.200907122) show that a close interaction between the clathrin adaptor AP-1 and a kinesin motor KIF13A is essential for delivering melanogenic enzymes from recycling endosomes to nascent melanosomes and for organelle biogenesis. PMID:19841135

Distinct conformations of the protein complex p97-Ufd1-Npl4 revealed by electron cryomicroscopy

PubMed Central

Bebeacua, Cecilia; Förster, Andreas; McKeown, Ciarán; Meyer, Hemmo H.; Zhang, Xiaodong; Freemont, Paul S.

2012-01-01

p97 is a key regulator of numerous cellular pathways and associates with ubiquitin-binding adaptors to remodel ubiquitin-modified substrate proteins. How adaptor binding to p97 is coordinated and how adaptors contribute to substrate remodeling is unclear. Here we present the 3D electron cryomicroscopy reconstructions of the major Ufd1-Npl4 adaptor in complex with p97. Our reconstructions show that p97-Ufd1-Npl4 is highly dynamic and that Ufd1-Npl4 assumes distinct positions relative to the p97 ring upon addition of nucleotide. Our results suggest a model for substrate remodeling by p97 and also explains how p97-Ufd1-Npl4 could form other complexes in a hierarchical model of p97-cofactor assembly. PMID:22232657

Regulation of synaptic activity by snapin-mediated endolysosomal transport and sorting

PubMed Central

Di Giovanni, Jerome; Sheng, Zu-Hang

2015-01-01

Recycling synaptic vesicles (SVs) transit through early endosomal sorting stations, which raises a fundamental question: are SVs sorted toward endolysosomal pathways? Here, we used snapin mutants as tools to assess how endolysosomal sorting and trafficking impact presynaptic activity in wild-type and snapin−/− neurons. Snapin acts as a dynein adaptor that mediates the retrograde transport of late endosomes (LEs) and interacts with dysbindin, a subunit of the endosomal sorting complex BLOC-1. Expressing dynein-binding defective snapin mutants induced SV accumulation at presynaptic terminals, mimicking the snapin−/− phenotype. Conversely, over-expressing snapin reduced SV pool size by enhancing SV trafficking to the endolysosomal pathway. Using a SV-targeted Ca2+ sensor, we demonstrate that snapin–dysbindin interaction regulates SV positional priming through BLOC-1/AP-3-dependent sorting. Our study reveals a bipartite regulation of presynaptic activity by endolysosomal trafficking and sorting: LE transport regulates SV pool size, and BLOC-1/AP-3-dependent sorting fine-tunes the Ca2+ sensitivity of SV release. Therefore, our study provides new mechanistic insights into the maintenance and regulation of SV pool size and synchronized SV fusion through snapin-mediated LE trafficking and endosomal sorting. PMID:26108535

New Insights to Clathrin and Adaptor Protein 2 for the Design and Development of Therapeutic Strategies

PubMed Central

Poulsen, Ebbe Toftgaard; Larsen, Agnete; Zollo, Alen; Jørgensen, Arne L.; Sanggaard, Kristian W.; Enghild, Jan J.; Matrone, Carmela

2015-01-01

The Amyloid Precursor Protein (APP) has been extensively studied for its role as the precursor of the β-amyloid protein (Aβ) in Alzheimer’s disease (AD). However, our understanding of the normal function of APP is still patchy. Emerging evidence indicates that a dysfunction in APP trafficking and degradation can be responsible for neuronal deficits and progressive degeneration in humans. We recently reported that the Y682 mutation in the 682YENPTY687 domain of APP affects its binding to specific adaptor proteins and leads to its anomalous trafficking, to defects in the autophagy machinery and to neuronal degeneration. In order to identify adaptors that influence APP function, we performed pull-down experiments followed by quantitative mass spectrometry (MS) on hippocampal tissue extracts of three month-old mice incubated with either the 682YENPTY687 peptide, its mutated form, 682GENPTY687 or its phosphorylated form, 682pYENPTY687. Our experiments resulted in the identification of two proteins involved in APP internalization and trafficking: Clathrin heavy chain (hc) and its Adaptor Protein 2 (AP-2). Overall our results consolidate and refine the importance of Y682 in APP normal functions from an animal model of premature aging and dementia. Additionally, they open the perspective to consider Clathrin hc and AP-2 as potential targets for the design and development of new therapeutic strategies. PMID:26690411

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification

PubMed Central

Dejonghe, Wim; Kuenen, Sabine; Mylle, Evelien; Vasileva, Mina; Keech, Olivier; Viotti, Corrado; Swerts, Jef; Fendrych, Matyáš; Ortiz-Morea, Fausto Andres; Mishev, Kiril; Delang, Simon; Scholl, Stefan; Zarza, Xavier; Heilmann, Mareike; Kourelis, Jiorgos; Kasprowicz, Jaroslaw; Nguyen, Le Son Long; Drozdzecki, Andrzej; Van Houtte, Isabelle; Szatmári, Anna-Mária; Majda, Mateusz; Baisa, Gary; Bednarek, Sebastian York; Robert, Stéphanie; Audenaert, Dominique; Testerink, Christa; Munnik, Teun; Van Damme, Daniël; Heilmann, Ingo; Schumacher, Karin; Winne, Johan; Friml, Jiří; Verstreken, Patrik; Russinova, Eugenia

2016-01-01

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. PMID:27271794

HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2

PubMed Central

Lubben, Nienke B.; Sahlender, Daniela A.; Motley, Alison M.; Lehner, Paul J.; Benaroch, Philippe

2007-01-01

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2–depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. PMID:17581864

In vitro binding of the asialoglycoprotein receptor to the beta adaptin of plasma membrane coated vesicles.

PubMed Central

Beltzer, J P; Spiess, M

1991-01-01

The asialoglycoprotein (ASGP) receptor was used to probe total clathrin-coated vesicle proteins and purified adaptor proteins (APs) which had been fractionated by gel electrophoresis and transferred to nitrocellulose. The receptor was found to interact with proteins of approximately 100 kDa. The cytoplasmic domain of the ASGP receptor subunit H1 fused to dihydrofolate reductase competed for receptor binding to the 100 kDa polypeptide in the plasma membrane-type AP complexes (AP-2). A fusion protein containing the cytoplasmic domain of the endocytic mutant haemagglutinin HA-Y543 also competed, but a protein with the wild-type haemagglutinin sequence did not. This indicates that the observed interaction is specific for the cytoplasmic domain of the receptor and involves the tyrosine signal for endocytosis. When fractionated by gel electrophoresis in the presence of urea, the ASGP receptor binding polypeptide displayed a characteristic shift in electrophoretic mobility identifying it as the beta adaptin. Partial proteolysis of the AP-2 preparation followed by the receptor binding assay revealed that the aminoterminal domain of the beta adaptin contains the binding site for receptors. Images PMID:1935897

Altered distribution of ATG9A and accumulation of axonal aggregates in neurons from a mouse model of AP-4 deficiency syndrome

PubMed Central

De Pace, Raffaella; Damme, Markus; Mattera, Rafael; Jarnik, Michal; Hoffmann, Victoria; Morris, H. Douglas; Han, Tae-Un; Mancini, Grazia M. S.; Buonanno, Andrés

2018-01-01

The hereditary spastic paraplegias (HSP) are a clinically and genetically heterogeneous group of disorders characterized by progressive lower limb spasticity. Mutations in subunits of the heterotetrameric (ε-β4-μ4-σ4) adaptor protein 4 (AP-4) complex cause an autosomal recessive form of complicated HSP referred to as “AP-4 deficiency syndrome”. In addition to lower limb spasticity, this syndrome features intellectual disability, microcephaly, seizures, thin corpus callosum and upper limb spasticity. The pathogenetic mechanism, however, remains poorly understood. Here we report the characterization of a knockout (KO) mouse for the AP4E1 gene encoding the ε subunit of AP-4. We find that AP-4 ε KO mice exhibit a range of neurological phenotypes, including hindlimb clasping, decreased motor coordination and weak grip strength. In addition, AP-4 ε KO mice display a thin corpus callosum and axonal swellings in various areas of the brain and spinal cord. Immunohistochemical analyses show that the transmembrane autophagy-related protein 9A (ATG9A) is more concentrated in the trans-Golgi network (TGN) and depleted from the peripheral cytoplasm both in skin fibroblasts from patients with mutations in the μ4 subunit of AP-4 and in various neuronal types in AP-4 ε KO mice. ATG9A mislocalization is associated with increased tendency to accumulate mutant huntingtin (HTT) aggregates in the axons of AP-4 ε KO neurons. These findings indicate that the AP-4 ε KO mouse is a suitable animal model for AP-4 deficiency syndrome, and that defective mobilization of ATG9A from the TGN and impaired autophagic degradation of protein aggregates might contribute to neuroaxonal dystrophy in this disorder. PMID:29698489

A Distributed Set of Interactions Controls μ2 Functionality in the Role of AP-2 as a Sorting Adaptor in Synaptic Vesicle Endocytosis*♦

PubMed Central

Kim, Sung Hyun; Ryan, Timothy A.

2009-01-01

The mechanisms of how, following exocytosis, the approximately nine types of synaptic vesicle (SV) transmembrane proteins are accurately resorted to form SVs are poorly understood. The time course of SV endocytosis is very sensitive to perturbations in clathrin and dynamin, supporting the model that SV endocytosis occurs through a clathrin-mediated pathway. We recently demonstrated that removal of the clathrin adaptor protein AP-2, the key protein thought to coordinate cargo selection into clathrin-coated pits, results in a significant impairment in endocytosis kinetics. Endocytosis, however, still proceeds in the absence of AP-2, bringing into question the role of AP-2 in cargo sorting in this process. Using quantitative endocytosis assays at nerve terminals, we examined how endocytosis depends on the integrity of μ2 function. Our experiments indicate that no single perturbation in μ2 prevents restoration of endocytic function when mutated μ2 replaces native μ2, whereas introduction of multiple distributed mutations significantly impairs endocytosis. We also examined whether the presence of AP-2 is important for the functionality of the previously identified endocytic motif in an SV cargo protein, the dileucine motif in vGlut-1. These data show that while mutations in the dileucine motif slow the retrieval of vGlut-1, they only do so in the presence of AP-2. These data thus indicate that AP-2 plays a role in cargo selection but that no single aspect of μ2 function is critical, implying that a more distributed network of interactions supports AP-2 function in SV endocytosis. PMID:19762466

Nuclear relocation of the nephrin and CD2AP-binding protein dendrin promotes apoptosis of podocytes

PubMed Central

Asanuma, Katsuhiko; Campbell, Kirk Nicholas; Kim, Kwanghee; Faul, Christian; Mundel, Peter

2007-01-01

Kidney podocytes and their slit diaphragms (SDs) form the final barrier to urinary protein loss. There is mounting evidence that SD proteins also participate in intracellular signaling pathways. The SD protein nephrin serves as a component of a signaling complex that directly links podocyte junctional integrity to actin cytoskeletal dynamics. Another SD protein, CD2-associated protein (CD2AP), is an adaptor molecule involved in podocyte homeostasis that can repress proapoptotic TGF-β signaling in podocytes. Here we show that dendrin, a protein originally identified in telencephalic dendrites, is a constituent of the SD complex, where it directly binds to nephrin and CD2AP. In experimental glomerulonephritis, dendrin relocates from the SD to the nucleus of injured podocytes. High-dose, proapoptotic TGF-β1 directly promotes the nuclear import of dendrin, and nuclear dendrin enhances both staurosporine- and TGF-β1-mediated apoptosis. In summary, our results identify dendrin as an SD protein with proapoptotic signaling properties that accumulates in the podocyte nucleus in response to glomerular injury and provides a molecular target to tackle proteinuric kidney diseases. Nuclear relocation of dendrin may provide a mechanism whereby changes in SD integrity could translate into alterations of podocyte survival under pathological conditions. PMID:17537921

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

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

PubMed

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.

Crystal structure of the C-terminal SH3 domain of the adaptor protein GADS in complex with SLP-76 motif peptide reveals a unique SH3-SH3 interaction.

PubMed

Dimasi, Nazzareno

2007-01-01

The Grb2-like adaptor protein GADS is essential for tyrosine kinase-dependent signaling in T lymphocytes. Following T cell receptor ligation, GADS interacts through its C-terminal SH3 domain with the adaptors SLP-76 and LAT, to form a multiprotein signaling complex that is crucial for T cell activation. To understand the structural basis for the selective recognition of GADS by SLP-76, herein is reported the crystal structure at 1.54 Angstrom of the C-terminal SH3 domain of GADS bound to the SLP-76 motif 233-PSIDRSTKP-241, which represents the minimal binding site. In addition to the unique structural features adopted by the bound SLP-76 peptide, the complex structure reveals a unique SH3-SH3 interaction. This homophilic interaction, which is observed in presence of the SLP-76 peptide and is present in solution, extends our understanding of the molecular mechanisms that could be employed by modular proteins to increase their signaling transduction specificity.

Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy.

PubMed

Zhou, Changqian; Ma, Kaili; Gao, Ruize; Mu, Chenglong; Chen, Linbo; Liu, Qiangqiang; Luo, Qian; Feng, Du; Zhu, Yushan; Chen, Quan

2017-02-01

Autophagy requires diverse membrane sources and involves membrane trafficking of mATG9, the only membrane protein in the ATG family. However, the molecular regulation of mATG9 trafficking for autophagy initiation remains unclear. Here we identified two conserved classic adaptor protein sorting signals within the cytosolic N-terminus of mATG9, which mediate trafficking of mATG9 from the plasma membrane and trans-Golgi network (TGN) via interaction with the AP1/2 complex. Src phosphorylates mATG9 at Tyr8 to maintain its endocytic and constitutive trafficking in unstressed conditions. In response to starvation, phosphorylation of mATG9 at Tyr8 by Src and at Ser14 by ULK1 functionally cooperate to promote interactions between mATG9 and the AP1/2 complex, leading to redistribution of mATG9 from the plasma membrane and juxta-nuclear region to the peripheral pool for autophagy initiation. Our findings uncover novel mechanisms of mATG9 trafficking and suggest a coordination of basal and stress-induced autophagy.

Disruption of AP3B1 by a chromosome 5 inversion: a new disease mechanism in Hermansky-Pudlak syndrome type 2.

PubMed

Jones, Matthew L; Murden, Sherina L; Brooks, Claire; Maloney, Viv; Manning, Richard A; Gilmour, Kimberly C; Bharadwaj, Vandana; de la Fuente, Josu; Chakravorty, Subarna; Mumford, Andrew D

2013-04-04

Hermansky-Pudlak syndrome 2 (HPS2; OMIM #608233) is a rare, autosomal recessive disorder caused by loss-of-function genetic variations affecting AP3B1, which encodes the β3A subunit of the adaptor-related protein complex 3 (AP3). Phenotypic characteristics include reduced pigmentation, absent platelet dense granule secretion, neutropenia and reduced cytotoxic T lymphocyte (CTL) and natural killer (NK) cell function. To date HPS2 has been associated with non-synonymous, stop-gain or deletion-insertion nucleotide variations within the coding region of AP3B1. We describe a consanguineous female infant with reduced pigmentation, neutropenia and recurrent infections. Platelets displayed reduced aggregation and absent ATP secretion in response to collagen and ADP, indicating a platelet dense granule defect. There was increased basal surface expression of CD107a (lysosome-associated membrane protein 1(LAMP-1)) on NK cells and CTLs from the study subject and a smaller increase in the percentage of CD107a positive cells after stimulation compared to most healthy controls. Immunoblotting of protein extracts from EBV-transformed lymphoblasts from the index case showed absent expression of full-length AP-3 β3A subunit protein, confirming a phenotypic diagnosis of HPS2.The index case displayed a homozygous pericentric inv(5)(p15.1q14.1), which was also detected as a heterozygous defect in both parents of the index case. No loss of genetic material was demonstrated by microarray comparative genome hybridisation at 60kb resolution. Fluorescence in-situ hybridisation using the 189.6kb probe RP11-422I12, which maps to 5q14.1, demonstrated dual hybridisation to both 5q14.1 and 5p15.1 regions of the inverted Chr5. The RP11-422I12 probe maps from intron 1 to intron 16 of AP3B1, thus localising the 5q inversion breakpoint to within AP3B1. The probe RP11-211K15, which corresponds to an intergenic region on 5p also showed dual hybridisation, enabling localisation of the 5p inversion breakpoint. This case report extends the phenotypic description of the very rare disorder HPS2. Our demonstration of a homozygous Chr5 inversion predicted to disrupt AP3B1 gene provides a novel pathogenic mechanism for this disorder.

Utilising the EGFR interactome to identify mechanisms of drug resistance in non-small cell lung cancer - Proof of concept towards a systems pharmacology approach.

PubMed

Saafan, Hisham; Foerster, Sarah; Parra-Guillen, Zinnia P; Hammer, Elke; Michaelis, Martin; Cinatl, Jindrich; Völker, Uwe; Fröhlich, Holger; Kloft, Charlotte; Ritter, Christoph A

2016-10-30

Drug treatment of epidermal growth factor receptor (EGFR) positive non-small cell lung cancer has improved substantially by targeting activating mutations within the receptor tyrosine kinase domain. However, the development of drug resistance still limits this approach. As root causes, large heterogeneity between tumour entities but also within tumour cells have been suggested. Therefore, approaches to identify these multitude and complex mechanisms are urgently required. Affinity purification coupled with high resolution mass spectrometry was applied to isolate and characterise the EGFR interactome from HCC4006 non-small cell lung cancer cells and their variant HCC4006 r ERLO 0.5 adapted to grow in the presence of therapeutically relevant concentrations of erlotinib. Bioinformatics analyses were carried out to identify proteins and their related molecular functions that interact differentially with EGFR in the untreated state or when incubated with erlotinib prior to EGFR activation. Across all experimental conditions 375 proteins were detected to participate in the EGFR interactome, 90% of which constituted a complex protein interaction network that was bioinformatically reconstructed from literature data. Treatment of HCC4006 r ERLO 0.5 cells carrying a resistance phenotype to erlotinib was associated with an increase of protein levels of members of the clathrin-associated adaptor protein family AP2 (AP2A1, AP2A2, AP2B1), structural proteins of cytoskeleton rearrangement as well as signalling molecules such as Shc. Validation experiments confirmed activation of the Ras-Raf-Mek-Erk (MAPK)-pathway, of which Shc is an initiating adaptor molecule, in HCC4006 r ERLO 0.5 cells. Taken together, differential proteins in the EGFR interactome of HCC4006 r ERLO 0.5 cells were identified that could be related to multiple resistance mechanisms including alterations in growth factor receptor expression, cellular remodelling processes suggesting epithelial-to-mesenchymal transition as well as alterations in downstream signalling. Knowledge of these mechanisms is a pivotal step to build an integrative model of drug resistance in a systems pharmacology manner and to be able to investigate the interplay of these mechanisms and ultimately recommend combinatorial treatment strategies to overcome drug resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

Arabidopsis thaliana BTB/ POZ-MATH proteins interact with members of the ERF/AP2 transcription factor family.

PubMed

Weber, Henriette; Hellmann, Hanjo

2009-11-01

In Arabidopsis thaliana, the BTB/POZ-MATH (BPM) proteins comprise a small family of six members. They have been described previously to use their broad complex, tram track, bric-a-brac/POX virus and zinc finger (BTB/POZ) domain to assemble with CUL3a and CUL3b and potentially to serve as substrate adaptors to cullin-based E3-ligases in plants. In this article, we show that BPMs can also assemble with members of the ethylene response factor/Apetala2 transcription factor family, and that this is mediated by their meprin and TRAF (tumor necrosis factor receptor-associated factor) homology (MATH) domain. In addition, we provide a detailed description of BPM gene expression patterns in different tissues and on abiotic stress treatments, as well as their subcellular localization. This work connects, for the first time, BPM proteins with ethylene response factor/Apetala2 family members, which is likely to represent a novel regulatory mechanism of transcriptional control.

BLOC-3 Mutated in Hermansky-Pudlak Syndrome Is a Rab32/38 Guanine Nucleotide Exchange Factor

PubMed Central

Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A.

2012-01-01

Summary Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting [1–3]. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles [2]. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes [4–6]. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious [7–9]. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38 [10–12]. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. PMID:23084991

Compartment-specific control of signaling from a DNA-sensing immune receptor.

PubMed

Engel, Alex; Barton, Gregory M

2010-11-30

Many cell signaling events are spatially organized, enabling control of specificity, amplitude, and duration. Toll-like receptor 9 (TLR9) binds to nucleic acid sequences present in bacteria or DNA viruses and initiates a signaling pathway that culminates in the transcriptional induction of genes important for host defense, such as those encoding proinflammatory cytokines and type I interferon. A specialized membrane trafficking pathway has been described that is required for a specific branch of TLR9 signaling: the production of type I interferon. Cells deficient for the clathrin adaptor complex AP-3 failed to traffic TLR9 to a specific endosomal compartment and were unable to produce type I interferon despite normal increases in the abundance of interleukin-12p40, a proinflammatory cytokine. These findings support a model in which the targets of TLR9 engagement are controlled by the compartment in which TLR9 is activated.

Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro.

PubMed

Sullivan, James A; Lewis, Michael J; Nikko, Elina; Pelham, Hugh R B

2007-07-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1.

Multiple Interactions Drive Adaptor-Mediated Recruitment of the Ubiquitin Ligase Rsp5 to Membrane Proteins In Vivo and In Vitro

PubMed Central

Sullivan, James A.; Lewis, Michael J.; Nikko, Elina

2007-01-01

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain “PY” motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY–WW interactions is required for the ubiquitination of Smf1. PMID:17429078

Triphenylmethane dye activation of beta-arrestin.

PubMed

Barak, Larry S; Bai, Yushi; Snyder, Joshua C; Wang, Jiangbo; Chen, Wei; Caron, Marc G

2013-08-13

β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy.

Triphenylmethane Dye Activation of Beta-Arrestin

PubMed Central

2013-01-01

β-Arrestins regulate G protein-coupled receptor signaling as competitive inhibitors and protein adaptors. Low molecular weight biased ligands that bind receptors and discriminate between the G protein dependent arm and β-arrestin, clathrin-associated arm of receptor signaling are considered therapeutically valuable as a result of this distinctive pharmacological behavior. Other than receptor agonists, compounds that activate β-arrestins are not available. We show that within minutes of exposure to the cationic triphenylmethane dyes malachite green and brilliant green, tissue culture cells recruit β-arrestins to clathrin scaffolds in a receptor-activation independent manner. In the presence of these compounds, G protein signaling is inhibited, ERK and GSK3β signaling are preserved, and the recruitment of the beta2-adaptin, AP2 adaptor complex to clathrin as well as transferrin internalization is reduced. Moreover, malachite green binds β-arrestin2-GFP coated immunotrap beads relative to GFP only coated beads. Triphenylmethane dyes are FDA approved for topical use on newborns as components of triple-dye preparations and are not approved but used effectively as aqueous antibiotics in fish husbandry. As possible carcinogens, their chronic ingestion in food preparations, particularly through farmed fish, is discouraged in the U.S. and Europe. Our results indicate triphenylmethane dyes as a result of novel pharmacology may have additional roles as β-arrestin/clathrin pathway signaling modulators in both pharmacology research and clinical therapy. PMID:23865508

Chromosome localization of human genes for clathrin adaptor polypeptides AP2{beta} and AP50 and the clathrin-binding protein, VCP

DOE Office of Scientific and Technical Information (OSTI.GOV)

Druck, T.; Gu, Y.; Prabhala, G.

1995-11-01

Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed {open_quotes}assembly proteins,{close_quotes} or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2{alpha} and AP2{beta}, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2{beta} (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with A1`s. Chromosomal in situ hybridization of a human genomic clonemore » demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2{beta} fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. 13 refs., 2 figs.« less

Structural basis for the binding of tryptophan-based motifs by δ-COP

PubMed Central

Suckling, Richard J.; Poon, Pak Phi; Travis, Sophie M.; Majoul, Irina V.; Hughson, Frederick M.; Evans, Philip R.; Duden, Rainer; Owen, David J.

2015-01-01

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing. PMID:26578768

CVAK104 is a Novel Regulator of Clathrin-mediated SNARE Sorting

PubMed Central

Borner, Georg H H; Rana, Amer A; Forster, Rebecca; Harbour, Michael; Smith, James C; Robinson, Margaret S

2007-01-01

Clathrin-coated vesicles (CCVs) mediate transport between the plasma membrane, endosomes and the trans Golgi network. Using comparative proteomics, we have identified coated-vesicle-associated kinase of 104 kDa (CVAK104) as a candidate accessory protein for CCV-mediated trafficking. Here, we demonstrate that the protein colocalizes with clathrin and adaptor protein-1 (AP-1), and that it is associated with a transferrin-positive endosomal compartment. Consistent with these observations, clathrin as well as the cargo adaptors AP-1 and epsinR can be coimmunoprecipitated with CVAK104. Small interfering RNA (siRNA) knockdown of CVAK104 in HeLa cells results in selective loss of the SNARE proteins syntaxin 8 and vti1b from CCVs. Morpholino-mediated knockdown of CVAK104 in Xenopus tropicalis causes severe developmental defects, including a bent body axis and ventral oedema. Thus, CVAK104 is an evolutionarily conserved protein involved in SNARE sorting that is essential for normal embryonic development. PMID:17587408

HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2.

PubMed

Metzler, M; Legendre-Guillemin, V; Gan, L; Chopra, V; Kwok, A; McPherson, P S; Hayden, M R

2001-10-19

Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

ATP6V1H regulates the growth and differentiation of bone marrow stromal cells.

PubMed

Li, Lin; Yang, Shaoqing; Zhang, Yanli; Ji, Dongrui; Jin, Zuolin; Duan, Xiaohong

2018-05-18

ATP6V1H encodes subunit H of vacuolar ATPase (V-ATPase) and may regulate osteoclastic function. The deficiency of ATP6V1H caused bone loss in human, mouse and zebrafish. In this report, we identified the mechanisms by which ATP6V1H regulates proliferation and differentiation of bone marrow stromal cells (BMSCs). We found that ATP6V1H was expressed in BMSCs, andAtp6v1h +/- BMSCs exhibited the lower proliferation rate, cell cycle arrest and reduced osteogenic differentiation capacity, as well as the increased adipogenic potentials. Histologic analysis confirmed less bone formation and more fatty degeneration in Atp6v1h +/- mice in the different age groups. Q-PCR analysis revealed that loss of ATP6V1H function downregulated the mRNA level of TGF-β1 receptor, and its binding molecule, subunit β of adaptor protein complex 2 (AP-2), suggesting ATP6V1H regulates the proliferation and differentiation of BMSCs by interacting with TGF-β receptor I and AP-2 complex. Copyright © 2018. Published by Elsevier Inc.

The antiporter-like subunit constituent of the universal adaptor of complex I, group 4 membrane-bound [NiFe]-hydrogenases and related complexes.

PubMed

Batista, Ana P; Marreiros, Bruno C; Pereira, Manuela M

2013-05-01

We have recently investigated the long-recognized relationship between complex I and group 4 [NiFe] hydrogenases and we have established the so-called Energy-converting hydrogenase related (Ehr) complex as a new member of the family. We have also observed that four subunits, homologues to NuoB, D, H and L, are common to the members of the family. We have designated this common group of subunits the universal adaptor. Taking into account the similarity of the Na(+)/H(+) antiporter-like subunits of complex I (NuoL, NuoM and NuoN) and the unique structural characteristic of the long amphipathic α helix part of NuoL, the nature of the antiporter-like subunit of the universal adaptor was questioned. Thus, in this work we further explore the properties of the universal adaptor, investigating which antiporter-like subunit is part of the universal adaptor. We observe that the universal adaptor contains an antiporter-like subunit with a long amphipathic α helix, similar to NuoL. Consequently, the long helix is a common denominator that has been conserved in all members of the family. Such conservation surely reflects the key role of such helix in the energy transduction mechanism of this family of enzymes.

Getting to the core of cadherin complex function in Caenorhabditis elegans.

PubMed

Hardin, Jeff

2015-01-01

The classic cadherin-catenin complex (CCC) mediates cell-cell adhesion in metazoans. Although substantial insights have been gained by studying the CCC in vertebrate tissue culture, analyzing requirements for and regulation of the CCC in vertebrates remains challenging. Caenorhabditis elegans is a powerful system for connecting the molecular details of CCC function with functional requirements in a living organism. Recent data, using an "angstroms to embryos" approach, have elucidated functions for key residues, conserved across all metazoans, that mediate cadherin/β-catenin binding. Other recent work reveals a novel, potentially ancestral, role for the C. elegans p120ctn homologue in regulating polarization of blastomeres in the early embryo via Cdc42 and the partitioning-defective (PAR)/atypical protein kinase C (aPKC) complex. Finally, recent work suggests that the CCC is trafficked to the cell surface via the clathrin adaptor protein complex 1 (AP-1) in surprising ways. These studies continue to underscore the value of C. elegans as a model system for identifying conserved molecular mechanisms involving the CCC.

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress*

PubMed Central

Tong, Jiefei; Taylor, Paul; Moran, Michael F.

2014-01-01

Aberrant expression, activation, and stabilization of epidermal growth factor receptor (EGFR) are causally associated with several human cancers. Post-translational modifications and protein-protein interactions directly modulate the signaling and trafficking of the EGFR. Activated EGFR is internalized by endocytosis and then either recycled back to the cell surface or degraded in the lysosome. EGFR internalization and recycling also occur in response to stresses that activate p38 MAP kinase. Mass spectrometry was applied to comprehensively analyze the phosphorylation, ubiquitination, and protein-protein interactions of wild type and endocytosis-defective EGFR variants before and after internalization in response to EGF ligand and stress. Prior to internalization, EGF-stimulated EGFR accumulated ubiquitin at 7 K residues and phosphorylation at 7 Y sites and at S1104. Following internalization, these modifications diminished and there was an accumulation of S/T phosphorylations. EGFR internalization and many but not all of the EGF-induced S/T phosphorylations were also stimulated by anisomycin-induced cell stress, which was not associated with receptor ubiquitination or elevated Y phosphorylation. EGFR protein interactions were dramatically modulated by ligand, internalization, and stress. In response to EGF, different E3 ubiquitin ligases became maximally associated with EGFR before (CBL, HUWE1, and UBR4) or after (ITCH) internalization, whereas CBLB was distinctively most highly EGFR associated following anisomycin treatment. Adaptin subunits of AP-1 and AP-2 clathrin adaptor complexes also became EGFR associated in response to EGF and anisomycin stress. Mutations preventing EGFR phosphorylation at Y998 or in the S1039 region abolished or greatly reduced EGFR interactions with AP-2 and AP-1, and impaired receptor trafficking. These results provide new insight into spatial, temporal, and mechanistic aspects of EGFR regulation. PMID:24797263

Cdk5 regulates PSD-95 ubiquitination in neurons

PubMed Central

Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

2011-01-01

The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

AP1S2 is mutated in X-linked Dandy-Walker malformation with intellectual disability, basal ganglia disease and seizures (Pettigrew syndrome).

PubMed

Cacciagli, Pierre; Desvignes, Jean-Pierre; Girard, Nadine; Delepine, Marc; Zelenika, Diana; Lathrop, Mark; Lévy, Nicolas; Ledbetter, David H; Dobyns, William B; Villard, Laurent

2014-03-01

MRXS5 or Pettigrew syndrome was described 20 years ago in a four generation family including nine affected individuals presenting with facial dysmorphism, intellectual disability, Dandy-Walker malformation and inconstant choreoathetosis. Four individuals had iron deposition in the basal ganglia seen on MRI or at autopsy. The mutation causing Pettigrew has remained elusive since the initial description of the condition. We report the identification of a mutation in the X-linked AP1S2 gene in the original Pettigrew syndrome family using X-chromosome exome sequencing. We report additional phenotype details for several of the affected individuals, allowing us to further refine the phenotype corresponding to this X-linked intellectual disability syndrome. The AP1S2 c.426+1 G>T mutation segregates with the disease in the Pettigrew syndrome family and results in loss of 46 amino acids in the clathrin adaptor complex small chain domain that spans most of the AP1S2 protein sequence. The mutation reported here in AP1S2 is the first mutation that is not predicted to cause a premature termination of the coding sequence or absence of the AP1S2 protein. Although most of the families affected by a mutation in AP1S2 were initially described as having different disorders assigned to at least three different OMIM numbers (MIM 300629, 300630 and 304340), our analysis of the phenotype shows that they are all the same syndrome with recognition complicated by highly variable expressivity that is seen within as well as between families and is probably not explained by differences in mutation severity.

AP1S2 is mutated in X-linked Dandy–Walker malformation with intellectual disability, basal ganglia disease and seizures (Pettigrew syndrome)

PubMed Central

Cacciagli, Pierre; Desvignes, Jean-Pierre; Girard, Nadine; Delepine, Marc; Zelenika, Diana; Lathrop, Mark; Lévy, Nicolas; Ledbetter, David H; Dobyns, William B; Villard, Laurent

2014-01-01

MRXS5 or Pettigrew syndrome was described 20 years ago in a four generation family including nine affected individuals presenting with facial dysmorphism, intellectual disability, Dandy–Walker malformation and inconstant choreoathetosis. Four individuals had iron deposition in the basal ganglia seen on MRI or at autopsy. The mutation causing Pettigrew has remained elusive since the initial description of the condition. We report the identification of a mutation in the X-linked AP1S2 gene in the original Pettigrew syndrome family using X-chromosome exome sequencing. We report additional phenotype details for several of the affected individuals, allowing us to further refine the phenotype corresponding to this X-linked intellectual disability syndrome. The AP1S2 c.426+1 G>T mutation segregates with the disease in the Pettigrew syndrome family and results in loss of 46 amino acids in the clathrin adaptor complex small chain domain that spans most of the AP1S2 protein sequence. The mutation reported here in AP1S2 is the first mutation that is not predicted to cause a premature termination of the coding sequence or absence of the AP1S2 protein. Although most of the families affected by a mutation in AP1S2 were initially described as having different disorders assigned to at least three different OMIM numbers (MIM 300629, 300630 and 304340), our analysis of the phenotype shows that they are all the same syndrome with recognition complicated by highly variable expressivity that is seen within as well as between families and is probably not explained by differences in mutation severity. PMID:23756445

Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes.

PubMed

Swamy, Mahima; Kulathu, Yogesh; Ernst, Sandra; Reth, Michael; Schamel, Wolfgang W A

2006-04-15

SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.

Expression, Refolding and Crystallizations of the Grb2-like (GADS) C-Terminal SH3 Domain Complexed with a SLP-76 Motif Peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli,A.; Dimasi, N.

The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.

Clathrin-mediated endocytosis is a candidate entry sorting mechanism for Bombyx mori cypovirus.

PubMed

Chen, Fei; Zhu, Liyuan; Zhang, Yiling; Kumar, Dhiraj; Cao, Guangli; Hu, Xiaolong; Liang, Zi; Kuang, Sulan; Xue, Renyu; Gong, Chengliang

2018-05-08

Bombyx mori cypovirus (BmCPV), a member of the Reoviridae, specifically infects silkworms and causes extensive economic losses to the sericulture industry. To date, the entry mechanism of BmCPV into cells is unclear. Here we used electron microscopy to study the route of entry of BmCPV into cells, and the results demonstrated that the entry of BmCPV into BmN cells was mediated by endocytosis. Blocking the entry pathway with four endocytosis inhibitors, including dansylcadaverine, chlorpromazine, genistein, and PP2, significantly decreased the infectivity of BmCPV. This indicates that BmCPV enters BmN cells via endocytosis, and that clathrin-mediated sorting is the predominant entry method. After the relative expression levels of clathrin heavy chain (clathrin, GenBank accession No. NM_001142971.1) and the adaptor protein complex-1 gamma subunit AP-1 (AP-1, GenBank accession No. JQ824201.1), which are involved in clathrin-mediated endocytosis, were inhibited by RNA interference or abolishing the functions of clathrin and AP-1 with their corresponding antibodies, the infectivity of BmCPV was reduced significantly, which suggests that clathrin-mediated endocytosis contributed to the entry of BmCPV into cells. Our findings suggest that the clathrin-mediated endocytosis pathway is a candidate for the development of therapeutics for silkworm cytoplasmic polyhedrosis.

CALM Regulates Clathrin-Coated Vesicle Size and Maturation by Directly Sensing and Driving Membrane Curvature

PubMed Central

Miller, Sharon E.; Mathiasen, Signe; Bright, Nicholas A.; Pierre, Fabienne; Kelly, Bernard T.; Kladt, Nikolay; Schauss, Astrid; Merrifield, Christien J.; Stamou, Dimitrios; Höning, Stefan; Owen, David J.

2015-01-01

Summary The size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake. PMID:25898166

DNA Origami Scaffolds as Templates for Functional Tetrameric Kir3 K+ Channels.

PubMed

Kurokawa, Tatsuki; Kiyonaka, Shigeki; Nakata, Eiji; Endo, Masayuki; Koyama, Shohei; Mori, Emiko; Tran, Nam Ha; Dinh, Huyen; Suzuki, Yuki; Hidaka, Kumi; Kawata, Masaaki; Sato, Chikara; Sugiyama, Hiroshi; Morii, Takashi; Mori, Yasuo

2018-03-01

In native systems, scaffolding proteins play important roles in assembling proteins into complexes to transduce signals. This concept is yet to be applied to the assembly of functional transmembrane protein complexes in artificial systems. To address this issue, DNA origami has the potential to serve as scaffolds that arrange proteins at specific positions in complexes. Herein, we report that Kir3 K + channel proteins are assembled through zinc-finger protein (ZFP)-adaptors at specific locations on DNA origami scaffolds. Specific binding of the ZFP-fused Kir3 channels and ZFP-based adaptors on DNA origami were confirmed by atomic force microscopy and gel electrophoresis. Furthermore, the DNA origami with ZFP binding sites nearly tripled the K + channel current activity elicited by heterotetrameric Kir3 channels in HEK293T cells. Thus, our method provides a useful template to control the oligomerization states of membrane protein complexes in vitro and in living cells. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faravelli, Alessandro; Dimasi, Nazzareno, E-mail: ndimasi@gmail.com

Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli,more » refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized.« less

Identification and Targeting of an Interaction between a Tyrosine Motif within Hepatitis C Virus Core Protein and AP2M1 Essential for Viral Assembly

PubMed Central

Ziv-Av, Amotz; Gerber, Doron; Jacob, Yves; Einav, Shirit

2012-01-01

Novel therapies are urgently needed against hepatitis C virus infection (HCV), a major global health problem. The current model of infectious virus production suggests that HCV virions are assembled on or near the surface of lipid droplets, acquire their envelope at the ER, and egress through the secretory pathway. The mechanisms of HCV assembly and particularly the role of viral-host protein-protein interactions in mediating this process are, however, poorly understood. We identified a conserved heretofore unrecognized YXXΦ motif (Φ is a bulky hydrophobic residue) within the core protein. This motif is homologous to sorting signals within host cargo proteins known to mediate binding of AP2M1, the μ subunit of clathrin adaptor protein complex 2 (AP-2), and intracellular trafficking. Using microfluidics affinity analysis, protein-fragment complementation assays, and co-immunoprecipitations in infected cells, we show that this motif mediates core binding to AP2M1. YXXΦ mutations, silencing AP2M1 expression or overexpressing a dominant negative AP2M1 mutant had no effect on HCV RNA replication, however, they dramatically inhibited intra- and extracellular infectivity, consistent with a defect in viral assembly. Quantitative confocal immunofluorescence analysis revealed that core's YXXΦ motif mediates recruitment of AP2M1 to lipid droplets and that the observed defect in HCV assembly following disruption of core-AP2M1 binding correlates with accumulation of core on lipid droplets, reduced core colocalization with E2 and reduced core localization to trans-Golgi network (TGN), the presumed site of viral particles maturation. Furthermore, AAK1 and GAK, serine/threonine kinases known to stimulate binding of AP2M1 to host cargo proteins, regulate core-AP2M1 binding and are essential for HCV assembly. Last, approved anti-cancer drugs that inhibit AAK1 or GAK not only disrupt core-AP2M1 binding, but also significantly inhibit HCV assembly and infectious virus production. These results validate viral-host interactions essential for HCV assembly and yield compounds for pharmaceutical development. PMID:22916011

The MYO6 interactome reveals adaptor complexes coordinating early endosome and cytoskeletal dynamics.

PubMed

O'Loughlin, Thomas; Masters, Thomas A; Buss, Folma

2018-04-01

The intracellular functions of myosin motors requires a number of adaptor molecules, which control cargo attachment, but also fine-tune motor activity in time and space. These motor-adaptor-cargo interactions are often weak, transient or highly regulated. To overcome these problems, we use a proximity labelling-based proteomics strategy to map the interactome of the unique minus end-directed actin motor MYO6. Detailed biochemical and functional analysis identified several distinct MYO6-adaptor modules including two complexes containing RhoGEFs: the LIFT (LARG-Induced F-actin for Tethering) complex that controls endosome positioning and motility through RHO-driven actin polymerisation; and the DISP (DOCK7-Induced Septin disPlacement) complex, a novel regulator of the septin cytoskeleton. These complexes emphasise the role of MYO6 in coordinating endosome dynamics and cytoskeletal architecture. This study provides the first in vivo interactome of a myosin motor protein and highlights the power of this approach in uncovering dynamic and functionally diverse myosin motor complexes. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Src-like adaptor protein regulates TCR expression on thymocytes by linking the ubiquitin ligase c-Cbl to the TCR complex.

PubMed

Myers, Margaret D; Sosinowski, Tomasz; Dragone, Leonard L; White, Carmen; Band, Hamid; Gu, Hua; Weiss, Arthur

2006-01-01

The adaptor molecule SLAP and E3 ubiquitin ligase c-Cbl each regulate expression of T cell receptor (TCR)-CD3 on thymocytes. Here we provide genetic and biochemical evidence that both molecules function in the same pathway. TCR-CD3 expression was similar in the absence of SLAP and/or c-Cbl. SLAP and c-Cbl were found to interact, and their expression together downregulated CD3epsilon. This required multiple domains in SLAP and the ring finger of c-Cbl. Furthermore, expression of SLAP and c-Cbl together induced TCRzeta ubiquitination and degradation, preventing the accumulation of fully assembled recycling TCR complexes. These studies indicate that SLAP links the E3 ligase activity of c-Cbl to the TCR, allowing for stage-specific regulation of TCR expression.

Structure-Guided Design of Peptides as Tools to Probe the Protein-Protein Interaction between Cullin-2 and Elongin BC Substrate Adaptor in Cullin RING E3 Ubiquitin Ligases.

PubMed

Cardote, Teresa A F; Ciulli, Alessio

2017-09-21

Cullin RING E3 ubiquitin ligases (CRLs) are large dynamic multi-subunit complexes that control the fate of many proteins in cells. CRLs are attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. Herein we describe a structure-guided biophysical approach to probe the protein-protein interaction (PPI) between the Cullin-2 scaffold protein and the adaptor subunits Elongin BC within the context of the von Hippel-Lindau complex (CRL2 VHL ) using peptides. Two peptides were shown to bind at the targeted binding site on Elongin C, named the "EloC site", with micromolar dissociation constants, providing a starting point for future optimization. Our results suggest ligandability of the EloC binding site to short linear peptides, unveiling the opportunity and challenges to develop small molecules that have the potential to target selectively the Cul2-adaptor PPI within CRLs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Trinuclear alkyl hydrido rare-earth complexes supported by amidopyridinato ligands: synthesis, structures, C-Si bond activation and catalytic activity in ethylene polymerization.

PubMed

Lyubov, Dmitry M; Cherkasov, Anton V; Fukin, Georgy K; Ketkov, Sergey Yu; Shavyrin, Andrey S; Trifonov, Alexander A

2014-10-14

The reaction of Ap(9Me)Lu(CH2SiMe3)2(thf) (Ap(9Me) = (2,4,6-trimethylphenyl)[6-(2,4,6-triisopropylphenyl)pyridine-2-yl]amido ligand) with two molar equivalents of PhSiH3 affords a trinuclear alkyl-hydrido cluster [(Ap(9Me)Lu)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2]. The analogous reactions with Ap(9Me)Ln(CH2SiMe3)2(thf) (Ln = Y, Yb) are more complex and result in the formation of mixtures of two types of trinuclear alkyl-hydrido complexes [(Ap(9Me)Ln)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2] and [(Ap(9Me)Ln)3(μ(2)-H)3(μ(3)-H)2(CH2SiH2Ph)(thf)2] differing in the alkyl group. The DFT calculations of [(Ap*Y)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2] (Ap* = (2,6-diisopropylphenyl)[6-(2,4,6-triisopropylphenyl)pyridine-2-yl]amido ligand) confirm localization of the HOMO on the Ap*-Y(1A)-CH2SiMe3 fragment, thus explaining its enhanced reactivity. Analysis of the electron density distribution reveals the Y-H and H-H bonding interactions in the (Y)3(μ(2)-H)3(μ(3)-H)2 moiety. The NMR studies of diamagnetic complexes [(Ap(9Me)Lu)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2] and [(Ap*Y)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2] demonstrated that the trinuclear cores are retained in the solution and revealed exchange between μ(3)- and μ(2)-bridging hydrido ligands. Complexes [(Ap*Ln)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2], the cationic yttrium hydrido cluster [(Ap*Y)3(μ(2)-H)3(μ(3)-H)2(thf)3](+)[B(C6F5)4](-) as well as [(Ap(9Me)Ln)3(μ(2)-H)3(μ(3)-H)2(CH2SiMe3)(thf)2] proved to be active in catalysis of ethylene polymerization under mild conditions.

Internal amino acids promote Gap1 permease ubiquitylation via TORC1/Npr1/14-3-3-dependent control of the Bul arrestin-like adaptors.

PubMed

Merhi, Ahmad; André, Bruno

2012-11-01

Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.

Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells.

PubMed

Gillard, Ghislain; Shafaq-Zadah, Massiullah; Nicolle, Ophélie; Damaj, Raghida; Pécréaux, Jacques; Michaux, Grégoire

2015-05-01

E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis. © 2015. Published by The Company of Biologists Ltd.

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth.

PubMed

Loyer, Nicolas; Kolotuev, Irina; Pinot, Mathieu; Le Borgne, Roland

2015-10-13

Intercellular bridges called "ring canals" (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs' anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin-containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs' anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin-dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth.

Drosophila E-cadherin is required for the maintenance of ring canals anchoring to mechanically withstand tissue growth

PubMed Central

Loyer, Nicolas; Kolotuev, Irina; Pinot, Mathieu; Le Borgne, Roland

2015-01-01

Intercellular bridges called “ring canals” (RCs) resulting from incomplete cytokinesis play an essential role in intercellular communication in somatic and germinal tissues. During Drosophila oogenesis, RCs connect the maturing oocyte to nurse cells supporting its growth. Despite numerous genetic screens aimed at identifying genes involved in RC biogenesis and maturation, how RCs anchor to the plasma membrane (PM) throughout development remains unexplained. In this study, we report that the clathrin adaptor protein 1 (AP-1) complex, although dispensable for the biogenesis of RCs, is required for the maintenance of the anchorage of RCs to the PM to withstand the increased membrane tension associated with the exponential tissue growth at the onset of vitellogenesis. Here we unravel the mechanisms by which AP-1 enables the maintenance of RCs’ anchoring to the PM during size expansion. We show that AP-1 regulates the localization of the intercellular adhesion molecule E-cadherin and that loss of AP-1 causes the disappearance of the E-cadherin–containing adhesive clusters surrounding the RCs. E-cadherin itself is shown to be required for the maintenance of the RCs’ anchorage, a function previously unrecognized because of functional compensation by N-cadherin. Scanning block-face EM combined with transmission EM analyses reveals the presence of interdigitated, actin- and Moesin-positive, microvilli-like structures wrapping the RCs. Thus, by modulating E-cadherin trafficking, we show that the sustained E-cadherin–dependent adhesion organizes the microvilli meshwork and ensures the proper attachment of RCs to the PM, thereby counteracting the increasing membrane tension induced by exponential tissue growth. PMID:26424451

A Structure-Based Mechanism for Arf1-Dependent Recruitment of Coatomer to Membranes

PubMed Central

Yu, Xinchao; Breitman, Marianna; Goldberg, Jonathan

2012-01-01

Summary Budding of COPI-coated vesicles from Golgi membranes requires an Arf-family G protein and the coatomer complex recruited from cytosol. Arf is also required with coatomer-related clathrin adaptor complexes to bud vesicles from the trans-Golgi network and endosomal compartments. To understand the structural basis for Arf-dependent recruitment of a vesicular coat to the membrane, we determined the structure of Arf1 bound to the γζ-COP subcomplex of coatomer. Structure-guided biochemical analysis reveals that a second Arf1-GTP molecule binds to βδ-COP at a site common to the γ- and β-COP subunits. The Arf1-binding sites on coatomer are spatially related to PtdIns4,5P2-binding sites on the endocytic AP2 complex, providing evidence that the orientation of membrane binding is general for this class of vesicular coat proteins. A bivalent GTP-dependent binding mode has implications for the dynamics of coatomer interaction with the Golgi and for the selection of cargo molecules. PMID:22304919

A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

PubMed

Lo, Wen-Ting; Vujičić Žagar, Andreja; Gerth, Fabian; Lehmann, Martin; Puchkov, Dymtro; Krylova, Oxana; Freund, Christian; Scapozza, Leonardo; Vadas, Oscar; Haucke, Volker

2017-11-20

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P 2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

Structural and functional analysis of the GABARAP interaction motif (GIM)

DOE PAGES

Rogov, Vladimir V.; Stolz, Alexandra; Ravichandran, Arvind C.; ...

2017-06-27

Through the canonical LC3 interaction motif (LIR), [W/F/Y]–X 1–X 2[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a GABARAP Interaction Motif (GIM) sequence ([W/F]–[V/I]–X 2–V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1–LIR is indeed 11–fold more specific for GABARAP than LC3B. Selective mutation of themore » X 1 and X 2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1–GIM selectivity 20–fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. In conclusion, the identification of a GABARAP–specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.« less

UNC93B1 mediates differential trafficking of endosomal TLRs

PubMed Central

Lee, Bettina L; Moon, Joanne E; Shu, Jeffrey H; Yuan, Lin; Newman, Zachary R; Schekman, Randy; Barton, Gregory M

2013-01-01

UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases. DOI: http://dx.doi.org/10.7554/eLife.00291.001 PMID:23426999

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif

PubMed Central

Tikhonova, Irina G.; Ivetic, Aleksandar; Schu, Peter

2017-01-01

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans-Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues (356RR357, 359KK360, and 362KK363) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues (369DD370) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo. Molecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans-Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin. PMID:28235798

The cytoplasmic tail of L-selectin interacts with the adaptor-protein complex AP-1 subunit μ1A via a novel basic binding motif.

PubMed

Dib, Karim; Tikhonova, Irina G; Ivetic, Aleksandar; Schu, Peter

2017-04-21

L-selectin regulates leukocyte adhesion and rolling along the endothelium. Proteins binding to the cytoplasmic tail of L-selectin regulate L-selectin functions. We used L-selectin cytoplasmic tail peptide pulldown assays combined with high sensitivity liquid chromatography/mass spectrometry to identify novel L-selectin tail-binding proteins. Incubation of the L-selectin tail with cell extracts from phorbol 12-myristate 13-acetate-stimulated Raw 264.7 macrophages resulted in the binding of μ1A of the clathrin-coated vesicle AP-1 complex. Furthermore, full-length GST-μ1A and the GST-μ1A C-terminal domain, but not the GST-μ1A N-terminal domain, bind to L-selectin tail peptide, and the intracellular pool of L-selectin colocalizes with AP-1 at the trans -Golgi network. We identified a novel basic protein motif consisting of a cluster of three dibasic residues ( 356 RR 357 , 359 KK 360 , and 362 KK 363 ) in the membrane-proximal domain of the L-selectin tail as well as a doublet of aspartic acid residues ( 369 DD 370 ) in the membrane-distal end of the L-selectin tail involved in μ1A binding. Stimulation of Raw 264.7 macrophages with PMA augmented the amount of μ1A associated with anti-L-selectin immunoprecipitates. However, full-length GST-μ1A did not bind to the phospho-L-selectin tail or phospho-mimetic S364D L-selectin tail. Accordingly, we propose that phosphorylation of μ1A is required for interaction with the L-selectin tail and that L-selectin tail phosphorylation may regulate this interaction in vivo Molecular docking of the L-selectin tail to μ1A was used to identify the μ1A surface domain binding the L-selectin tail and to explain how phosphorylation of the L-selectin tail abrogates μ1A interaction. Our findings indicate that L-selectin is transported constitutively by the AP-1 complex, leading to the formation of a trans -Golgi network reserve pool and that phosphorylation of the L-selectin tail blocks AP-1-dependent retrograde transport of L-selectin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Src-like adaptor protein down-regulates T cell receptor (TCR)-CD3 expression by targeting TCRzeta for degradation.

PubMed

Myers, Margaret D; Dragone, Leonard L; Weiss, Arthur

2005-07-18

Src-like adaptor protein (SLAP) down-regulates expression of the T cell receptor (TCR)-CD3 complex during a specific stage of thymocyte development when the TCR repertoire is selected. Consequently, SLAP-/- thymocytes display alterations in thymocyte development. Here, we have studied the mechanism of SLAP function. We demonstrate that SLAP-deficient thymocytes have increased TCRzeta chain expression as a result of a defect in TCRzeta degradation. Failure to degrade TCRzeta leads to an increased pool of fully assembled TCR-CD3 complexes that are capable of recycling back to the cell surface. We also provide evidence that SLAP functions in a pathway that requires the phosphorylated TCRzeta chain and the Src family kinase Lck, but not ZAP-70 (zeta-associated protein of 70 kD). These studies reveal a unique mechanism by which SLAP contributes to the regulation of TCR expression during a distinct stage of thymocyte development.

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

PubMed

Selvakumar, Dakshnamurthy; Drescher, Marian J; Deckard, Nathan A; Ramakrishnan, Neeliyath A; Morley, Barbara J; Drescher, Dennis G

2017-01-01

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca 2+ ] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin. Surface plasmon resonance (SPR) analysis, competitive pull-downs, and co-immunoprecipitation indicated that these interactions were promoted by Ca 2+ and occur together. D1A was also found to separately interact with NSF, but with an inverse dependence on Ca 2+ Evidence was obtained, for the first time, that otoferlin domains C2A, C2B, C2D, and C2F interact with NSF and AP2mu1, whereas C2C or C2E do not bind to either protein, representing binding characteristics consistent with respective inclusion or omission in individual C2 domains of the tyrosine motif YXXΦ. In competitive pull-down assays, as predicted by K D values from SPR (+Ca 2+ ), C2F pulled down primarily NSF as opposed to AP2mu1. Phosphorylation of AP2mu1 gave rise to a reversal: an increase in binding by C2F to phosphorylated AP2mu1 was accompanied by a decrease in binding to NSF, consistent with a molecular switch for otoferlin from membrane fusion (NSF) to endocytosis (AP2mu1). An increase in phosphorylated AP2mu1 at the base of the cochlear inner hair cell was the observed response elicited by a dopamine D1A agonist, as predicted. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

PubMed Central

Arnold, Miranda; Cross, Rebecca; Singleton, Kaela S.; Zlatic, Stephanie; Chapleau, Christopher; Mullin, Ariana P.; Rolle, Isaiah; Moore, Carlene C.; Theibert, Anne; Pozzo-Miller, Lucas; Faundez, Victor; Larimore, Jennifer

2016-01-01

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function. PMID:27713690

AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis

PubMed Central

Delevoye, Cédric; Hurbain, Ilse; Tenza, Danièle; Sibarita, Jean-Baptiste; Uzan-Gafsou, Stéphanie; Ohno, Hiroshi; Geerts, Willie J.C.; Verkleij, Arie J.; Salamero, Jean; Marks, Michael S.

2009-01-01

Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation. PMID:19841138

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

PubMed Central

Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

2012-01-01

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

Role of the AP2 β-Appendage Hub in Recruiting Partners for Clathrin-Coated Vesicle Assembly

PubMed Central

Burtey, Anne; Praefcke, Gerrit J. K; Peak-Chew, Sew-Yeu; Mills, Ian G; Benmerah, Alexandre; McMahon, Harvey T

2006-01-01

Adaptor protein complex 2 α and β-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of β-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the β-appendage (the “top” and “side” sites) that bind motifs distinct from those previously identified on the α-appendage. We solved the structure of the β-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor β-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the β-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability (“matricity”). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as β-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors. PMID:16903783

Human kidney anion exchanger 1 interacts with kinesin family member 3B (KIF3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duangtum, Natapol; Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700; Junking, Mutita

Highlights: {yields} Impaired trafficking of kAE1 causes distal renal tubular acidosis (dRTA). {yields} The interaction between kAE1 and kinesin family member 3B (KIF3B) is reported. {yields} The co-localization between kAE and KIF3B was detected in human kidney tissues. {yields} A marked reduction of kAE1 on the cell membrane was observed when KIF3B was knockdown. {yields} KFI3B plays an important role in trafficking of kAE1 to the plasma membrane. -- Abstract: Impaired trafficking of human kidney anion exchanger 1 (kAE1) to the basolateral membrane of {alpha}-intercalated cells of the kidney collecting duct leads to the defect of the Cl{sup -}/HCO{sub 3}{supmore » -} exchange and the failure of proton (H{sup +}) secretion at the apical membrane of these cells, causing distal renal tubular acidosis (dRTA). In the sorting process, kAE1 interacts with AP-1 mu1A, a subunit of AP-1A adaptor complex. However, it is not known whether kAE1 interacts with motor proteins in its trafficking process to the plasma membrane or not. We report here that kAE1 interacts with kinesin family member 3B (KIF3B) in kidney cells and a dileucine motif at the carboxyl terminus of kAE1 contributes to this interaction. We have also demonstrated that kAE1 co-localizes with KIF3B in human kidney tissues and the suppression of endogenous KIF3B in HEK293T cells by small interfering RNA (siRNA) decreases membrane localization of kAE1 but increases its intracellular accumulation. All results suggest that KIF3B is involved in the trafficking of kAE1 to the plasma membrane of human kidney {alpha}-intercalated cells.« less

The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding*

PubMed Central

Ewens, Caroline A.; Panico, Silvia; Kloppsteck, Patrik; McKeown, Ciaran; Ebong, Ima-Obong; Robinson, Carol; Zhang, Xiaodong; Freemont, Paul S.

2014-01-01

p97, also known as valosin-containing protein, is a versatile participant in the ubiquitin-proteasome system. p97 interacts with a large network of adaptor proteins to process ubiquitylated substrates in different cellular pathways, including endoplasmic reticulum-associated degradation and transcription factor activation. p97 and its adaptor Fas-associated factor-1 (FAF1) both have roles in the ubiquitin-proteasome system during NF-κB activation, although the mechanisms are unknown. FAF1 itself also has emerging roles in other cell-cycle pathways and displays altered expression levels in various cancer cell lines. We have performed a detailed study the p97-FAF1 interaction. We show that FAF1 binds p97 stably and in a stoichiometry of 3 to 6. Cryo-EM analysis of p97-FAF1 yielded a 17 Å reconstruction of the complex with FAF1 above the p97 ring. Characteristics of p97-FAF1 uncovered in this study reveal common features in the interactions of p97, providing mechanistic insight into how p97 mediates diverse functionalities. PMID:24619421

P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

PubMed Central

Liu, Ke; Surendhran, Kavitha; Nothwehr, Steven F.

2008-01-01

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN. PMID:18508916

Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

PubMed Central

Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, Kürşad

2017-01-01

Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics. PMID:28748186

Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis.

PubMed

Elsholz, Alexander K W; Birk, Marlene S; Charpentier, Emmanuelle; Turgay, Kürşad

2017-01-01

Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis . We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

The Hippo component YAP localizes in the nucleus of human papilloma virus positive oropharyngeal squamous cell carcinoma.

PubMed

Alzahrani, Faisal; Clattenburg, Leanne; Muruganandan, Shanmugam; Bullock, Martin; MacIsaac, Kaitlyn; Wigerius, Michael; Williams, Blair A; Graham, M Elise R; Rigby, Matthew H; Trites, Jonathan R B; Taylor, S Mark; Sinal, Christopher J; Fawcett, James P; Hart, Robert D

2017-02-22

HPV infection causes cervical cancer, mediated in part by the degradation of Scribble via the HPV E6 oncoprotein. Recently, Scribble has been shown to be an important regulator of the Hippo signaling cascade. Deregulation of the Hippo pathway induces an abnormal cellular transformation, epithelial to mesenchymal transition, which promotes oncogenic progression. Given the recent rise in oropharyngeal HPV squamous cell carcinoma we sought to determine if Hippo signaling components are implicated in oropharyngeal squamous cell carcinoma. Molecular and cellular techniques including immunoprecipiations, Western blotting and immunocytochemistry were used to identify the key Hippo pathway effector Yes-Associated Protein (YAP)1. Oropharyngeal tissue was collected from CO 2 laser resections, and probed with YAP1 antibody in tumor and pre-malignant regions of HPV positive OPSCC tissue. This study reveals that the Scribble binding protein Nitric Oxide Synthase 1 Adaptor Protein (NOS1AP) forms a complex with YAP. Further, the NOS1APa and NOS1APc isoforms show differential association with activated and non-activated YAP, and impact cellular proliferation. Consistent with deregulated Hippo signaling in OPSCC HPV tumors, we see a delocalization of Scribble and increased nuclear accumulation of YAP1 in an HPV-positive OPSCC. Our preliminary data indicates that NOS1AP isoforms differentially associate with YAP1, which, together with our previous findings, predicts that loss of YAP1 enhances cellular transformation. Moreover, YAP1 is highly accumulated in the nucleus of HPV-positive OPSCC, implying that Hippo signaling and possibly NOS1AP expression are de-regulated in OPSCC. Further studies will help determine if NOS1AP isoforms, Scribble and Hippo components will be useful biomarkers in OPSCC tumor biology.

The effect of clozapine on mRNA expression for genes encoding G protein-coupled receptors and the protein components of clathrin-mediated endocytosis

PubMed Central

Hu, Ying; Weymer, Jon F.; Rizig, Mie; McQuillin, Andrew; Hunt, Stephen P.; Gurling, Hugh M.D.

2013-01-01

Objectives Clathrin-mediated endocytosis (CME) is an intracellular trafficking mechanism for packaging cargo, including G protein-coupled receptors (GPCRs), into clathrin-coated vesicles (CCVs). The antipsychotic chlorpromazine inhibits CCV assembly of adaptor protein AP2 whereas clozapine increases serotonin2A receptor internalization. We hypothesized that clozapine alters the expression of CME genes modulating vesicle turnover and GPCR internalization. Materials and methods SH-SY5Y human neuroblastoma cells were incubated with clozapine (1–20 µmol/l) for 24–72 h. GPCR and CME-related gene mRNA expression was measured using RT-PCR. We quantified changes in the same genes using expression data from a microarray study of mice brains after 12 weeks of treatment with 12 mg/kg/day clozapine. Results The expression of genes encoding adaptor and clathrin assembly proteins, AP2A2, AP2B1, AP180, CLINT1, HIP1, ITSN2, and PICALM, increased relative to the control in SH-SY5Y cells incubated with 5–10 µmol/l clozapine for 24–72 h. The microarray study showed significantly altered expression of the above CME-related genes, with a marked 641-fold and 17-fold increase in AP180 and the serotonin1A GPCR, respectively. The expression of three serotonergic receptor and lysophosphatidic acid receptor 2 (EDG4) GPCR genes was upregulated in SH-SY5Y cells incubated with 5 µmol/l clozapine for 24 h. EDG4 expression was also increased with 10–20 µmol/l clozapine treatment at 48–72 h. Clozapine significantly decreased the expression of β-arrestin, involved in GPCR desensitization, both in vitro and vivo. Conclusion The changes we report in CME and GPCR mRNAs implicate CCV-mediated internalization of GPCRs and the serotonergic system in clozapine’s mechanism of action, which may be useful in the design of more effective and less toxic antipsychotic therapies. PMID:23811784

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

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.

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

PubMed

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4+ T cell apoptosis.

PubMed

Jacob, Rajesh Abraham; Johnson, Aaron L; Pawlak, Emily N; Dirk, Brennan S; Van Nynatten, Logan R; Haeryfar, S M Mansour; Dikeakos, Jimmy D

2017-09-01

Acquired Immune Deficiency Syndrome is characterized by a decline in CD4 + T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4 + T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (Nef M20A or Nef EEEE62-65AAAA ) or Nef:AP-2 (Nef LL164/165AA ), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (Nef H89A or Nef F191A ) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Molecular and electronic structure of octahedral o-aminophenolato and o-iminobenzosemiquinonato complexes of V(V), Cr(III), Fe(III), and Co(III). Experimental determination of oxidation levels of ligands and metal ions.

PubMed

Chun, H; Verani, C N; Chaudhuri, P; Bothe, E; Bill, E; Weyhermüller, T; Wieghardt, K

2001-08-13

The coordination chemistry of the ligands 2-anilino-4,6-di-tert-butylphenol, H[L(AP)], and N,N"'-bis[2-(4,6-di-tert-butylphenol]diethylenetriamine, H(2)[(L(AP))N(L(AP))], has been studied with the first-row transition metal ions V, Cr, Fe, and Co. The ligands are noninnocent in the sense that the aminophenolato parts, [L(AP)](-) and [L(AP)-H](2)(-), can be readily oxidized to their o-iminobenzosemiquinonato, [L(ISQ)](-), and o-iminobenzoquinone, [L(ISB)], forms. The following neutral octahedral complexes have been isolated as crystalline materials, and their crystal structures have been determined by X-ray crystallography at 100 K: [Cr(III)(L(ISQ))(3)] (1), [Fe(III)(L(ISQ))(3)] (2), [Co(III)(L(ISQ))(3)] (3), [V(V)(L(ISQ))(L(AP)-H)(2)] (4), [V(V)(L(AP)-H)(2)(L(AP))] (5), and [V(V)O[(L(AP))N(L(AP)-H)

The Listeriolysin O PEST-like Sequence Co-opts AP-2-Mediated Endocytosis to Prevent Plasma Membrane Damage during Listeria Infection.

PubMed

Chen, Chen; Nguyen, Brittney N; Mitchell, Gabriel; Margolis, Shally R; Ma, Darren; Portnoy, Daniel A

2018-06-13

Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of Listeria monocytogenes from a phagosome, enabling growth of the bacteria in the host cell cytosol. LLO contains a PEST-like sequence that prevents it from killing infected cells, but the mechanism involved is unknown. We found that the LLO PEST-like sequence was necessary to mediate removal of LLO from the interior face of the plasma membrane, where it coalesces into discrete puncta. LLO interacts with Ap2a2, an adaptor protein involved in endocytosis, via its PEST-like sequence, and Ap2a2-dependent endocytosis is required to prevent LLO-induced cytotoxicity. An unrelated PEST-like sequence from a human G protein-coupled receptor (GPCR), which also interacts with Ap2a2, could functionally complement the PEST-like sequence in L. monocytogenes LLO. These data revealed that LLO co-opts the host endocytosis machinery to protect the integrity of the host plasma membrane during L. monocytogenes infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

PubMed

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2011-02-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Conformational changes in the AAA ATPase p97–p47 adaptor complex

PubMed Central

Beuron, Fabienne; Dreveny, Ingrid; Yuan, Xuemei; Pye, Valerie E; Mckeown, Ciaran; Briggs, Louise C; Cliff, Matthew J; Kaneko, Yayoi; Wallis, Russell; Isaacson, Rivka L; Ladbury, John E; Matthews, Steve J; Kondo, Hisao; Zhang, Xiaodong; Freemont, Paul S

2006-01-01

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at ∼20 Å resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes. PMID:16601695

The sorting nexin, DSH3PX1, connects the axonal guidance receptor, Dscam, to the actin cytoskeleton.

PubMed

Worby, C A; Simonson-Leff, N; Clemens, J C; Kruger, R P; Muda, M; Dixon, J E

2001-11-09

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, we used the SH2 domain of Dock to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which was subsequently shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system (Schmucker, D., Clemens, J. C., Shu, H., Worby, C. A., Xiao, J., Muda, M., Dixon, J. E., and Zipursky, S. L. (2000) Cell 101, 671-684). The smallest protein in this complex (p63) has now been identified. We have named p63 DSH3PX1 because it appears to be the Drosophila orthologue of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. We demonstrate that DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. We further show that DSH3PX1 interacts with the Drosophila orthologue of Wasp, a protein component of actin polymerization machinery, and that DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking.

Simultaneous interaction with base and phosphate moieties modulates the phosphodiester cleavage of dinucleoside 3',5'-monophosphates by dinuclear Zn2+ complexes of di(azacrown) ligands.

PubMed

Wang, Qi; Lönnberg, Harri

2006-08-23

Five dinucleating ligands (1-5) and one trinucleating ligand (6) incorporating 1,5,9-triazacyclododecan-3-yloxy groups attached to an aromatic scaffold have been synthesized. The ability of the Zn(2+) complexes of these ligands to promote the transesterification of dinucleoside 3',5'-monophosphates to a 2',3'-cyclic phosphate derived from the 3'-linked nucleoside by release of the 5'-linked nucleoside has been studied over a narrow pH range, from pH 5.8 to 7.2, at 90 degrees C. The dinuclear complexes show marked base moiety selectivity. Among the four dinucleotide 3',5'-phosphates studied, viz. adenylyl-3',5'-adenosine (ApA), adenylyl-3',5'-uridine (ApU), uridylyl-3',5'-adenosine (UpA), and uridylyl-3',5'-uridine (UpU), the dimers containing one uracil base (ApU and UpA) are cleaved up to 2 orders of magnitude more readily than those containing either two uracil bases (UpU) or two adenine bases (ApA). The trinuclear complex (6), however, cleaves UpU as readily as ApU and UpA, while the cleavage of ApA remains slow. UV spectrophotometric and (1)H NMR spectroscopic studies with one of the dinucleating ligands (3) verify binding to the bases of UpU and ApU at less than millimolar concentrations, while no interaction with the base moieties of ApA is observed. With ApU and UpA, one of the Zn(2+)-azacrown moieties in all likelihood anchors the cleaving agent to the uracil base of the substrate, while the other azacrown moiety serves as a catalyst for the phosphodiester transesterification. With UpU, two azacrown moieties are engaged in the base moiety binding. The catalytic activity is, hence, lost, but it can be restored by addition of a third azacrown group on the cleaving agent.

Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

PubMed Central

Augustine, G.J.; Morgan, J.R.; Villalba-Galea, C.A.; Jin, S.; Prasad, K.; Lafer, E.M.

2015-01-01

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions. PMID:16417485

HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain.

PubMed

Legendre-Guillemin, Valerie; Metzler, Martina; Charbonneau, Martine; Gan, Lu; Chopra, Vikramjit; Philie, Jacynthe; Hayden, Michael R; McPherson, Peter S

2002-05-31

Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis.

mda-9/Syntenin protein positively regulates the activation of Akt protein by facilitating integrin-linked kinase adaptor function during adhesion to type I collagen.

PubMed

Hwangbo, Cheol; Park, Juhee; Lee, Jeong-Hyung

2011-09-23

The integrin-linked kinase (ILK)-PINCH1-α-parvin (IPP) complex functions as a signaling platform for integrins that modulates various cellular processes. ILK functions as a central adaptor for the assembly of IPP complex. We report here that mda-9/syntenin, a positive regulator of cancer metastasis, regulates the activation of Akt (also known as protein kinase B) by facilitating ILK adaptor function during adhesion to type I collagen (COL-I) in human breast cancer cells. COL-I stimulation induced the phosphorylation and plasma membrane translocation of Akt. Inhibition of mda-9/syntenin or expression of mutant ILK (E359K) significantly blocked the translocation of both ILK and Akt to the plasma membrane. mda-9/syntenin associated with ILK, and this association was increased at the plasma membrane by COL-I stimulation. Knockdown of mda-9/syntenin impaired COL-I-induced association of ILK with Akt and plasma membrane targeting of ILK-Akt complex. These results demonstrated that mda-9/syntenin regulates the activation of Akt by controlling the plasma membrane targeting of Akt via a mechanism that facilitates the association of Akt with ILK at the plasma membrane during adhesion to COL-I. On a striking note, inhibition of mda-9/syntenin impaired COL-I-induced plasma membrane translocation of the IPP complex and assembly of integrin β1-IPP signaling complexes. Thus, our study defines the role of mda-9/syntenin in ILK adaptor function and describes a new mechanism of mda-9/syntenin for regulation of cell migration.

One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system

PubMed Central

Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

2008-01-01

The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Jun × 4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems. PMID:18329890

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells

PubMed Central

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W.; Rudd, Christopher E.

2015-01-01

Summary While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. PMID:26321253

The Immune Adaptor SLP-76 Binds to SUMO-RANGAP1 at Nuclear Pore Complex Filaments to Regulate Nuclear Import of Transcription Factors in T Cells.

PubMed

Liu, Hebin; Schneider, Helga; Recino, Asha; Richardson, Christine; Goldberg, Martin W; Rudd, Christopher E

2015-09-03

While immune cell adaptors regulate proximal T cell signaling, direct regulation of the nuclear pore complex (NPC) has not been reported. NPC has cytoplasmic filaments composed of RanGAP1 and RanBP2 with the potential to interact with cytoplasmic mediators. Here, we show that the immune cell adaptor SLP-76 binds directly to SUMO-RanGAP1 of cytoplasmic fibrils of the NPC, and that this interaction is needed for optimal NFATc1 and NF-κB p65 nuclear entry in T cells. Transmission electron microscopy showed anti-SLP-76 cytoplasmic labeling of the majority of NPCs in anti-CD3 activated T cells. Further, SUMO-RanGAP1 bound to the N-terminal lysine 56 of SLP-76 where the interaction was needed for optimal RanGAP1-NPC localization and GAP exchange activity. While the SLP-76-RanGAP1 (K56E) mutant had no effect on proximal signaling, it impaired NF-ATc1 and p65/RelA nuclear entry and in vivo responses to OVA peptide. Overall, we have identified SLP-76 as a direct regulator of nuclear pore function in T cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

δ-COP contains a helix C-terminal to its longin domain key to COPI dynamics and function

PubMed Central

Arakel, Eric C.; Richter, Kora P.; Clancy, Anne; Schwappach, Blanche

2016-01-01

Membrane recruitment of coatomer and formation of coat protein I (COPI)-coated vesicles is crucial to homeostasis in the early secretory pathway. The conformational dynamics of COPI during cargo capture and vesicle formation is incompletely understood. By scanning the length of δ-COP via functional complementation in yeast, we dissect the domains of the δ-COP subunit. We show that the μ-homology domain is dispensable for COPI function in the early secretory pathway, whereas the N-terminal longin domain is essential. We map a previously uncharacterized helix, C-terminal to the longin domain, that is specifically required for the retrieval of HDEL-bearing endoplasmic reticulum-luminal residents. It is positionally analogous to an unstructured linker that becomes helical and membrane-facing in the open form of the AP2 clathrin adaptor complex. Based on the amphipathic nature of the critical helix it may probe the membrane for lipid packing defects or mediate interaction with cargo and thus contribute to stabilizing membrane-associated coatomer. PMID:27298352

Synthesis and spectroscopic studies on the new Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol with 5-aminouracil (BDF5AU) and its transition metal complexes. Influence on biologically active peptides-regulating aminopeptidases.

PubMed

Hueso-Ureña, Francisco; Illán-Cabeza, Nuria A; Moreno-Carretero, Miguel N; Martínez-Martos, José M; Ramírez-Expósito, María J

2003-04-01

The synthesis, spectroscopic (IR, 1H and 13C NMR, UV-Vis-NIR, EPR), magnetic measurements and biological studies of a number of complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Au(III) and Hg(II) of the Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol and 5-aminouracil, ((5-[[(3-[[(2,4-dioxopyrimidin-5(1H,3H)-yl)imino]methyl]-2-hydroxy-5-methylphenyl)methylene]amino]pyrimidine-2,4(1H,3H)-dione, hereafter denoted as BDF5AU) are reported. In all cases, the complexes appear to be monomeric. The deprotonated ligand in the phenolic oxygen atom shows a tridentate coordination mode through the two azomethine nitrogen atoms and the phenolic oxygen atom. The coordination of the neutral ligand takes place through the phenolic oxygen atom and one azomethine nitrogen atom and the carbonylic oxygen atom in fourth position of one uracil ring. The biological properties of some perchlorate complexes on the activity of some neutral, acid, basic and omega aminopeptidases (AP) are assayed, demonstrating a general inhibitory effect. Neutral and basic AP are mainly inhibited by Cu(II), Ni(II) and Cd(II) complexes, although tyrosyl-AP is activated by Zn(II) complex. Glutamyl-AP but not aspartyl-AP is inhibited by all the complexes assayed excepting Zn(II) complex. Finally, omega AP is inhibited by Ni(II) and Cd(II) complexes. Copyright 2003 Elsevier Science Inc.

TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

PubMed

Hennigar, Stephen R; Kelleher, Shannon L

2015-10-01

Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; P<0.05) to lysosomes (0.292 ± 0.03 and 0.649 ± 0.03; P<0.0001), which increased lysosomal Zn (P<0.0001) and activated LCD (P<0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNFα to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (P<0.05) AP-3 binding to wt ZnT2 but not to L294V immunoprecipitates. Finally, using phospho- and dephospho-mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. © 2015 Wiley Periodicals, Inc.

Co-assembly of Viral Envelope Glycoproteins Regulates Their Polarized Sorting in Neurons

PubMed Central

Mardones, Gonzalo A.; Bonifacino, Juan S.

2014-01-01

Newly synthesized envelope glycoproteins of neuroinvasive viruses can be sorted in a polarized manner to the somatodendritic and/or axonal domains of neurons. Although critical for transneuronal spread of viruses, the molecular determinants and interregulation of this process are largely unknown. We studied the polarized sorting of the attachment (NiV-G) and fusion (NiV-F) glycoproteins of Nipah virus (NiV), a paramyxovirus that causes fatal human encephalitis, in rat hippocampal neurons. When expressed individually, NiV-G exhibited a non-polarized distribution, whereas NiV-F was specifically sorted to the somatodendritic domain. Polarized sorting of NiV-F was dependent on interaction of tyrosine-based signals in its cytosolic tail with the clathrin adaptor complex AP-1. Co-expression of NiV-G with NiV-F abolished somatodendritic sorting of NiV-F due to incorporation of NiV-G•NiV-F complexes into axonal transport carriers. We propose that faster biosynthetic transport of unassembled NiV-F allows for its proteolytic activation in the somatodendritic domain prior to association with NiV-G and axonal delivery of NiV-G•NiV-F complexes. Our study reveals how interactions of viral glycoproteins with the host's transport machinery and between themselves regulate their polarized sorting in neurons. PMID:24831812

CIKS, a connection to IκB kinase and stress-activated protein kinase

PubMed Central

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Scandium(III) complexes of monophosphorus acid DOTA analogues: a thermodynamic and radiolabelling study with (44)Sc from cyclotron and from a (44)Ti/(44)Sc generator.

PubMed

Kerdjoudj, R; Pniok, M; Alliot, C; Kubíček, V; Havlíčková, J; Rösch, F; Hermann, P; Huclier-Markai, S

2016-01-28

The complexation ability of DOTA analogs bearing one methylenephosphonic (DO3AP) or methylenephosphinic (DO3AP(PrA) and DO3AP(ABn)) acid pendant arm toward scandium was evaluated. Stability constants of their scandium(iii) complexes were determined by potentiometry combined with (45)Sc NMR spectroscopy. The stability constants of the monophosphinate analogues are somewhat lower than that of the Sc-DOTA complex. The phosphorus acid moiety interacts with trivalent scandium even in very acidic solutions forming out-of-cage complexes; the strong affinity of the phosphonate group to Sc(iii) precludes stability constant determination of the Sc-DO3AP complex. These results were compared with those obtained by the free-ion selective radiotracer extraction (FISRE) method which is suitable for trace concentrations. FISRE underestimated the stability constants but their relative order was preserved. Nonetheless, as this method is experimentally simple, it is suitable for a quick relative comparison of stability constant values under trace concentrations. Radiolabelling of the ligands with (44)Sc was performed using the radioisotope from two sources, a (44)Ti/(44)Sc generator and (44m)Sc/(44)Sc from a cyclotron. The best radiolabelling conditions for the ligands were pH = 4, 70 °C and 20 min which were, however, not superior to those of the parent DOTA. Nonetheless, in vitro behaviour of the Sc(iii) complexes in the presence of hydroxyapatite and rat serum showed sufficient stability of (44)Sc complexes of these ligands for in vivo applications. PET images and ex vivo biodistribution of the (44)Sc-DO3AP complex performed on healthy Wistar male rats showed no specific bone uptake and rapid clearance through urine.

Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88.

PubMed

Dunne, Aisling; Ejdeback, Mikael; Ludidi, Phumzile L; O'Neill, Luke A J; Gay, Nicholas J

2003-10-17

The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.

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

Structural elements and organization of the ancestral translational machinery

NASA Technical Reports Server (NTRS)

Rein, R.; Srinivasan, S.; Mcdonald, J.; Raghunathan, G.; Shibata, M.

1987-01-01

The molecular mechanisms of the primitive translational apparatus are discussed in the framework of present-day protein biosynthesis. The structural necessities of an early adaptor and the multipoint recognition properties of such an adaptor are investigated on the basis of structure/function relationships found in a contemporary system and a molecular model of the contemporary transpeptidation complex. A model of the tRNA(Tyr)-tyrosyl tRNA synthetase complex including the positioning of the disordered region is proposed; the model is used to illustrate the required recognition properties of the ancestor aminoacyl synthetase.

μ2-Dependent endocytosis of N-cadherin is regulated by β-catenin to facilitate neurite outgrowth.

PubMed

Chen, Yi-Ting; Tai, Chin-Yin

2017-05-01

Circuit formation in the brain requires neurite outgrowth throughout development to establish synaptic contacts with target cells. Active endocytosis of several adhesion molecules facilitates the dynamic exchange of these molecules at the surface and promotes neurite outgrowth in developing neurons. The endocytosis of N-cadherin, a calcium-dependent adhesion molecule, has been implicated in the regulation of neurite outgrowth, but the mechanism remains unclear. Here, we identified that a fraction of N-cadherin internalizes through clathrin-mediated endocytosis (CME). Two tyrosine-based motifs in the cytoplasmic domain of N-cadherin recognized by the μ2 subunit of the AP-2 adaptor complex are responsible for CME of N-cadherin. Moreover, β-catenin, a core component of the N-cadherin adhesion complex, inhibits N-cadherin endocytosis by masking the 2 tyrosine-based motifs. Removal of β-catenin facilitates μ2 binding to N-cadherin, thereby increasing clathrin-mediated N-cadherin endocytosis and neurite outgrowth without affecting the steady-state level of surface N-cadherin. These results identify and characterize the mechanism controlling N-cadherin endocytosis through β-catenin-regulated μ2 binding to modulate neurite outgrowth. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Functional properties of internalization-deficient P2X4 receptors reveal a novel mechanism of ligand-gated channel facilitation by ivermectin.

PubMed

Toulmé, Estelle; Soto, Florentina; Garret, Maurice; Boué-Grabot, Eric

2006-02-01

Although P2X receptors within the central nervous system mediate excitatory ATP synaptic transmission, the identity of central ATP-gated channels has not yet been elucidated. P2X(4), the most widely expressed subunit in the brain, was previously shown to undergo clathrin-dependent constitutive internalization by direct interaction between activator protein (AP)2 adaptors and a tyrosine-based sorting signal specifically present in the cytosolic C-terminal tail of mammalian P2X(4) sequences. In this study, we first used internalization-deficient P2X(4) receptor mutants to show that suppression of the endocytosis motif significantly increased the apparent sensitivity to ATP and the ionic permeability of P2X(4) channels. These unique properties, observed at low channel density, suggest that interactions with AP2 complexes may modulate the function of P2X(4) receptors. In addition, ivermectin, an allosteric modulator of several receptor channels, including mammalian P2X(4), did not potentiate the maximal current of internalization-deficient rat or human P2X(4) receptors. We demonstrated that binding of ivermectin onto wild-type P2X(4) channels increased the fraction of plasma membrane P2X(4) receptors, whereas surface expression of internalization-deficient P2X(4) receptors remained unchanged. Disruption of the clathrin-mediated endocytosis with the dominant-negative mutants Eps15 or AP-50 abolished the ivermectin potentiation of wild-type P2X(4) channel currents. Likewise, ivermectin increased the membrane fraction of nicotinic alpha7 acetylcholine (nalpha7ACh) receptors and the potentiation of acetylcholine current by ivermectin was suppressed when the same dominant-negative mutants were expressed. These data showed that potentiation by ivermectin of both P2X(4) and nalpha7ACh receptors was primarily caused by an increase in the number of cell surface receptors resulting from a mechanism dependent on clathrin/AP2-mediated endocytosis.

Physiological factors that regulate skin pigmentation

PubMed Central

Yamaguchi, Yuji; Hearing, Vincent J.

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1 and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2 and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including α-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. PMID:19449448

Synthesis and Reactivity toward H2 of (η(5)-C5Me5)Rh(III) Complexes with Bulky Aminopyridinate Ligands.

PubMed

Zamorano, Ana; Rendón, Nuria; Valpuesta, José E V; Álvarez, Eleuterio; Carmona, Ernesto

2015-07-06

Electrophilic, cationic Rh(III) complexes of composition [(η(5)-C5Me5)Rh(Ap)](+), (1(+)), were prepared by reaction of [(η(5)-C5Me5)RhCl2]2 and LiAp (Ap = aminopyridinate ligand) followed by chloride abstraction with NaBArF (BArF = B[3,5-(CF3)2C6H3]4). Reactions of cations 1(+) with different Lewis bases (e.g., NH3, 4-dimethylaminopyridine, or CNXyl) led in general to monoadducts 1·L(+) (L = Lewis base; Xyl = 2,6-Me2C6H3), but carbon monoxide provided carbonyl-carbamoyl complexes 1·(CO)2(+) as a result of metal coordination and formal insertion of CO into the Rh-Namido bond of complexes 1(+). Arguably, the most relevant observation reported in this study stemmed from the reactions of complexes 1(+) with H2. (1)H NMR analyses of the reactions demonstrated a H2-catalyzed isomerization of the aminopyridinate ligand in cations 1(+) from the ordinary κ(2)-N,N' coordination to a very uncommon, formally tridentate κ-N,η(3) pseudoallyl bonding mode (complexes 3(+)) following benzylic C-H activation within the xylyl substituent of the pyridinic ring of the aminopyridinate ligand. The isomerization entailed in addition H-H and N-H bond activation and mimicked previous findings with the analogous iridium complexes. However, in dissimilarity with iridium, rhodium complexes 1(+) reacted stoichiometrically at 20 °C with excess H2. The transformations resulted in the hydrogenation of the C5Me5 and Ap ligands with concurrent reduction to Rh(I) and yielded complexes [(η(4)-C5Me5H)Rh(η(6)-ApH)](+), (2(+)), in which the pyridinic xylyl substituent is η(6)-bonded to the rhodium(I) center. New compounds reported were characterized by microanalysis and NMR spectroscopy. Representative complexes were additionally investigated by X-ray crystallography.

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

PubMed Central

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. PMID:24282540

Probing the Energetics of Dynactin Filament Assembly and the Binding of Cargo Adaptor Proteins Using Molecular Dynamics Simulation and Electrostatics-Based Structural Modeling.

PubMed

Zheng, Wenjun

2017-01-10

Dynactin, a large multiprotein complex, binds with the cytoplasmic dynein-1 motor and various adaptor proteins to allow recruitment and transportation of cellular cargoes toward the minus end of microtubules. The structure of the dynactin complex is built around an actin-like minifilament with a defined length, which has been visualized in a high-resolution structure of the dynactin filament determined by cryo-electron microscopy (cryo-EM). To understand the energetic basis of dynactin filament assembly, we used molecular dynamics simulation to probe the intersubunit interactions among the actin-like proteins, various capping proteins, and four extended regions of the dynactin shoulder. Our simulations revealed stronger intersubunit interactions at the barbed and pointed ends of the filament and involving the extended regions (compared with the interactions within the filament), which may energetically drive filament termination by the capping proteins and recruitment of the actin-like proteins by the extended regions, two key features of the dynactin filament assembly process. Next, we modeled the unknown binding configuration among dynactin, dynein tails, and a number of coiled-coil adaptor proteins (including several Bicaudal-D and related proteins and three HOOK proteins), and predicted a key set of charged residues involved in their electrostatic interactions. Our modeling is consistent with previous findings of conserved regions, functional sites, and disease mutations in the adaptor proteins and will provide a structural framework for future functional and mutational studies of these adaptor proteins. In sum, this study yielded rich structural and energetic information about dynactin and associated adaptor proteins that cannot be directly obtained from the cryo-EM structures with limited resolutions.

Tripartite assembly of RND multidrug efflux pumps

NASA Astrophysics Data System (ADS)

Daury, Laetitia; Orange, François; Taveau, Jean-Christophe; Verchère, Alice; Monlezun, Laura; Gounou, Céline; Marreddy, Ravi K. R.; Picard, Martin; Broutin, Isabelle; Pos, Klaas M.; Lambert, Olivier

2016-02-01

Tripartite multidrug efflux systems of Gram-negative bacteria are composed of an inner membrane transporter, an outer membrane channel and a periplasmic adaptor protein. They are assumed to form ducts inside the periplasm facilitating drug exit across the outer membrane. Here we present the reconstitution of native Pseudomonas aeruginosa MexAB-OprM and Escherichia coli AcrAB-TolC tripartite Resistance Nodulation and cell Division (RND) efflux systems in a lipid nanodisc system. Single-particle analysis by electron microscopy reveals the inner and outer membrane protein components linked together via the periplasmic adaptor protein. This intrinsic ability of the native components to self-assemble also leads to the formation of a stable interspecies AcrA-MexB-TolC complex suggesting a common mechanism of tripartite assembly. Projection structures of all three complexes emphasize the role of the periplasmic adaptor protein as part of the exit duct with no physical interaction between the inner and outer membrane components.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells

PubMed Central

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2013-01-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non–DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex–binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs. PMID:21186366

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15*

PubMed Central

Oberg, Elizabeth A.; Nifoussi, Shanna K.; Gingras, Anne-Claude; Strack, Stefan

2012-01-01

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events. PMID:23135275

The light gene of Drosophila melanogaster encodes a homologue of VPS41, a yeast gene involved in cellular-protein trafficking.

PubMed

Warner, T S; Sinclair, D A; Fitzpatrick, K A; Singh, M; Devlin, R H; Honda, B M

1998-04-01

Mutations in a number of genes affect eye colour in Drosophila melanogaster; some of these "eye-colour" genes have been shown to be involved in various aspects of cellular transport processes. In addition, combinations of viable mutant alleles of some of these genes, such as carnation (car) combined with either light (lt) or deep-orange (dor) mutants, show lethal interactions. Recently, dor was shown to be homologous to the yeast gene PEP3 (VPS18), which is known to be involved in intracellular trafficking. We have undertaken to extend our earlier work on the lt gene, in order to examine in more detail its expression pattern and to characterize its gene product via sequencing of a cloned cDNA. The gene appears to be expressed at relatively high levels in all stages and tissues examined, and shows strong homology to VPS41, a gene involved in cellular-protein trafficking in yeast and higher eukaryotes. Further genetic experiments also point to a role for lt in transport processes: we describe lethal interactions between viable alleles of lt and dor, as well as phenotypic interactions (reductions in eye pigment) between allels of lt and another eye-colour gene, garnet (g), whose gene product has close homology to a subunit of the human adaptor complex, AP-3.

[Association of Schizophrenia and its Clinical Implications with the NOS1AP Gene in the Colombian Population].

PubMed

Valencia, Jenny García; Duarte, Ana Victoria Valencia; Vila, Ana Lucía Páez; Kremeyer, Bárbara; Montoya, María Patricia Arbeláez; Linares, Andrés Ruiz; Acosta, Carlos Alberto Palacio; Duque, Jorge Ospina; Berrío, Gabriel Bedoya

2012-06-01

The nitric oxide synthase 1 adaptor protein (NOS1AP) gene is possibly implicated in schizophrenia etiopathogenesis. To determine the association of NOS1AP gene variants with schizophrenia and the relationship of variants with the clinical dimensions of the disorder in the Colombian population. It is a case-control study with 255 subjects per group. Markers within the NOS1AP gene were typified as well as other informative material of genetic origin so as to adjust by population stratification. A factorial analysis of the main components for each item in the Scales for Evaluating Negative Symptoms (SENS) together with the Scales for Evaluating Positive Symptoms (SEPS) to determine clinical dimensions. Association between the C/C genotype of the rs945713 marker with schizophrenia (OR = 1.79, 95% CI: 1.13 - 2.84) was found. The C/C genotype of the rs945713 was related to higher scores in the "affective flattening and alogia" dimension; and the A/A genotype of the rs4657181 marker was associated to lower scores in the same dimension. Significant associations of markers inside the NOS1AP gene with schizophrenia and the "affective flattening and alogia" clinical dimension were found. These results are consistent with previous studies and support the possibility that NOS1AP influences schizophrenia susceptibility. Furthermore, NOS1AP might be a modifier of schizophrenia clinical characteristics. Copyright © 2012 Asociación Colombiana de Psiquiatría. Publicado por Elsevier España. All rights reserved.

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

Systematic proteomics of the VCP-UBXD adaptor network identifies a role for UBXN10 in regulating ciliogenesis.

PubMed

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

2015-10-01

The AAA-ATPase VCP (also known as p97 or CDC48) uses ATP hydrolysis to 'segregate' ubiquitylated proteins from their binding partners. VCP acts through UBX-domain-containing adaptors that provide target specificity, but the 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.

Complexity and Synchronicity of Resting State BOLD FMRI in Normal Aging and Cognitive Decline

PubMed Central

Liu, Collin Y; Krishnan, Anitha P; Yan, Lirong; Smith, Robert X; Kilroy, Emily; Alger, Jeffery R; Ringman, John M; Wang, Danny JJ

2012-01-01

Purpose To explore the use of approximate entropy (ApEn) as an index of the complexity and the synchronicity of resting state BOLD fMRI in normal aging and cognitive decline associated with familial Alzheimer’s disease (fAD). Materials and Methods Resting state BOLD fMRI data were acquired at 3T from 2 independent cohorts of subjects consisting of healthy young (age 23±2 years, n=8) and aged volunteers (age 66±3 years, n=8), as well as 22 fAD associated subjects (14 mutation carriers, age 41.2±15.8 years; and 8 non-mutation carrying family members, age 28.8±5.9 years). Mean ApEn values were compared between the two age groups, and correlated with cognitive performance in the fAD group. Cross-ApEn (C-ApEn) was further calculated to assess the asynchrony between precuneus and the rest of the brain. Results Complexity of brain activity measured by mean ApEn in gray and white matter decreased with normal aging. In the fAD group, cognitive impairment was associated with decreased mean ApEn in gray matter as well as decreased regional ApEn in right precuneus, right lateral parietal regions, left precentral gyrus, and right paracentral gyrus. A pattern of asynchrony between BOLD fMRI series emerged from C-ApEn analysis, with significant regional anti-correlation with cross-correlation coefficient of functional connectivity analysis. Conclusion ApEn and C-ApEn may be useful for assessing the complexity and synchronicity of brain activity in normal aging and cognitive decline associated with neurodegenerative diseases PMID:23225622

Sulfate-activating enzymes of Penicillium chrysogenum. The ATP sulfurylase. adenosine 5'-phosphosulfate complex does not serve as a substrate for adenosine 5'-phosphosulfate kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Renosto, F.; Martin, R.L.; Segel, I.H.

1989-06-05

At a noninhibitory steady state concentration of adenosine 5'-phosphosulfate (APS), increasing the concentration of Penicillium chrysogenum ATP sulfurylase drives the rate of the APS kinase-catalyzed reaction toward zero. The result indicates that the ATP sulfurylase.APS complex does not serve as a substrate for APS kinase, i.e. there is no ''substrate channeling'' of APS between the two sulfate-activating enzymes. APS kinase had no effect on the (S)0.5 values, nH values, or maximum isotope trapping in the single turnover of ATP sulfurylase-bound (/sup 35/S)APS. Equimolar APS kinase (+/- MgATP or APS) also had no effect on the rate constants for the inactivationmore » of ATP sulfurylase by phenylglyoxal, diethylpyrocarbonate, or N-ethylmaleimide. Similarly, ATP sulfurylase (+/- ligands) had no effect on the inactivation of equimolar APS kinase by trinitrobenzene sulfonate, diethylpyrocarbonate, or heat. (The last promotes the dissociation of dimeric APS kinase to inactive monomers.) ATP sulfurylase also had no effect on the reassociation of APS kinase subunits at low temperature. The cumulative results suggest that the two sulfate activating enzymes do not associate to form a ''3'-phosphoadenosine 5'-phosphosulfate synthetase'' complex.« less

A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing

PubMed Central

Umasankar, Perunthottathu K; Ma, Li; Thieman, James R; Jha, Anupma; Doray, Balraj; Watkins, Simon C; Traub, Linton M

2014-01-01

Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001 PMID:25303365

A Hereditary Spastic Paraplegia Mouse Model Supports a Role of ZFYVE26/SPASTIZIN for the Endolysosomal System

PubMed Central

Khundadze, Mukhran; Kollmann, Katrin; Koch, Nicole; Biskup, Christoph; Nietzsche, Sandor; Zimmer, Geraldine; Hennings, J. Christopher; Huebner, Antje K.; Symmank, Judit; Jahic, Amir; Ilina, Elena I.; Karle, Kathrin; Schöls, Ludger; Kessels, Michael; Braulke, Thomas; Qualmann, Britta; Kurth, Ingo; Beetz, Christian; Hübner, Christian A.

2013-01-01

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells. PMID:24367272

A hereditary spastic paraplegia mouse model supports a role of ZFYVE26/SPASTIZIN for the endolysosomal system.

PubMed

Khundadze, Mukhran; Kollmann, Katrin; Koch, Nicole; Biskup, Christoph; Nietzsche, Sandor; Zimmer, Geraldine; Hennings, J Christopher; Huebner, Antje K; Symmank, Judit; Jahic, Amir; Ilina, Elena I; Karle, Kathrin; Schöls, Ludger; Kessels, Michael; Braulke, Thomas; Qualmann, Britta; Kurth, Ingo; Beetz, Christian; Hübner, Christian A

2013-01-01

Hereditary spastic paraplegias (HSPs) are characterized by progressive weakness and spasticity of the legs because of the degeneration of cortical motoneuron axons. SPG15 is a recessively inherited HSP variant caused by mutations in the ZFYVE26 gene and is additionally characterized by cerebellar ataxia, mental decline, and progressive thinning of the corpus callosum. ZFYVE26 encodes the FYVE domain-containing protein ZFYVE26/SPASTIZIN, which has been suggested to be associated with the newly discovered adaptor protein 5 (AP5) complex. We show that Zfyve26 is broadly expressed in neurons, associates with intracellular vesicles immunopositive for the early endosomal marker EEA1, and co-fractionates with a component of the AP5 complex. As the function of ZFYVE26 in neurons was largely unknown, we disrupted Zfyve26 in mice. Zfyve26 knockout mice do not show developmental defects but develop late-onset spastic paraplegia with cerebellar ataxia confirming that SPG15 is caused by ZFYVE26 deficiency. The morphological analysis reveals axon degeneration and progressive loss of both cortical motoneurons and Purkinje cells in the cerebellum. Importantly, neuron loss is preceded by accumulation of large intraneuronal deposits of membrane-surrounded material, which co-stains with the lysosomal marker Lamp1. A density gradient analysis of brain lysates shows an increase of Lamp1-positive membrane compartments with higher densities in Zfyve26 knockout mice. Increased levels of lysosomal enzymes in brains of aged knockout mice further support an alteration of the lysosomal compartment upon disruption of Zfyve26. We propose that SPG15 is caused by an endolysosomal membrane trafficking defect, which results in endolysosomal dysfunction. This appears to be particularly relevant in neurons with highly specialized neurites such as cortical motoneurons and Purkinje cells.

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

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance.

PubMed

Worby, Carolyn A; Simonson-Leff, Nancy; Clemens, James C; Huddler, Donald; Muda, Marco; Dixon, Jack E

2002-03-15

Dock, the Drosophila orthologue of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, we purified proteins in a complex with the SH2 domain of Dock from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. We demonstrate that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. We conclude that DSH3PX1 is a substrate for DAck in vivo and in vitro and define one of the major in vitro sites of DSH3PX1 phosphorylation to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. We demonstrate that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance.

Non-T cell activation linker (NTAL): a transmembrane adaptor protein involved in immunoreceptor signaling.

PubMed

Brdicka, Tomás; Imrich, Martin; Angelisová, Pavla; Brdicková, Nadezda; Horváth, Ondrej; Spicka, Jirí; Hilgert, Ivan; Lusková, Petra; Dráber, Petr; Novák, Petr; Engels, Niklas; Wienands, Jürgen; Simeoni, Luca; Osterreicher, Jan; Aguado, Enrique; Malissen, Marie; Schraven, Burkhart; Horejsí, Václav

2002-12-16

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non-T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcgamma- and Fc epsilon -receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non-T cells.

Phosphoinositides and membrane curvature switch the mode of actin polymerization via selective recruitment of toca-1 and Snx9

PubMed Central

Gallop, Jennifer L.; Walrant, Astrid; Cantley, Lewis C.; Kirschner, Marc W.

2013-01-01

The membrane–cytosol interface is the major locus of control of actin polymerization. At this interface, phosphoinositides act as second messengers to recruit membrane-binding proteins. We show that curved membranes, but not flat ones, can use phosphatidylinositol 3-phosphate [PI(3)P] along with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] to stimulate actin polymerization. In this case, actin polymerization requires the small GTPase cell cycle division 42 (Cdc42), the nucleation-promoting factor neural Wiskott–Aldrich syndrome protein (N-WASP) and the actin nucleator the actin-related protein (Arp) 2/3 complex. In liposomes containing PI(4,5)P2 as the sole phosphoinositide, actin polymerization requires transducer of Cdc42 activation-1 (toca-1). In the presence of phosphatidylinositol 3-phosphate, polymerization is both more efficient and independent of toca-1. Under these conditions, sorting nexin 9 (Snx9) can be implicated as a specific adaptor that replaces toca-1 to mobilize neural Wiskott–Aldrich syndrome protein and the Arp2/3 complex. This switch in phosphoinositide and adaptor specificity for actin polymerization from membranes has implications for how different types of actin structures are generated at precise times and locations in the cell. PMID:23589871

p130Cas scaffolds the signalosome to direct adaptor-effector cross talk during Kaposi's sarcoma-associated herpesvirus trafficking in human microvascular dermal endothelial cells.

PubMed

Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy; Chandran, Bala

2014-12-01

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. Eukaryotic cell adaptor molecules, without any intrinsic enzymatic activity, are well known to allow a great diversity of specific and coordinated protein-protein interactions imparting signal amplification to different networks for physiological and pathological signaling. They are involved in integrating signals from growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. The present study identifies human microvascular dermal endothelial (HMVEC-d) cellular scaffold protein p130Cas (Crk-associated substrate) as a platform to promote Kaposi's sarcoma-associated herpesvirus (KSHV) trafficking. Early during KSHV de novo infection, p130Cas associates with lipid rafts and scaffolds EphrinA2 (EphA2)-associated critical adaptor members to downstream effector molecules, promoting successful nuclear delivery of the KSHV genome. Hence, simultaneous targeting of the receptor EphA2 and scaffolding action of p130Cas can potentially uncouple the signal cross talk of the KSHV entry-associated upstream signal complex from the immediate downstream trafficking-associated signalosome, consequently routing KSHV toward lysosomal degradation and eventually blocking KSHV infection and associated malignancies. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The innate immune sensor NLRC3 attenuates Toll-like receptor signaling via modification of the signaling adaptor TRAF6 and transcription factor NF-κB.

PubMed

Schneider, Monika; Zimmermann, Albert G; Roberts, Reid A; Zhang, Lu; Swanson, Karen V; Wen, Haitao; Davis, Beckley K; Allen, Irving C; Holl, Eda K; Ye, Zhengmao; Rahman, Adeeb H; Conti, Brian J; Eitas, Timothy K; Koller, Beverly H; Ting, Jenny P-Y

2012-09-01

Several members of the NLR family of sensors activate innate immunity. In contrast, we found here that NLRC3 inhibited Toll-like receptor (TLR)-dependent activation of the transcription factor NF-κB by interacting with the TLR signaling adaptor TRAF6 to attenuate Lys63 (K63)-linked ubiquitination of TRAF6 and activation of NF-κB. We used bioinformatics to predict interactions between NLR and TRAF proteins, including interactions of TRAF with NLRC3. In vivo, macrophage expression of Nlrc3 mRNA was diminished by the administration of lipopolysaccharide (LPS) but was restored when cellular activation subsided. To assess biologic relevance, we generated Nlrc3(-/-) mice. LPS-treated Nlrc3(-/-) macrophages had more K63-ubiquitinated TRAF6, nuclear NF-κB and proinflammatory cytokines. Finally, LPS-treated Nlrc3(-/-) mice had more signs of inflammation. Thus, signaling via NLRC3 and TLR constitutes a negative feedback loop. Furthermore, prevalent NLR-TRAF interactions suggest the formation of a 'TRAFasome' complex.

Physiological factors that regulate skin pigmentation.

PubMed

Yamaguchi, Yuji; Hearing, Vincent J

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes, and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells, and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A, and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2, and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va, and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including alpha-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins, and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines.

PubMed

Thaker, Youg R; Recino, Asha; Raab, Monika; Jabeen, Asma; Wallberg, Maja; Fernandez, Nelson; Rudd, Christopher E

2017-04-14

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, "3Y") as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4 + primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Activated Cdc42-associated kinase 1 (ACK1) binds the sterile α motif (SAM) domain of the adaptor SLP-76 and phosphorylates proximal tyrosines

PubMed Central

Thaker, Youg R.; Recino, Asha; Raab, Monika; Jabeen, Asma; Wallberg, Maja; Fernandez, Nelson; Rudd, Christopher E.

2017-01-01

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a crucial role in T cell activation by linking antigen receptor (T cell receptor, TCR) signals to downstream pathways. At its N terminus, SLP-76 has three key tyrosines (Tyr-113, Tyr-128, and Tyr-145, “3Y”) as well as a sterile α motif (SAM) domain whose function is unclear. We showed previously that the SAM domain has two binding regions that mediate dimer and oligomer formation. In this study, we have identified SAM domain-carrying non-receptor tyrosine kinase, activated Cdc42-associated tyrosine kinase 1 (ACK1; also known as Tnk2, tyrosine kinase non-receptor 2) as a novel binding partner of SLP-76. Co-precipitation, laser-scanning confocal microscopy, and in situ proximity analysis confirmed the binding of ACK1 to SLP-76. Further, the interaction was induced in response to the anti-TCR ligation and abrogated by the deletion of SLP-76 SAM domain (ΔSAM) or mutation of Tyr-113, Tyr-128, and Tyr-145 to phenylalanine (3Y3F). ACK1 induced phosphorylation of the SLP-76 N-terminal tyrosines (3Y) dependent on the SAM domain. Further, ACK1 promoted calcium flux and NFAT-AP1 promoter activity and decreased the motility of murine CD4+ primary T cells on ICAM-1-coated plates, an event reversed by a small molecule inhibitor of ACK1 (AIM-100). These findings identify ACK1 as a novel SLP-76-associated protein-tyrosine kinase that modulates early activation events in T cells. PMID:28188290

DOTA analogues with a phosphinate-iminodiacetate pendant arm: modification of the complex formation rate with a strongly chelating pendant.

PubMed

Procházková, Soňa; Kubíček, Vojtěch; Böhmová, Zuzana; Holá, Kateřina; Kotek, Jan; Hermann, Petr

2017-08-08

The new ligand H 6 do3aP ida combines the macrocyclic DOTA-like cavity and the open-chain iminodiacetate group connected through a coordinating phosphinate spacer. Its acid-base and coordination properties in solution were studied by potentiometry. Thermodynamic coordination characteristics of both chelating units are similar to those reported for H 4 dota and iminodiacetic acid themselves, respectively, so, macrocyclic and iminodiacetate units behave independently. The formation kinetics of the Ce(iii)-H 6 do3aP ida complex was studied by UV-Vis spectrophotometry. Various out-of-cage intermediates were identified with 1 : 1, 1 : 2 and 2 : 1 ligand-to-metal ratios. The presence of the strongly coordinating iminodiacetate group significantly slows down the metal ion transfer into the macrocyclic cavity and, so, the formation of the in-cage complex is two orders of magnitude slower than that reported for the Ce(iii)-H 4 dota system. The kinetic inertness of the [Ce(do3aP ida )] 3- complex towards acid-assisted dissociation is comparable to that of the [Ce(dota)] - complex. The coordination modes of the ligand are demonstrated in the solid-state structure of [Cu 4 (do3aP ida )(OH)(H 2 O) 4 ]Cl·7.5H 2 O.

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

Inhibition of the Jun N-Terminal Protein Kinase Pathway by SHIP-1, a Lipid Phosphatase That Interacts with the Adaptor Molecule Dok-3

PubMed Central

Robson, Jeffrey D.; Davidson, Dominique; Veillette, André

2004-01-01

Dok-3 is a Dok-related adaptor expressed in B cells and macrophages. Previously, we reported that Dok-3 is an inhibitor of B-cell activation in A20 B cells and that it associates with SHIP-1, a 5′ inositol-specific lipid phosphatase, as well as Csk, a negative regulator of Src kinases. Here, we demonstrate that Dok-3 suppresses B-cell activation by way of its interaction with SHIP-1, rather than Csk. Our biochemical analyses showed that the Dok-3-SHIP-1 complex acts by selectively inhibiting the B-cell receptor (BCR)-evoked activation of the Jun N-terminal protein kinase (JNK) cascade without affecting overall protein tyrosine phosphorylation or activation of previously described SHIP-1 targets like Btk and Akt/PKB. Studies of B cells derived from SHIP-1-deficient mice showed that BCR-triggered activation of JNK is enhanced in the absence of SHIP-1, implying that the Dok-3-SHIP-1 complex (or a related mechanism) is a physiological negative regulator of the JNK cascade in normal B cells. Together, these data elucidate the mechanism by which Dok-3 inhibits B-cell activation. Furthermore, they provide evidence that SHIP-1 can be a negative regulator of JNK signaling in B cells. PMID:14993273

Multiple Dileucine-like Motifs Direct VGLUT1 Trafficking

PubMed Central

Foss, Sarah M.; Li, Haiyan; Santos, Magda S.; Edwards, Robert H.

2013-01-01

The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation. PMID:23804088

Multiple dileucine-like motifs direct VGLUT1 trafficking.

PubMed

Foss, Sarah M; Li, Haiyan; Santos, Magda S; Edwards, Robert H; Voglmaier, Susan M

2013-06-26

The vesicular glutamate transporters (VGLUTs) package glutamate into synaptic vesicles, and the two principal isoforms VGLUT1 and VGLUT2 have been suggested to influence the properties of release. To understand how a VGLUT isoform might influence transmitter release, we have studied their trafficking and previously identified a dileucine-like endocytic motif in the C terminus of VGLUT1. Disruption of this motif impairs the activity-dependent recycling of VGLUT1, but does not eliminate its endocytosis. We now report the identification of two additional dileucine-like motifs in the N terminus of VGLUT1 that are not well conserved in the other isoforms. In the absence of all three motifs, rat VGLUT1 shows limited accumulation at synaptic sites and no longer responds to stimulation. In addition, shRNA-mediated knockdown of clathrin adaptor proteins AP-1 and AP-2 shows that the C-terminal motif acts largely via AP-2, whereas the N-terminal motifs use AP-1. Without the C-terminal motif, knockdown of AP-1 reduces the proportion of VGLUT1 that responds to stimulation. VGLUT1 thus contains multiple sorting signals that engage distinct trafficking mechanisms. In contrast to VGLUT1, the trafficking of VGLUT2 depends almost entirely on the conserved C-terminal dileucine-like motif: without this motif, a substantial fraction of VGLUT2 redistributes to the plasma membrane and the transporter's synaptic localization is disrupted. Consistent with these differences in trafficking signals, wild-type VGLUT1 and VGLUT2 differ in their response to stimulation.

Identification of Novel Molecular Targets for Pleckstrin Homology (PH) Domains Found in Oncogenes Implicated in Breast Cancer

DTIC Science & Technology

2005-03-01

iný a Milia n, rra ihzed ait:r! PH dumairn w.ith ul pgnfvdiir SDAPPI PH Orraai is, ~rlose rffpl<P, AtIrl l --.- P dSark~ly r;h ~~’ hincing spperiftkil...Phosphoinositide Binding Domain in the Clathrin Adaptor AP-2 oc Subunit. J. Biol. Chem. 271(34), 20922-20929. Godi, A, Di Campli, A, Konstantakopoulos, Di ...Ummersen, L , Binger, K, Volkman, J, Mamocha, R, Tutsch, K, Kolesar, J, Arzoomanian, R, Alberti, D, and Wilding, G. (2004) A Phase I Trial of Perifosine (NSC

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

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects.

PubMed

Bekerman, Elena; Neveu, Gregory; Shulla, Ana; Brannan, Jennifer; Pu, Szu-Yuan; Wang, Stanley; Xiao, Fei; Barouch-Bentov, Rina; Bakken, Russell R; Mateo, Roberto; Govero, Jennifer; Nagamine, Claude M; Diamond, Michael S; De Jonghe, Steven; Herdewijn, Piet; Dye, John M; Randall, Glenn; Einav, Shirit

2017-04-03

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses.

Anticancer kinase inhibitors impair intracellular viral trafficking and exert broad-spectrum antiviral effects

PubMed Central

Bekerman, Elena; Shulla, Ana; Brannan, Jennifer; Wang, Stanley; Barouch-Bentov, Rina; Bakken, Russell R.; Mateo, Roberto; Govero, Jennifer; Nagamine, Claude M.; Diamond, Michael S.; De Jonghe, Steven; Herdewijn, Piet; Dye, John M.; Randall, Glenn

2017-01-01

Global health is threatened by emerging viral infections, which largely lack effective vaccines or therapies. Targeting host pathways that are exploited by multiple viruses could offer broad-spectrum solutions. We previously reported that AAK1 and GAK, kinase regulators of the host adaptor proteins AP1 and AP2, are essential for hepatitis C virus (HCV) infection, but the underlying mechanism and relevance to other viruses or in vivo infections remained unknown. Here, we have discovered that AP1 and AP2 cotraffic with HCV particles in live cells. Moreover, we found that multiple viruses, including dengue and Ebola, exploit AAK1 and GAK during entry and infectious virus production. In cultured cells, treatment with sunitinib and erlotinib, approved anticancer drugs that inhibit AAK1 or GAK activity, or with more selective compounds inhibited intracellular trafficking of HCV and multiple unrelated RNA viruses with a high barrier to resistance. In murine models of dengue and Ebola infection, sunitinib/erlotinib combination protected against morbidity and mortality. We validated sunitinib- and erlotinib-mediated inhibition of AAK1 and GAK activity as an important mechanism of antiviral action. Additionally, we revealed potential roles for additional kinase targets. These findings advance our understanding of virus-host interactions and establish a proof of principle for a repurposed, host-targeted approach to combat emerging viruses. PMID:28240606

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor*

PubMed Central

Clayton, Cecilea C.; Donthamsetti, Prashant; Lambert, Nevin A.; Javitch, Jonathan A.; Neve, Kim A.

2014-01-01

Arrestins mediate desensitization and internalization of G protein-coupled receptors and also direct receptor signaling toward heterotrimeric G protein-independent signaling pathways. We previously identified a four-residue segment (residues 212–215) of the dopamine D2 receptor that is necessary for arrestin binding in an in vitro heterologous expression system but that also impairs receptor expression. We now describe the characterization of additional mutations at that arrestin binding site in the third intracellular loop. Mutating two (residues 214 and 215) or three (residues 213–215) of the four residues to alanine partially decreased agonist-induced recruitment of arrestin3 without altering activation of a G protein. Arrestin-dependent receptor internalization, which requires arrestin binding to β2-adaptin (the β2 subunit of the clathrin-associated adaptor protein AP2) and clathrin, was disproportionately affected by the three-residue mutation, with no agonist-induced internalization observed even in the presence of overexpressed arrestin or G protein-coupled receptor kinase 2. The disjunction between arrestin recruitment and internalization could not be explained by alterations in the time course of the receptor-arrestin interaction, the recruitment of G protein-coupled receptor kinase 2, or the receptor-induced interaction between arrestin and β2-adaptin, suggesting that the mutation impairs a property of the internalization complex that has not yet been identified. PMID:25336643

SLP-76 sterile α motif (SAM) and individual H5 α helix mediate oligomer formation for microclusters and T-cell activation.

PubMed

Liu, Hebin; Thaker, Youg Raj; Stagg, Loren; Schneider, Helga; Ladbury, John E; Rudd, Christopher E

2013-10-11

Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation. Dynamic light scattering and fluorescent microscale thermophoresis of the isolated SAM domain (residues 1-78) revealed evidence of dimers and tetramers. Consistently, deletion of the SAM region eliminated SLP-76 co-precipitation of itself, concurrent with a loss of microcluster formation, nuclear factor of activated T-cells (NFAT) transcription, and interleukin-2 production in Jurkat or primary T-cells. Furthermore, the H5 α helix within the SAM domain contributed to self-association. Retention of H5 in the absence of H1-4 sufficed to support SLP-76 self-association with smaller microclusters that nevertheless enhanced anti-CD3-driven AP1/NFAT transcription and IL-2 production. By contrast, deletion of the H5 α helix impaired self-association and anti-CD3 induced AP1/NFAT transcription. Our data identified for the first time a role for the SAM domain in mediating SLP-76 self-association for T-cell function.

SLP-76 Sterile α Motif (SAM) and Individual H5 α Helix Mediate Oligomer Formation for Microclusters and T-cell Activation*

PubMed Central

Liu, Hebin; Thaker, Youg Raj; Stagg, Loren; Schneider, Helga; Ladbury, John E.; Rudd, Christopher E.

2013-01-01

Despite the importance of the immune adaptor SLP-76 in T-cell immunity, it has been unclear whether SLP-76 directly self-associates to form higher order oligomers for T-cell activation. In this study, we show that SLP-76 self-associates in response to T-cell receptor ligation as mediated by the N-terminal sterile α motif (SAM) domain. SLP-76 co-precipitated alternately tagged SLP-76 in response to anti-CD3 ligation. Dynamic light scattering and fluorescent microscale thermophoresis of the isolated SAM domain (residues 1–78) revealed evidence of dimers and tetramers. Consistently, deletion of the SAM region eliminated SLP-76 co-precipitation of itself, concurrent with a loss of microcluster formation, nuclear factor of activated T-cells (NFAT) transcription, and interleukin-2 production in Jurkat or primary T-cells. Furthermore, the H5 α helix within the SAM domain contributed to self-association. Retention of H5 in the absence of H1–4 sufficed to support SLP-76 self-association with smaller microclusters that nevertheless enhanced anti-CD3-driven AP1/NFAT transcription and IL-2 production. By contrast, deletion of the H5 α helix impaired self-association and anti-CD3 induced AP1/NFAT transcription. Our data identified for the first time a role for the SAM domain in mediating SLP-76 self-association for T-cell function. PMID:23935094

NOS1AP is associated with increased severity of PTSD and depression in untreated combat veterans.

PubMed

Lawford, Bruce R; Morris, Charles P; Swagell, Christopher D; Hughes, Ian P; Young, Ross McD; Voisey, Joanne

2013-05-01

Posttraumatic stress disorder (PTSD) and depressive disorder are over represented in combat veterans. Veterans with both disorders have an increased risk of suicide. The nitric oxide synthase 1 adaptor protein (NOS1AP) gene, which modulates stress-evoked N-methyl-d-aspartate (NMDA) activity, was investigated in combat veterans. A comprehensive genetic analysis of NOS1AP and its association with PTSD was investigated in Vietnam combat veterans with PTSD (n=121) and a group of healthy control individuals (n=237). PTSD patients were assessed for symptom severity and level of depression using the Mississippi Scale for Combat-Related PTSD and the Beck Depression Inventory-II (BDI). The G allele of NOS1AP SNP rs386231 was significantly associated with PTSD (p=0.002). Analysis of variance revealed significant differences in BDI-II and Mississippi scores between genotypes for rs386231 with the GG genotype associated with increased severity of depression (p=0.002 F=6.839) and higher Mississippi Scale for Combat-Related PTSD scores (p=0.033). Haplotype analysis revealed that the C/G haplotype (rs451275/rs386231) was significantly associated with PTSD (p=0.001). The sample sizes in our study were not sufficient to detect SNP associations with very small effects. In addition the study was limited by its cross sectional design. This is the first study reporting that a variant of the NOS1AP gene is associated with PTSD. Our data also suggest that a genetic variant in NOS1AP may increase the susceptibility to severe depression in patients with PTSD and increased risk for suicide. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

The Adaptor Molecule Nck Localizes the WAVE Complex to Promote Actin Polymerization during CEACAM3-Mediated Phagocytosis of Bacteria

PubMed Central

Delgado Tascón, Julia; Nyffenegger-Jann, Naja J.; Hauck, Christof R.

2012-01-01

Background CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. Principal Findings In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Conclusions Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment. PMID:22448228

The adaptor molecule Nck localizes the WAVE complex to promote actin polymerization during CEACAM3-mediated phagocytosis of bacteria.

PubMed

Pils, Stefan; Kopp, Kathrin; Peterson, Lisa; Delgado Tascón, Julia; Nyffenegger-Jann, Naja J; Hauck, Christof R

2012-01-01

CEACAM3 is a granulocyte receptor mediating the opsonin-independent recognition and phagocytosis of human-restricted CEACAM-binding bacteria. CEACAM3 function depends on an intracellular immunoreceptor tyrosine-based activation motif (ITAM)-like sequence that is tyrosine phosphorylated by Src family kinases upon receptor engagement. The phosphorylated ITAM-like sequence triggers GTP-loading of Rac by directly associating with the guanine nucleotide exchange factor (GEF) Vav. Rac stimulation in turn is critical for actin cytoskeleton rearrangements that generate lamellipodial protrusions and lead to bacterial uptake. In our present study we provide biochemical and microscopic evidence that the adaptor proteins Nck1 and Nck2, but not CrkL, Grb2 or SLP-76, bind to tyrosine phosphorylated CEACAM3. The association is phosphorylation-dependent and requires the Nck SH2 domain. Overexpression of the isolated Nck1 SH2 domain, RNAi-mediated knock-down of Nck1, or genetic deletion of Nck1 and Nck2 interfere with CEACAM3-mediated bacterial internalization and with the formation of lamellipodial protrusions. Nck is constitutively associated with WAVE2 and directs the actin nucleation promoting WAVE complex to tyrosine phosphorylated CEACAM3. In turn, dominant-negative WAVE2 as well as shRNA-mediated knock-down of WAVE2 or the WAVE-complex component Nap1 reduce internalization of bacteria. Our results provide novel mechanistic insight into CEACAM3-initiated phagocytosis. We suggest that the CEACAM3 ITAM-like sequence is optimized to co-ordinate a minimal set of cellular factors needed to efficiently trigger actin-based lamellipodial protrusions and rapid pathogen engulfment.

SLP-65 signal transduction requires Src homology 2 domain-mediated membrane anchoring and a kinase-independent adaptor function of Syk.

PubMed

Abudula, Abulizi; Grabbe, Annika; Brechmann, Markus; Polaschegg, Christian; Herrmann, Nadine; Goldbeck, Ingo; Dittmann, Kai; Wienands, Jürgen

2007-09-28

The family of SLPs (Src homology 2 domain-containing leukocyte adaptor proteins) are cytoplasmic signal effectors of lymphocyte antigen receptors. A main function of SLP is to orchestrate the assembly of Ca(2+)-mobilizing enzymes at the inner leaflet of the plasma membrane. For this purpose, SLP-76 in T cells utilizes the transmembrane adaptor LAT, but the mechanism of SLP-65 membrane anchoring in B cells remains an enigma. We now employed two genetic reconstitution systems to unravel structural requirements of SLP-65 for the initiation of Ca(2+) mobilization and subsequent activation of gene transcription. First, mutational analysis of SLP-65 in DT40 B cells revealed that its C-terminal Src homology 2 domain controls efficient tyrosine phosphorylation by the kinase Syk, plasma membrane recruitment, as well as downstream signaling to NFAT activation. Second, we dissected these processes by expressing SLP-65 in SLP-76-deficient T cells and found that a kinase-independent adaptor function of Syk is required to link phosphorylated SLP-65 to Ca(2+) mobilization. These approaches unmask a mechanistic complexity of SLP-65 activation and coupling to signaling cascades in that Syk is upstream as well as downstream of SLP-65. Moreover, membrane anchoring of the SLP-65-assembled Ca(2+) initiation complex, which appears to be fundamentally different from that of closely related SLP-76, does not necessarily involve a B cell-specific component.

Coordination of receptor signaling in multiple hematopoietic cell lineages by the adaptor protein SLP-76.

PubMed

Jordan, Martha S; Koretzky, Gary A

2010-04-01

The adaptor protein SLP-76 is expressed in multiple hematopoietic lineages including T cells, platelets, and neutrophils. SLP-76 mediated signaling is dependent on its multiple protein interaction domains, as it creates a scaffold on which key signaling complexes are built. SLP-76 is critical for supporting signaling downstream of both immunoreceptors and integrins. The signaling molecules used both upstream and downstream of SLP-76 are similar among these receptors and across cell types; however, important differences exist. Appreciating how SLP-76 coordinates signal transduction across different cell and receptor types provides insights into the complex interplay of pathways critical for activation of cells of the immune system that are essential for host defense.

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

Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B.

PubMed

Rivera, G M; Antoku, S; Gelkop, S; Shin, N Y; Hanks, S K; Pawson, T; Mayer, B J

2006-06-20

The Nck family of Src homology (SH) 2/SH3 domain adaptors functions to link tyrosine phosphorylation induced by extracellular signals with downstream regulators of actin dynamics. We investigated the role of mammalian Nck adaptors in signaling from the activated platelet-derived growth factor (PDGF) receptor (PDGFbetaR) to the actin cytoskeleton. We report here that Nck adaptors are required for cytoskeletal reorganization and chemotaxis stimulated by PDGF-B. Analysis of tyrosine-phosphorylated proteins demonstrated that Crk-associated substrate (p130(Cas)), not the activated PDGFbetaR itself, is the major Nck SH2 domain-binding protein in PDGF-B-stimulated cells. Both Nck- and p130(Cas)-deficient cells fail to display cytoskeletal rearrangements, including the formation of membrane ruffles and the disassembly of actin bundles, typically shown by their WT counterparts in response to PDGF-B. Furthermore, Nck and p130(Cas) colocalize in phosphotyrosine-enriched membrane ruffles induced by PDGF-B in NIH 3T3 cells. These results suggest that Nck adaptors play an essential role in linking the activated PDGFbetaR with actin dynamics through a pathway that involves p130(Cas).

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. Copyright © 2015 Elsevier Inc. All rights reserved.

Dock/Nck facilitates PTP61F/PTP1B regulation of insulin signalling.

PubMed

Wu, Chia-Lun; Buszard, Bree; Teng, Chun-Hung; Chen, Wei-Lin; Warr, Coral G; Tiganis, Tony; Meng, Tzu-Ching

2011-10-01

PTP1B (protein tyrosine phosphatase 1B) is a negative regulator of IR (insulin receptor) activation and glucose homoeostasis, but the precise molecular mechanisms governing PTP1B substrate selectivity and the regulation of insulin signalling remain unclear. In the present study we have taken advantage of Drosophila as a model organism to establish the role of the SH3 (Src homology 3)/SH2 adaptor protein Dock (Dreadlocks) and its mammalian counterpart Nck in IR regulation by PTPs. We demonstrate that the PTP1B orthologue PTP61F dephosphorylates the Drosophila IR in S2 cells in vitro and attenuates IR-induced eye overgrowth in vivo. Our studies indicate that Dock forms a stable complex with PTP61F and that Dock/PTP61F associate with the IR in response to insulin. We report that Dock is required for effective IR dephosphorylation and inactivation by PTP61F in vitro and in vivo. Furthermore, we demonstrate that Nck interacts with PTP1B and that the Nck/PTP1B complex inducibly associates with the IR for the attenuation of IR activation in mammalian cells. Our studies reveal for the first time that the adaptor protein Dock/Nck attenuates insulin signalling by recruiting PTP61F/PTP1B to its substrate, the IR.

Characterization of complexes of nucleoside-5'-phosphorothioate analogues with zinc ions.

PubMed

Sayer, Alon Haim; Itzhakov, Yehudit; Stern, Noa; Nadel, Yael; Fischer, Bilha

2013-10-07

On the basis of the high affinity of Zn(2+) to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP3(β-S)A, as potential biocompatible Zn(2+)-chelators. The thiophosphate moiety enhanced the stability of the Zn(2+)-nucleotide complex by about 0.7 log units. ATP-α,β-CH2-γ-S formed the most stable Zn(2+)-complex studied here, log K 6.50, being ~0.8 and ~1.1 log units more stable than ATP-γ-S-Zn(2+) and ATP-Zn(2+) complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn(2+) complexes were more stable by 0.3-0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP3(β-S)A-zinc complex was ~0.5 log units more stable than AP3A complex. (1)H- and (31)P NMR monitored Zn(2+) titration showed that Zn(2+) coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn(2+)-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn(2+) complexes than a bridging phosphorothioate.

Loss of endocytic clathrin-coated pits upon acute depletion of phosphatidylinositol 4,5-bisphosphate.

PubMed

Zoncu, Roberto; Perera, Rushika M; Sebastian, Rafael; Nakatsu, Fubito; Chen, Hong; Balla, Tamas; Ayala, Guillermo; Toomre, Derek; De Camilli, Pietro V

2007-03-06

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], a phosphoinositide concentrated predominantly in the plasma membrane, binds endocytic clathrin adaptors, many of their accessory factors, and a variety of actin-regulatory proteins. Here we have used fluorescent fusion proteins and total internal reflection fluorescence microscopy to investigate the effect of acute PI(4,5)P(2) breakdown on the dynamics of endocytic clathrin-coated pit components and of the actin regulatory complex, Arp2/3. PI(4,5)P(2) breakdown was achieved by the inducible recruitment to the plasma membrane of an inositol 5-phosphatase module through the rapamycin/FRB/FKBP system or by treatment with ionomycin. PI(4,5)P(2) depletion resulted in a dramatic loss of clathrin puncta, which correlated with a massive dissociation of endocytic adaptors from the plasma membrane. Remaining clathrin spots at the cell surface had only weak fluorescence and were static over time. Dynamin and the p20 subunit of the Arp2/3 actin regulatory complex, which were concentrated at late-stage clathrin-coated pits and in lamellipodia, also dissociated from the plasma membrane, and these changes correlated with an arrest of motility at the cell edge. These findings demonstrate the critical importance of PI(4,5)P(2) in clathrin coat dynamics and Arp2/3-dependent actin regulation.

Interactive Nonlinear Structural Analysis: Enhancement.

DTIC Science & Technology

1981-07-31

3251 Hanover Street Palo Alto, California 94304 i "඙ 8 17 040 . G IS T GRAPHICS- INTERACTIVE STRUCTURAL ANALYSIS VIA THE GIFTS /STAGS SOFTWARE ASSEMBLY...GIST Software Components Page Section 3.0 Introduction * 2 3.1 GIFTS Architecture . . . . . . . . . . . . . 4 3.2 STAGS Architecture . . . 5 3.3 The... GIFTS ->STAGS Adaptor . . . . . . . . . . 6 3.4 The STAGS-> GIFTS Adaptor . . . . . . . . . . 37 3.5 The GIST Control Module . . . . . . . . . . 55 GIST

Gene structure, cDNA characterization and RNAi-based functional analysis of a myeloid differentiation factor 88 homolog in Tenebrio molitor larvae exposed to Staphylococcus aureus infection.

PubMed

Patnaik, Bharat Bhusan; Patnaik, Hongray Howrelia; Seo, Gi Won; Jo, Yong Hun; Lee, Yong Seok; Lee, Bok Luel; Han, Yeon Soo

2014-10-01

Myeloid differentiation factor 88 (MyD88), an intracellular adaptor protein involved in Toll/Toll-like receptor (TLR) signal processing, triggers activation of nuclear factor-kappaB (NF-κB) transcription factors. In the present study, we analyzed the gene structure and biological function of MyD88 in a coleopteran insect, Tenebrio molitor (TmMyD88). The TmMyD88 gene was 1380 bp in length and consisted of five exons and four introns. The 5'-flanking sequence revealed several putative transcription factor binding sites, such as STAT-4, AP-1, cJun, cfos, NF-1 and many heat shock factor binding elements. The cDNA contained a typical death domain, a conservative Toll-like interleukin-1 receptor (TIR) domain, and a C-terminal extension (CTE). The TmMyD88 TIR domain showed three significantly conserved motifs for interacting with the TIR domain of TLRs. TmMyD88 was grouped within the invertebrate cluster of the phylogenetic tree and shared 75% sequence identity with the TIR domain of Tribolium castaneum MyD88. Homology modeling of the TmMyD88 TIR domain revealed five parallel β-strands surrounded by five α-helices that adopted loop conformations to function as an adaptor. TmMyD88 expression was upregulated 7.3- and 4.79-fold after 12 and 6h, respectively, of challenge with Staphylococcus aureus and fungal β-1,3 glucan. Silencing of the TmMyD88 transcript by RNA interference led to reduced resistance of the host to infection by S. aureus. These results indicate that TmMyD88 is required for survival against Staphylococcus infection. Copyright © 2014 Elsevier Ltd. All rights reserved.

Parallel SCF Adaptor Capture Proteomics Reveals a Role for SCFFBXL17 in NRF2 Activation via BACH1 Repressor Turnover

PubMed Central

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J.; Shi, Yang; Harper, J. Wade

2014-01-01

Modular Cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of Parallel Adaptor Capture (PAC) proteomics, and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCFFBXL17 in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. PMID:24035498

Parallel SCF adaptor capture proteomics reveals a role for SCFFBXL17 in NRF2 activation via BACH1 repressor turnover.

PubMed

Tan, Meng-Kwang Marcus; Lim, Hui-Jun; Bennett, Eric J; Shi, Yang; Harper, J Wade

2013-10-10

Modular cullin-RING E3 ubiquitin ligases (CRLs) use substrate binding adaptor proteins to specify target ubiquitylation. Many of the ~200 human CRL adaptor proteins remain poorly studied due to a shortage of efficient methods to identify biologically relevant substrates. Here, we report the development of parallel adaptor capture (PAC) proteomics and its use to systematically identify candidate targets for the leucine-rich repeat family of F-box proteins (FBXLs) that function with SKP1-CUL1-F-box protein (SCF) E3s. In validation experiments, we identify the unstudied F-box protein FBXL17 as a regulator of the NFR2 oxidative stress pathway. We demonstrate that FBXL17 controls the transcription of the NRF2 target HMOX1 via turnover of the transcriptional repressor BACH1 in the absence or presence of extrinsic oxidative stress. This work identifies a role for SCF(FBXL17) in controlling the threshold for NRF2-dependent gene activation and provides a framework for elucidating the functions of CRL adaptor proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

Myosin 7 and its adaptors link cadherins to actin.

PubMed

Yu, I-Mei; Planelles-Herrero, Vicente J; Sourigues, Yannick; Moussaoui, Dihia; Sirkia, Helena; Kikuti, Carlos; Stroebel, David; Titus, Margaret A; Houdusse, Anne

2017-06-29

Cadherin linkages between adjacent stereocilia and microvilli are essential for mechanotransduction and maintaining their organization. They are anchored to actin through interaction of their cytoplasmic domains with related tripartite complexes consisting of a class VII myosin and adaptor proteins: Myo7a/SANS/Harmonin in stereocilia and Myo7b/ANKS4B/Harmonin in microvilli. Here, we determine high-resolution structures of Myo7a and Myo7b C-terminal MyTH4-FERM domain (MF2) and unveil how they recognize harmonin using a novel binding mode. Systematic definition of interactions between domains of the tripartite complex elucidates how the complex assembles and prevents possible self-association of harmonin-a. Several Myo7a deafness mutants that map to the surface of MF2 disrupt harmonin binding, revealing the molecular basis for how they impact the formation of the tripartite complex and disrupt mechanotransduction. Our results also suggest how switching between different harmonin isoforms can regulate the formation of networks with Myo7a motors and coordinate force sensing in stereocilia.

FSGS3/CD2AP is a barbed-end capping protein that stabilizes actin and strengthens adherens junctions

PubMed Central

Brieher, William M.

2013-01-01

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as a novel actin barbed-end capping protein responsible for actin stability at the adherens junction. FSGS3/CD2AP colocalizes with E-cadherin and α-actinin-4 at the apical junction in polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of FSGS3/CD2AP compromised actin stability and decreased actin accumulation at the adherens junction. Using a novel apparatus to apply mechanical stress to cell–cell junctions, we showed that knockdown of FSGS3/CD2AP compromised adhesive strength, resulting in tearing between cells and disruption of barrier function. Our results reveal a novel function of FSGS3/CD2AP and a previously unrecognized role of barbed-end capping in junctional actin dynamics. Our study underscores the complexity of actin regulation at cell–cell contacts that involves actin activators, inhibitors, and stabilizers to control adhesive strength, epithelial behavior, and permeability barrier integrity. PMID:24322428

New La(III) complex immobilized on 3-aminopropyl-functionalized silica as an efficient and reusable catalyst for hydrolysis of phosphate ester bonds.

PubMed

Muxel, Alfredo A; Neves, Ademir; Camargo, Maryene A; Bortoluzzi, Adailton J; Szpoganicz, Bruno; Castellano, Eduardo E; Castilho, Nathalia; Bortolotto, Tiago; Terenzi, Hernán

2014-03-17

Described herein is the synthesis, structure, and monoesterase and diesterase activities of a new mononuclear [La(III)(L(1))(NO3)2] (1) complex (H2L(1) = 2-bis[{(2-pyridylmethyl)-aminomethyl}-6-[N-(2-pyridylmethyl) aminomethyl)])-4-methyl-6-formylphenol) in the hydrolysis of 2,4-bis(dinitrophenyl)phosphate (2,4-BDNPP). When covalently linked to 3-aminopropyl-functionalized silica, 1 undergoes disproportionation to form a dinuclear species (APS-1), whose catalytic efficiency is increased when compared to the homogeneous reaction due to second coordination sphere effects which increase the substrate to complex association constant. The anchored catalyst APS-1 can be recovered and reused for subsequent hydrolysis reactions (five times) with only a slight loss in activity. In the presence of DNA, we suggest that 1 is also converted into the dinuclear active species as observed with APS-1, and both were shown to be efficient in DNA cleavage.

MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1.

PubMed

Lim, Raelene; Winteringham, Louise N; Williams, James H; McCulloch, Ross K; Ingley, Evan; Tiao, Jim Y-H; Lalonde, Jean-Philippe; Tsai, Schickwann; Tilbrook, Peta A; Sun, Yi; Wu, Xiaohua; Morris, Stephan W; Klinken, S Peter

2002-10-25

A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function.

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. © 2016 Müller-McNicoll et al.; Published by Cold Spring Harbor Laboratory Press.

Evolutionary genomics suggests that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ortega, Davi R.; Zhulin, Igor B.; Punta, Marco

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

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

Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease 1

PubMed Central

Malina, Jaroslav; Scott, Peter; Brabec, Viktor

2015-01-01

Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo–helical ‘flexicate’ complexes, bimetallic triple-stranded ferro-helicates [Fe2(NN-NN)3]4+ incorporating the common NN–NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo–helical complexes for cancer chemotherapy. PMID:25940617

Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

PubMed Central

Honda, Kenya; Yanai, Hideyuki; Mizutani, Tatsuaki; Negishi, Hideo; Shimada, Naoya; Suzuki, Nobutaka; Ohba, Yusuke; Takaoka, Akinori; Yeh, Wen-Chen; Taniguchi, Tadatsugu

2004-01-01

Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-α/β) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal “input” can be processed through MyD88 to “output” the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor. PMID:15492225

A composite MyD88/CD40 switch synergistically activates mouse and human dendritic cells for enhanced antitumor efficacy

PubMed Central

Narayanan, Priyadharshini; Lapteva, Natalia; Seethammagari, Mamatha; Levitt, Jonathan M.; Slawin, Kevin M.; Spencer, David M.

2011-01-01

The in vivo therapeutic efficacy of DC-based cancer vaccines is limited by suboptimal DC maturation protocols. Although delivery of TLR adjuvants systemically boosts DC-based cancer vaccine efficacy, it could also increase toxicity. Here, we have engineered a drug-inducible, composite activation receptor for DCs (referred to herein as DC-CAR) comprising the TLR adaptor MyD88, the CD40 cytoplasmic region, and 2 ligand-binding FKBP12 domains. Administration of a lipid-permeant dimerizing ligand (AP1903) induced oligomerization and activation of this fusion protein, which we termed iMyD88/CD40. AP1903 administration to vaccinated mice enabled prolonged and targeted activation of iMyD88/CD40-modified DCs. Compared with conventionally matured DCs, AP1903-activated iMyD88/CD40-DCs had increased activation of proinflammatory MAPKs. AP1903-activated iMyD88/CD40-transduced human or mouse DCs also produced higher levels of Th1 cytokines, showed improved migration in vivo, and enhanced both antigen-specific CD8+ T cell responses and innate NK cell responses. Furthermore, treatment with AP1903 in vaccinated mice led to robust antitumor immunity against preestablished E.G7-OVA lymphomas and aggressive B16.F10 tumors. Thus, the iMyD88/CD40 unified “switch” effectively and safely replaced exogenous adjuvant cocktails, allowing remote and sustained DC activation in vivo. DC “licensing” through iMyD88/CD40 may represent a mechanism by which to exploit the natural synergy between the TLR and CD40 signaling pathways in DCs using a single small molecule drug and could augment the efficacy of antitumor DC-based vaccines. PMID:21383499

ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor.

PubMed

Lavin, Martin F; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W

2015-10-23

The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes.

Differential transcriptome analysis supports Rhodnius montenegrensis and Rhodnius robustus (Hemiptera, Reduviidae, Triatominae) as distinct species.

PubMed

de Carvalho, Danila Blanco; Congrains, Carlos; Chahad-Ehlers, Samira; Pinotti, Heloisa; Brito, Reinaldo Alves de; da Rosa, João Aristeu

2017-01-01

Chagas disease is one of the main parasitic diseases found in Latin America and it is estimated that between six and seven million people are infected worldwide. Its etiologic agent, the protozoan Trypanosoma cruzi, is transmitted by triatomines, some of which from the genus Rhodnius. Twenty species are currently recognized in this genus, including some closely related species with low levels of morphological differentiation, such as Rhodnius montenegrensis and Rhodnius robustus. In order to investigate genetic differences between these two species, we generated large-scale RNA-sequencing data (consisting of four RNA-seq libraries) from the heads and salivary glands of males of R. montenegrensis and R. robustus. Transcriptome assemblies produced for each species resulted in 64,952 contigs for R. montenegrensis and 70,894 contigs for R. robustus, with N50 of approximately 2,100 for both species. SNP calling based on the more complete R. robustus assembly revealed 3,055 fixed interspecific differences and 216 transcripts with high levels of divergence which contained only fixed differences between the two species. A gene ontology enrichment analysis revealed that these highly differentiated transcripts were enriched for eight GO terms related to AP-2 adaptor complex, as well as other interesting genes that could be involved in their differentiation. The results show that R. montenegrensis and R. robustus have a substantial quantity of fixed interspecific polymorphisms, which suggests a high degree of genetic divergence between the two species and likely corroborates the species status of R. montenegrensis.

Structural basis for recognition of the T cell adaptor protein SLP-76 by the SH3 domain of phospholipase Cgamma1.

PubMed

Deng, Lu; Velikovsky, C Alejandro; Swaminathan, Chittoor P; Cho, Sangwoo; Mariuzza, Roy A

2005-09-09

The enzyme phospholipase Cgamma1 (PLCgamma1) is essential for T cell signaling and activation. Following T cell receptor ligation, PLCgamma1 interacts through its SH2 and SH3 domains with the adaptors LAT and SLP-76, respectively, to form a multiprotein signaling complex that leads to activation of PLCgamma1 by Syk tyrosine kinases. To identify the binding site for PLCgamma1 in SLP-76, we used isothermal titration calorimetry to measure affinities for the interaction of PLCgamma1-SH3 with a set of overlapping peptides spanning the central proline-rich region of SLP-76. PLCgamma1-SH3 bound with high specificity to the SLP-76 motif 186PPVPPQRP193, which represents the minimal binding site. To understand the basis for selective recognition, we determined the crystal structures of PLCgamma1-SH3 in free form, and bound to a 10-mer peptide containing this site, to resolutions of 1.60 A and 1.81 A, respectively. The structures reveal that several key contacting residues of the SH3 shift toward the SLP-76 peptide upon complex formation, optimizing the fit and strengthening hydrophobic interactions. Selectivity results mainly from strict shape complementarity between protein and peptide, rather than sequence-specific hydrogen bonding. In addition, Pro193 of SLP-76 assists in positioning Arg192 into the compass pocket of PLCgamma1-SH3, which coordinates the compass residue through an unusual aspartate. The PLCgamma1-SH3/SLP-76 structure provides insights into ligand binding by SH3 domains related to PLCgamma1-SH3, as well as into recognition by PLCgamma1 of signaling partners other than SLP-76.

Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies.

PubMed

Omotuyi, Olaposi; Matsunaga, Hayato; Ueda, Hiroshi

2015-01-01

During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF-IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied. Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics. Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 - 111) superficially interacts with similar TLR4/MD-2 (KD = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 - 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS. ProTα via its C-terminal peptide (aa 91 - 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

DETAIL VIEW OF WINCH USED TO RAISE AND LOWER AP ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

DETAIL VIEW OF WINCH USED TO RAISE AND LOWER AP 93W, ROOF LEVEL, PLATFORM D-SOUTH, HB-3, FACING WEST - Cape Canaveral Air Force Station, Launch Complex 39, Vehicle Assembly Building, VAB Road, East of Kennedy Parkway North, Cape Canaveral, Brevard County, FL

Immunomodulatory therapies for acute pancreatitis

PubMed Central

Li, Jing; Yang, Wen-Juan; Huang, Lu-Ming; Tang, Cheng-Wei

2014-01-01

It is currently difficult for conventional treatments of acute pancreatitis (AP), which primarily consist of anti-inflammatory therapies, to prevent the progression of AP or to improve its outcome. This may be because the occurrence and progression of AP, which involves various inflammatory cells and cytokines, includes a series of complex immune events. Considering the complex immune system alterations during the course of AP, it is necessary to monitor the indicators related to immune cells and inflammatory mediators and to develop more individualized interventions for AP patients using immunomodulatory therapy. This review discusses the recent advances in immunomodulatory therapies. It has been suggested that overactive inflammatory responses should be inhibited and excessive immunosuppression should be avoided in the early stages of AP. The optimal duration of anti-inflammatory therapy may be shorter than previously expected (< 24 h), and appropriate immunostimulatory therapies should be administered during the period from the 3rd d to the 14th d in the course of AP. A combination therapy of anti-inflammatory and immune-stimulating drugs would hopefully constitute an alternative to anti-inflammatory drug monotherapy. Additionally, the detection of the genotypes of critical inflammatory mediators may be useful for screening populations of AP patients at high risk of severe infections to enable the administration of early interventions to improve their prognosis. PMID:25493006

Structure of the E6/E6AP/p53 complex required for HPV-mediated degradation of p53

PubMed Central

Martinez-Zapien, Denise; Ruiz, Francesc Xavier; Poirson, Juline; Mitschler, André; Ramirez-Ramos, Juan; Forster, Anne; Cousido-Siah, Alexandra; Masson, Murielle; Pol, Scott Vande; Podjarny, Alberto; Travé, Gilles; Zanier, Katia

2015-01-01

Summary The p53 pro-apoptotic tumor suppressor is mutated or functionally altered in most cancers. In epithelial tumors induced by “high-risk” mucosal Human Papillomaviruses (hrm-HPVs), including human cervical carcinoma and a growing number of head-and-neck cancers 1, p53 is degraded by the viral oncoprotein E6 2. In this process, E6 binds to a short LxxLL consensus sequence within the cellular ubiquitin ligase E6AP 3. Subsequently, the E6/E6AP heterodimer recruits and degrades p53 4. Neither E6 nor E6AP are separately able to recruit p53 3,5, and the precise mode of assembly of E6, E6AP and p53 is unknown. Here, we solved the crystal structure of a ternary complex comprising full-length HPV16 E6, the LxxLL motif of E6AP and the core domain of p53. The LxxLL motif of E6AP renders the conformation of E6 competent for interaction with p53 by structuring a p53-binding cleft on E6. Mutagenesis of critical positions at the E6-p53 interface disrupts p53 degradation. The E6-binding site of p53 is distal from previously described DNA- and protein-binding surfaces of the core domain. This suggests that, in principle, E6 may avoid competition with cellular factors by targeting both free and bound p53 molecules. The E6/E6AP/p53 complex represents a prototype of viral hijacking of both the ubiquitin-mediated protein degradation pathway and the p53 tumor suppressor pathway. The present structure provides a framework for the design of inhibitory therapeutic strategies against HPV-mediated oncogenesis. PMID:26789255

Matrix proteins of Nipah and Hendra viruses interact with beta subunits of AP-3 complexes.

PubMed

Sun, Weina; McCrory, Thomas S; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell; Schmitt, Anthony P

2014-11-01

Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Matrix Proteins of Nipah and Hendra Viruses Interact with Beta Subunits of AP-3 Complexes

PubMed Central

Sun, Weina; McCrory, Thomas S.; Khaw, Wei Young; Petzing, Stephanie; Myers, Terrell

2014-01-01

ABSTRACT Paramyxoviruses and other negative-strand RNA viruses encode matrix proteins that coordinate the virus assembly process. The matrix proteins link the viral glycoproteins and the viral ribonucleoproteins at virus assembly sites and often recruit host machinery that facilitates the budding process. Using a co-affinity purification strategy, we have identified the beta subunit of the AP-3 adapter protein complex, AP3B1, as a binding partner for the M proteins of the zoonotic paramyxoviruses Nipah virus and Hendra virus. Binding function was localized to the serine-rich and acidic Hinge domain of AP3B1, and a 29-amino-acid Hinge-derived polypeptide was sufficient for M protein binding in coimmunoprecipitation assays. Virus-like particle (VLP) production assays were used to assess the relationship between AP3B1 binding and M protein function. We found that for both Nipah virus and Hendra virus, M protein expression in the absence of any other viral proteins led to the efficient production of VLPs in transfected cells, and this VLP production was potently inhibited upon overexpression of short M-binding polypeptides derived from the Hinge region of AP3B1. Both human and bat (Pteropus alecto) AP3B1-derived polypeptides were highly effective at inhibiting the production of VLPs. VLP production was also impaired through small interfering RNA (siRNA)-mediated depletion of AP3B1 from cells. These findings suggest that AP-3-directed trafficking processes are important for henipavirus particle production and identify a new host protein-virus protein binding interface that could become a useful target in future efforts to develop small molecule inhibitors to combat paramyxoviral infections. IMPORTANCE Henipaviruses cause deadly infections in humans, with a mortality rate of about 40%. Hendra virus outbreaks in Australia, all involving horses and some involving transmission to humans, have been a continuing problem. Nipah virus caused a large outbreak in Malaysia in 1998, killing 109 people, and smaller outbreaks have since occurred in Bangladesh and India. In this study, we have defined, for the first time, host factors that interact with henipavirus M proteins and contribute to viral particle assembly. We have also defined a new host protein-viral protein binding interface that can potentially be targeted for the inhibition of paramyxovirus infections. PMID:25210190

Theoretical studies on the electronic and optoelectronic properties of [A.2AP(w)/A*.2AP(WC)/C.2AP(w)/C*.2AP(WC)/C.A(w)/C*.A(WC)]-Au8 mismatch nucleobase complexes

NASA Astrophysics Data System (ADS)

Srivastava, Ruby

2018-01-01

The electronic and optoelectronic properties of [A.2AP(w)/A*.2AP(WC)/C.2AP(w)/C*.2AP(WC)/C.A(w)/ C*.A(WC)]-Au8 metal-mismatch nucleobase complexes are investigated by means of density functional theory and time-dependent methods. We selected these mispairs as 2-aminopurine (2AP) produces incorporation errors when binding with cytosine (C) into the wobble (w) C.2AP(w) mispair, and is tautomerised into Watson-Crick (WC)-like base mispair C*.2AP(WC) and less effectively produces A.2AP(w)/A*.2AP(WC) mispairs. The vertical ionisation potential, vertical electron affinity, hardness and electrophilicity index of these complexes have also been discussed. The modifications of energy levels and charge density distributions of the frontier orbitals are also analysed. The absorption spectra of these complexes lie in the visible region, which suggests their application in fluorescent-bio imaging. The mechanism of cooperativity effect is studied by molecular orbital potential (MEP), atoms-in-molecules (AIM) and natural bond orbital analyses. Most metalated pairs have smaller HOMO-LUMO band gaps than the isolated mismatch nucleobases which suggest interesting consequences for electron transfer through DNA duplexes.

Adaptor assembly for coupling turbine blades to rotor disks

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 rootmore » 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.« less

Shape-selective recognition of DNA abasic sites by metallohelices: inhibition of human AP endonuclease 1.

PubMed

Malina, Jaroslav; Scott, Peter; Brabec, Viktor

2015-06-23

Loss of a base in DNA leading to creation of an abasic (AP) site leaving a deoxyribose residue in the strand, is a frequent lesion that may occur spontaneously or under the action of various physical and chemical agents. Progress in the understanding of the chemistry and enzymology of abasic DNA largely relies upon the study of AP sites in synthetic duplexes. We report here on interactions of diastereomerically pure metallo-helical 'flexicate' complexes, bimetallic triple-stranded ferro-helicates [Fe2(NN-NN)3](4+) incorporating the common NN-NN bis(bidentate) helicand, with short DNA duplexes containing AP sites in different sequence contexts. The results show that the flexicates bind to AP sites in DNA duplexes in a shape-selective manner. They preferentially bind to AP sites flanked by purines on both sides and their binding is enhanced when a pyrimidine is placed in opposite orientation to the lesion. Notably, the Λ-enantiomer binds to all tested AP sites with higher affinity than the Δ-enantiomer. In addition, the binding of the flexicates to AP sites inhibits the activity of human AP endonuclease 1, which is as a valid anticancer drug target. Hence, this finding indicates the potential of utilizing well-defined metallo-helical complexes for cancer chemotherapy. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Approximate Entropy Values Demonstrate Impaired Neuromotor Control of Spontaneous Leg Activity in Infants with Myelomeningocele

PubMed Central

Smith, Beth A.; Teulier, Caroline; Sansom, Jennifer; Stergiou, Nicholas; Ulrich, Beverly D.

2012-01-01

Purpose One obstacle to providing early intervention to infants with myelomeningocele (MMC) is the challenge of quantifying impaired neuromotor control of movements early in life. Methods We used the nonlinear analysis tool Approximate Entropy (ApEn) to analyze periodicity and complexity of supine spontaneous lower extremity movements of infants with MMC and typical development (TD) at 1, 3, 6 and 9 months of age. Results Movements of infants with MMC were more regular and repeatable (lower ApEn values) than movements of infants with TD indicating less adaptive and flexible movement patterns. For both groups ApEn values decreased with age, and the movements of infants with MMC were less complex than movements of infants with TD. Further, for infants with MMC, lesion level and age of walking onset correlated negatively with ApEn values. Conclusions Our study begins to demonstrate the feasibility of ApEn to identify impaired neuromotor control in infants with MMC. PMID:21829116

o-Iminobenzosemiquinonate and o-imino-p-methylbenzosemiquinonate anion radicals coupled VO2+ stabilization.

PubMed

Roy, Amit Saha; Saha, Pinaki; Adhikary, Nirmal Das; Ghosh, Prasanta

2011-03-21

The diamagnetic VO(2+)-iminobenzosemiquinonate anion radical (L(R)(IS)(•-), R = H, Me) complexes, (L(-))(VO(2+))(L(R)(IS)(•-)): (L(1)(-))(VO(2+))(L(H)(IS)(•-))•3/2MeOH (1•3/2MeOH), (L(2)(-))(VO(2+))(L(H)(IS)(•-)) (2), and (L(2)(-))(VO(2+))(L(Me)(IS)(•-))•1/2 L(Me)(AP) (3•1/2 L(Me)(AP)), incorporating tridentate monoanionic NNO-donor ligands {L = L(1)(-) or L(2)(-), L(1)H = (2-[(phenylpyridin-2-yl-methylene)amino]phenol; L(2)H = 1-(2-pyridylazo)-2-naphthol; L(H)(IS)(•-) = o-iminobenzosemiquinonate anion radical; L(Me)(IS)(•-) = o-imino-p-methylbenzosemiquinonate anion radical; and L(Me)(AP) = o-amino-p-methylphenol} have been isolated and characterized by elemental analyses, IR, mass, NMR, and UV-vis spectra, including the single-crystal X-ray structure determinations of 1•3/2MeOH and 3•1/2 L(Me)(AP). Complexes 1•3/2MeOH, 2, and 3•1/2 L(Me)(AP) absorb strongly in the visible region because of intraligand (IL) and ligand-to-metal charge transfers (LMCT). 1•3/2MeOH is luminescent (λ(ext), 333 nm; λ(em), 522, 553 nm) in frozen dichloromethane-toluene glass at 77 K due to π(diimine→)π(diimine)* transition. The V-O(phenolato) (cis to the V═O) lengths, 1.940(2) and 1.984(2) Å, respectively, in 1•3/2MeOH and 3•1/2 L(Me)(AP) are consistent with the VO(2+) description. The V-O(iminosemiquinonate) (trans to the V═O) lengths, 2.1324(19) in 1•3/2MeOH and 2.083(2) Å in 3•1/2 L(Me)(AP), are expectedly ∼0.20 Å longer due to the trans influence of the V═O bond. Because of the stronger affinity of the paramagnetic VO(2+) ion to the L(H)(IS)(•-) or L(Me)(IS)(•-), the V-N(iminosemiquinonate) lengths, 1.908(2) and 1.921(2) Å, respectively, in 1•3/2MeOH and 3•1/2 L(Me)(AP), are unexpectedly shorter. Density functional theory (DFT) calculations using B3LYP, B3PW91, and PBE1PBE functionals on 1 and 2 have established that the closed shell singlet (CSS) solutions (VO(3+)-amidophenolato (L(R)(AP)(2-)) coordination) of these complexes are unstable with respect to triplet perturbations. But BS (1,1) M(s) = 0 (VO(2+)-iminobenzosemiquinonate anion radical (L(R)(IS)(•-)) coordination) solutions of these species are stable and reproduce the experimental bond parameters well. Spin density distributions of one electron oxidized cations are consistent with the [(L(-))(VO(2+))(L(R)(IQ))](+) descriptions [VO(2+)-o-iminobenzoquinone (L(R)(IQ)) coordination], and one electron reduced anions are consistent with the [(L(•2-))(VO(3+))(L(R)(AP)(2-))](-) descriptions [VO(3+)-amidophenolato (L(R)(AP)(2-)) coordination], incorporating the diimine anion radical (L(1)(•2-)) or azo anion radical (L(2)(3-)). Although, cations and anions are not isolable, but electro-and spectro-electrochemical experiments have shown that 3(+) and 3(-) ions are more stable than 1(+), 2(+) and 1(-), 2(-) ions. In all cases, the reductions occur with simultaneous two electron transfer, may be due to formation of coupled diimine/azo anion radical-VO(2+) species as in [(L(•2-))(VO(2+))(L(R)(AP)(2-))](2-).

Study of an efficient conversion of 1,3-dimethyl-5-(Arylazo)-6-Amino-Uracils to 1,3-dimethyl-8-(Aryl)-Azapurin-2,6-Diones

NASA Astrophysics Data System (ADS)

Debnath, Diptanu; Purkayastha, Atanu; Kirillov, Alexander; Ganguly, Rakesh; Misra, Tarun Kumar

2017-12-01

6-Aminouracils have extensively been used as precursors for synthesizing numerous uracil derivatives of biological and pharmaceutical significance. This study describes an application of 1,3-dimethyl-5-(arylazo)-6-aminouracils (Uazo: Uazo1-Uazo4, precursors) for an efficient synthesis of a series of 8-substituted-azapurins (AP), namely 1,3-dimethyl-8-(aryl)-azapurin-2,6-diones (aryl = p-HC6H4 (AP1), -MeC6H4 (AP2), sbnd ClC6H4 (AP3), and sbnd SO2NH2C6H4 (AP4)) following an oxidation method in the presence of copper (II) nitrate and in alkaline medium. The obtained compounds were isolated in good yields as crystalline air-stable products and have been fully characterized in the solution by UV-vis and NMR spectroscopy, as well as in the solid state by FT-IR spectroscopy, elemental analysis, and single-crystal X-ray diffraction (for AP2 and AP4). UV-vis study evidences that the conversion of the 6-aminouracil precursors occurs via an intermediate, Cu(II)-complex and a plausible mechanism for the formation of AP1-AP4 has been proposed. Unlike AP2 the crystal structure of AP4 reveals the formation of interdigitated 1D H-bonded chains that has been topologically classified within the 2C1 type. The 1H NMR spectra of the products have proton signals that completely devoid of hydrazone (sbnd NHsbnd) and imine (=NH) signals of their parent Uazo derivatives, thus confirming their full conversion and a stability of the AP1-AP4 in solution. The excitation and emission spectra of AP1-AP4 were also recorded in solution, revealing electronic transitions between similar vibrational energy levels of S0 (singlet ground state) and S1 (singlet first excited state).

STAM Adaptor Proteins Interact with COPII Complexes and Function in ER-to-Golgi Trafficking

PubMed Central

Rismanchi, Neggy; Puertollano, Rosa; Blackstone, Craig

2009-01-01

Signal transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. Here we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein (VSVG)-GFP trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with COPII proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex. PMID:19054391

Specificity of the Human Frequency Following Response for Carrier and Modulation Frequency Assessed Using Adaptation.

PubMed

Gockel, Hedwig E; Krugliak, Alexandra; Plack, Christopher J; Carlyon, Robert P

2015-12-01

The frequency following response (FFR) is a scalp-recorded measure of phase-locked brainstem activity to stimulus-related periodicities. Three experiments investigated the specificity of the FFR for carrier and modulation frequency using adaptation. FFR waveforms evoked by alternating-polarity stimuli were averaged for each polarity and added, to enhance envelope, or subtracted, to enhance temporal fine structure information. The first experiment investigated peristimulus adaptation of the FFR for pure and complex tones as a function of stimulus frequency and fundamental frequency (F0). It showed more adaptation of the FFR in response to sounds with higher frequencies or F0s than to sounds with lower frequency or F0s. The second experiment investigated tuning to modulation rate in the FFR. The FFR to a complex tone with a modulation rate of 213 Hz was not reduced more by an adaptor that had the same modulation rate than by an adaptor with a different modulation rate (90 or 504 Hz), thus providing no evidence that the FFR originates mainly from neurons that respond selectively to the modulation rate of the stimulus. The third experiment investigated tuning to audio frequency in the FFR using pure tones. An adaptor that had the same frequency as the target (213 or 504 Hz) did not generally reduce the FFR to the target more than an adaptor that differed in frequency (by 1.24 octaves). Thus, there was no evidence that the FFR originated mainly from neurons tuned to the frequency of the target. Instead, the results are consistent with the suggestion that the FFR for low-frequency pure tones at medium to high levels mainly originates from neurons tuned to higher frequencies. Implications for the use and interpretation of the FFR are discussed.

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

PubMed

Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

2016-08-31

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Myosin 7 and its adaptors link cadherins to actin

PubMed Central

Yu, I-Mei; Planelles-Herrero, Vicente J.; Sourigues, Yannick; Moussaoui, Dihia; Sirkia, Helena; Kikuti, Carlos; Stroebel, David; Titus, Margaret A.; Houdusse, Anne

2017-01-01

Cadherin linkages between adjacent stereocilia and microvilli are essential for mechanotransduction and maintaining their organization. They are anchored to actin through interaction of their cytoplasmic domains with related tripartite complexes consisting of a class VII myosin and adaptor proteins: Myo7a/SANS/Harmonin in stereocilia and Myo7b/ANKS4B/Harmonin in microvilli. Here, we determine high-resolution structures of Myo7a and Myo7b C-terminal MyTH4-FERM domain (MF2) and unveil how they recognize harmonin using a novel binding mode. Systematic definition of interactions between domains of the tripartite complex elucidates how the complex assembles and prevents possible self-association of harmonin-a. Several Myo7a deafness mutants that map to the surface of MF2 disrupt harmonin binding, revealing the molecular basis for how they impact the formation of the tripartite complex and disrupt mechanotransduction. Our results also suggest how switching between different harmonin isoforms can regulate the formation of networks with Myo7a motors and coordinate force sensing in stereocilia. PMID:28660889

Crystal structures of Salmonella typhimurium propionate kinase and its complex with Ap4A: evidence for a novel Ap4A synthetic activity.

PubMed

Simanshu, Dhirendra K; Savithri, H S; Murthy, M R N

2008-03-01

Propionate kinase catalyses the last step in the anaerobic breakdown of L-threonine to propionate in which propionyl phosphate and ADP are converted to propionate and ATP. Here we report the structures of propionate kinase (TdcD) in the native form as well as in complex with diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A) by X-ray crystallography. Structure of TdcD obtained after cocrystallization with ATP showed Ap4A bound to the active site pocket suggesting the presence of Ap4A synthetic activity in TdcD. Binding of Ap4A to the enzyme was confirmed by the structure determination of a TdcD-Ap4A complex obtained after cocrystallization of TdcD with commercially available Ap4A. Mass spectroscopic studies provided further evidence for the formation of Ap4A by propionate kinase in the presence of ATP. In the TdcD-Ap4A complex structure, Ap4A is present in an extended conformation with one adenosine moiety present in the nucleotide binding site and other in the proposed propionate binding site. These observations tend to support direct in-line transfer of phosphoryl group during the kinase reaction. 2007 Wiley-Liss, Inc.

Aminopyridinate-FI hybrids, their hafnium and titanium complexes, and their application in the living polymerization of 1-hexene.

PubMed

Haas, Isabelle; Dietel, Thomas; Press, Konstantin; Kol, Moshe; Kempe, Rhett

2013-10-11

Based on two well-established ligand systems, the aminopyridinato (Ap) and the phenoxyimine (FI) ligand systems, new Ap-FI hybrid ligands were developed. Four different Ap-FI hybrid ligands were synthesized through a simple condensation reaction and fully characterized. The reaction of hafnium tetrabenzyl with all four Ap-FI hybrid ligands exclusively led to mono(Ap-FI) complexes of the type [(Ap-FI)HfBn2 ]. The ligands acted as tetradentate dianionic chelates. Upon activation with tris(pentafluorophenyl)borane, the hafnium-dibenzyl complexes led to highly active catalysts for the polymerization of 1-hexene. Ultrahigh molecular weights and extremely narrow polydispersities support the living nature of this polymerization process. A possible deactivation product of the hafnium catalysts was characterized by single-crystal X-ray analysis and is discussed. The coordination modes of these new ligands were studied with the help of model titanium complexes. The reaction of titanium(IV) isopropoxide with ligand 1 led to a mono(Ap-FI) complex, which showed the desired fac-mer coordination mode. Titanium (IV) isopropoxide reacted with ligand 4 to give a complex of the type [(ApH-FI)2 Ti(OiPr)2 ], which featured the ligand in its monoanionic form. The two titanium complexes were characterized by X-ray crystal-structure analysis. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of two adaptor molecules SLP-76 and LAT in the PI3K signaling pathway in activated T cells.

PubMed

Shim, Eun Kyung; Jung, Seung Hee; Lee, Jong Ran

2011-03-01

Previously, we identified p85, a subunit of PI3K, as one of the molecules that interacts with the N-terminal region of Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76). We also demonstrated that tyrosine phosphorylation either at the 113 and/or 128 position is sufficient for the association of SLP-76 with the Src homology 2 domain near the N terminus of p85. The present study further examines the role of the association of these two molecules on the activation of PI3K signaling cascade. Experiments were done to determine the role of SLP-76, either wild-type, tyrosine mutants, or membrane-targeted forms of various SLP-76 constructs, on the membrane localization and phosphorylation of Akt, which is an event downstream of PI3K activation. Reconstitution studies with these various SLP-76 constructs in a Jurkat variant cell line that lacks SLP-76 or linker for activation of T cells (LAT) show that the activation of PI3K pathway following TCR ligation requires both SLP-76 and LAT adaptor proteins. The results suggest that SLP-76 associates with p85 after T cell activation and that LAT recruits this complex to the membrane, leading to Akt activation.

The Amyloid Precursor Protein is rapidly transported from the Golgi apparatus to the lysosome and where it is processed into beta-amyloid

PubMed Central

2014-01-01

Background Alzheimer’s disease (AD) is characterized by cerebral deposition of β-amyloid peptide (Aβ). Aβ is produced by sequential cleavage of the Amyloid Precursor Protein (APP) by β- and γ-secretases. Many studies have demonstrated that the internalization of APP from the cell surface can regulate Aβ production, although the exact organelle in which Aβ is produced remains contentious. A number of recent studies suggest that intracellular trafficking also plays a role in regulating Aβ production, but these pathways are relatively under-studied. The goal of this study was to elucidate the intracellular trafficking of APP, and to examine the site of intracellular APP processing. Results We have tagged APP on its C-terminal cytoplasmic tail with photoactivatable Green Fluorescent Protein (paGFP). By photoactivating APP-paGFP in the Golgi, using the Golgi marker Galactosyltranferase fused to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to follow a population of nascent APP molecules from the Golgi to downstream compartments identified with compartment markers tagged with red fluorescent protein (mRFP or mCherry); including rab5 (early endosomes) rab9 (late endosomes) and LAMP1 (lysosomes). Because γ-cleavage of APP releases the cytoplasmic tail of APP including the photoactivated GFP, resulting in loss of fluorescence, we are able to visualize the cleavage of APP in these compartments. Using APP-paGFP, we show that APP is rapidly trafficked from the Golgi apparatus to the lysosome; where it is rapidly cleared. Chloroquine and the highly selective γ-secretase inhibitor, L685, 458, cause the accumulation of APP in lysosomes implying that APP is being cleaved by secretases in the lysosome. The Swedish mutation dramatically increases the rate of lysosomal APP processing, which is also inhibited by chloroquine and L685, 458. By knocking down adaptor protein 3 (AP-3; a heterotetrameric protein complex required for trafficking many proteins to the lysosome) using siRNA, we are able to reduce this lysosomal transport. Blocking lysosomal transport of APP reduces Aβ production by more than a third. Conclusion These data suggests that AP-3 mediates rapid delivery of APP to lysosomes, and that the lysosome is a likely site of Aβ production. PMID:25085554

In vivo disruption of T cell development by expression of a dominant-negative polypeptide designed to abolish the SLP-76/Gads interaction.

PubMed

Jordan, Martha S; Maltzman, Jonathan S; Kliche, Stefanie; Shabason, Jacob; Smith, Jennifer E; Obstfeld, Amrom; Schraven, Burkhart; Koretzky, Gary A

2007-10-01

Multi-molecular complexes nucleated by adaptor proteins play a central role in signal transduction. In T cells, one central axis consists of the assembly of several signaling proteins linked together by the adaptors linker of activated T cells (LAT), Src homology 2 domain-containing leukocyte-specific phosphoprotein of 76 kDa (SLP-76), and Grb2-related adaptor downstream of Shc (Gads). Each of these adaptors has been shown to be important for normal T cell development, and their proper sub-cellular localization is critical for optimal function in cell lines. We previously demonstrated in Jurkat T cells and a rat basophilic leukemic cell line that expression of a 50-amino acid polypeptide identical to the site on SLP-76 that binds to Gads blocks proper localization of SLP-76 and SLP-76-dependent signaling events. Here we extend these studies to investigate the ability of this polypeptide to inhibit TCR-induced integrin activity in Jurkat cells and to inhibit in vivo thymocyte development and primary T cell function. These data provide evidence for the in vivo function of a dominant-negative peptide based upon the biology of SLP-76 action and suggest the possibility of therapeutic potential of targeting the SLP-76/Gads interaction.

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation

PubMed Central

Zhang, Yi; Ng, Huck-Hui; Erdjument-Bromage, Hediye; Tempst, Paul; Bird, Adrian; Reinberg, Danny

1999-01-01

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation. PMID:10444591

The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

PubMed Central

Proust, Richard; Bertoglio, Jacques; Gesbert, Franck

2012-01-01

Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex. PMID:22912825

ATM-Dependent Phosphorylation of All Three Members of the MRN Complex: From Sensor to Adaptor

PubMed Central

Lavin, Martin F.; Kozlov, Sergei; Gatei, Magtouf; Kijas, Amanda W.

2015-01-01

The recognition, signalling and repair of DNA double strand breaks (DSB) involves the participation of a multitude of proteins and post-translational events that ensure maintenance of genome integrity. Amongst the proteins involved are several which when mutated give rise to genetic disorders characterised by chromosomal abnormalities, cancer predisposition, neurodegeneration and other pathologies. ATM (mutated in ataxia-telangiectasia (A-T) and members of the Mre11/Rad50/Nbs1 (MRN complex) play key roles in this process. The MRN complex rapidly recognises and locates to DNA DSB where it acts to recruit and assist in ATM activation. ATM, in the company of several other DNA damage response proteins, in turn phosphorylates all three members of the MRN complex to initiate downstream signalling. While ATM has hundreds of substrates, members of the MRN complex play a pivotal role in mediating the downstream signalling events that give rise to cell cycle control, DNA repair and ultimately cell survival or apoptosis. Here we focus on the interplay between ATM and the MRN complex in initiating signaling of breaks and more specifically on the adaptor role of the MRN complex in mediating ATM signalling to downstream substrates to control different cellular processes. PMID:26512707

Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA

PubMed Central

Göpel, Yvonne; Papenfort, Kai; Reichenbach, Birte; Vogel, Jörg; Görke, Boris

2013-01-01

Bacterial small RNAs (sRNAs) are well established to regulate diverse cellular processes, but how they themselves are regulated is less understood. Recently, we identified a regulatory circuit wherein the GlmY and GlmZ sRNAs of Escherichia coli act hierarchically to activate mRNA glmS, which encodes glucosamine-6-phosphate (GlcN6P) synthase. Although the two sRNAs are highly similar, only GlmZ is a direct activator that base-pairs with the glmS mRNA, aided by protein Hfq. GlmY, however, does not bind Hfq and activates glmS indirectly by protecting GlmZ from RNA cleavage. This complex regulation feedback controls the levels of GlmS protein in response to its product, GlcN6P, a key metabolite in cell wall biosynthesis. Here, we reveal the molecular basis for the regulated turnover of GlmZ, identifying RapZ (RNase adaptor protein for sRNA GlmZ; formerly YhbJ) as a novel type of RNA-binding protein that recruits the major endoribonuclease RNase E to GlmZ. This involves direct interaction of RapZ with the catalytic domain of RNase E. GlmY binds RapZ through a secondary structure shared by both sRNAs and therefore acts by molecular mimicry as a specific decoy for RapZ. Thus, in analogy to regulated proteolysis, RapZ is an adaptor, and GlmY is an anti-adaptor in regulated turnover of a regulatory small RNA. PMID:23475961

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

PubMed

Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.

PubMed

Takai, Hideki; Araki, Shouta; Mezawa, Masaru; Kim, Dong-Soon; Li, Xinyue; Yang, Li; Li, Zhengyang; Wang, Zhitao; Nakayama, Youhei; Ogata, Yorimasa

2008-02-29

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

VizieR Online Data Catalog: Spatial structure of young stellar clusters. III. (Kuhn+, 2015)

NASA Astrophysics Data System (ADS)

Kuhn, M. A.; Feigelson, E. D.; Getman, K. V.; Sills, A.; Bate, M. R.; Borissova, J.

2017-10-01

This paper uses the Paper I (Kuhn et al. 2014, J/ApJ/787/107) catalog of 142 subclusters of young stars in 17 MYStIX star-forming regions. These subclusters were found and characterized using the stars in the MYStIX Probable Complex Member (MPCM) catalogs from Broos et al. (2013, J/ApJS/209/32). The multiwavelength data analysis efforts that went into this catalog are described by Feigelson et al. (2013, J/ApJS/209/26), King et al. (2013, J/ApJS/209/28), Kuhn et al. (2013, J/ApJS/209/27; 2013, J/ApJS/209/29), Naylor et al. (2013, J/ApJS/209/30), Povich et al. (2013, J/ApJS/209/31), and Townsley et al. (2014, J/ApJS/213/1), which provided uniform data coverage across the 17 star-forming regions investigated here, including the most comprehensive and reliable lists of young stars in many of the nearest MSFRs. (3 data files).

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3-independent mechanism.

PubMed

Gwynn, B; Ciciotte, S L; Hunter, S J; Washburn, L L; Smith, R S; Andersen, S G; Swank, R T; Dell'Angelica, E C; Bonifacino, J S; Eicher, E M; Peters, L L

2000-12-15

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)

Hydrogen-Rich Saline Inhibits NLRP3 Inflammasome Activation and Attenuates Experimental Acute Pancreatitis in Mice

PubMed Central

Ma, Jie; Jin, Wei-Hua; Wu, Juan; Fan, Kai-Hua

2014-01-01

Increasing evidence has demonstrated that reactive oxygen species (ROS) induces oxidative stress and plays a crucial role in the pathogenesis of acute pancreatitis (AP). Hydrogen-rich saline (HRS), a well-known ROS scavenger, has been shown to possess therapeutic benefit on AP in many animal experiments. Recent findings have indicated that the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome, an intracellular multiprotein complex required for the maturation of interleukin- (IL-) 1β, may probably be a potential target of HRS in the treatment of AP. Therefore, in this study, we evaluated the activation of NLRP3 inflammasome and meanwhile assessed the degree of oxidative stress and inflammatory cascades, as well as the histological alterations in mice suffering from cerulein-induced AP after the treatment of HRS. The results showed that the activation of NLRP3 inflammasome in AP mice was substantially inhibited following the administration of HRS, which was paralleled with the decreased NF-κB activity and cytokines production, attenuated oxidative stress and the amelioration of pancreatic tissue damage. In conclusion, our study has, for the first time, revealed that inhibition of the activation of NLRP3 inflammasome probably contributed to the therapeutic potential of HRS in AP. PMID:25214720

Method and apparatus for staking optical elements

DOEpatents

Woods, Robert O.

1988-01-01

A method and apparatus for staking two optical elements together in order to retain their alignment is disclosed. The apparatus includes a removable adaptor made up of first and second adaptor bodies each having a lateral slot in their front and side faces. The adaptor also includes a system for releasably attaching each adaptor body to a respective optical element such that when the two optical elements are positioned relative to one another the adaptor bodies are adjacent and the lateral slots therein are aligned to form key slots. The adaptor includes keys which are adapted to fit into the key slots. A curable filler material is employed to retain the keys in the key slots and thereby join the first and second adaptor bodies to form the adaptor. Also disclosed is a method for staking together two optical elements employing the adaptor of the present invention.

Method and apparatus for staking optical elements

DOEpatents

Woods, Robert O.

1988-10-04

A method and apparatus for staking two optical elements together in order to retain their alignment is disclosed. The apparatus includes a removable adaptor made up of first and second adaptor bodies each having a lateral slot in their front and side faces. The adaptor also includes a system for releasably attaching each adaptor body to a respective optical element such that when the two optical elements are positioned relative to one another the adaptor bodies are adjacent and the lateral slots therein are aligned to form key slots. The adaptor includes keys which are adapted to fit into the key slots. A curable filler material is employed to retain the keys in the key slots and thereby join the first and second adaptor bodies to form the adaptor. Also disclosed is a method for staking together two optical elements employing the adaptor of the present invention.

A Rab5 GTPase module is important for autophagosome closure

PubMed Central

Lipatova, Zhanna; Sun, Dan; Zhu, Xiaolong; Li, Rui; Wu, Zulin; You, Weiming; Cong, Xiaoxia; Zhou, Yiting; Gyurkovska, Valeriya; Liu, Yutao; Li, Qunli; Li, Wenjing; Cheng, Jie; Segev, Nava

2017-01-01

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure. PMID:28934205

An Aberrant Phosphorylation of Amyloid Precursor Protein Tyrosine Regulates Its Trafficking and the Binding to the Clathrin Endocytic Complex in Neural Stem Cells of Alzheimer's Disease Patients

PubMed Central

Poulsen, Ebbe T.; Iannuzzi, Filomena; Rasmussen, Helle F.; Maier, Thorsten J.; Enghild, Jan J.; Jørgensen, Arne L.; Matrone, Carmela

2017-01-01

Alzheimer's disease (AD) is the most common cause of dementia and is likely caused by defective amyloid precursor protein (APP) trafficking and processing in neurons leading to amyloid plaques containing the amyloid-β (Aβ) APP peptide byproducts. Understanding how APP is targeted to selected destinations inside neurons and identifying the mechanisms responsible for the generation of Aβ are thus the keys for the advancement of new therapies. We previously developed a mouse model with a mutation at tyrosine (Tyr) 682 in the C-terminus of APP. This residue is needed for APP to bind to the coating protein Clathrin and to the Clathrin adaptor protein AP2 as well as for the correct APP trafficking and sorting in neurons. By extending these findings to humans, we found that APP binding to Clathrin is decreased in neural stem cells from AD sufferers. Increased APP Tyr phosphorylation alters APP trafficking in AD neurons and it is associated to Fyn Tyr kinase activation. We show that compounds affecting Tyr kinase activity and counteracting defects in AD neurons can control APP location and compartmentalization. APP Tyr phosphorylation is thus a potential therapeutic target for AD. PMID:28360834

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins*

PubMed Central

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-01

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. PMID:27927989

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

PubMed

Inaba, Satomi; Numoto, Nobutaka; Ogawa, Shuhei; Morii, Hisayuki; Ikura, Teikichi; Abe, Ryo; Ito, Nobutoshi; Oda, Masayuki

2017-01-20

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Recent advances in inflammasome biology.

PubMed

Place, David E; Kanneganti, Thirumala-Devi

2018-02-01

The inflammasome is a complex of proteins that through the activity of caspase-1 and the downstream substrates gasdermin D, IL-1β, and IL-18 execute an inflammatory form of cell death termed pyroptosis. Activation of this complex often involves the adaptor protein ASC and upstream sensors including NLRP1, NLRP3, NLRC4, AIM2, and pyrin, which are activated by different stimuli including infectious agents and changes in cell homeostasis. Here we discuss new regulatory mechanisms that have been identified for the canonical inflammasomes, the most recently identified NLRP9b inflammasome, and the new gasdermin family of proteins that mediate pyroptosis and other forms of regulated cell death. Copyright © 2017 Elsevier Ltd. All rights reserved.

Blade attachment assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia-Crespo, Andres Jose; Delvaux, John McConnell; Miller, Diane Patricia

An assembly and method for affixing a turbomachine rotor blade to a rotor wheel are disclosed. In an embodiment, an adaptor member is provided disposed between the blade and the rotor wheel, the adaptor member including an adaptor attachment slot that is complementary to the blade attachment member, and an adaptor attachment member that is complementary to the rotor wheel attachment slot. A coverplate is provided, having a coverplate attachment member that is complementary to the rotor wheel attachment slot, and a hook for engaging the adaptor member. When assembled, the coverplate member matingly engages with the adaptor member, andmore » retains the blade in the adaptor member, and the assembly in the rotor wheel.« less

Naturally occurring phenolic acids modulate TPA-induced activation of EGFR, AP-1, and STATs in mouse epidermis.

PubMed

Cichocki, Michał; Dałek, Miłosz; Szamałek, Mateusz; Baer-Dubowska, Wanda

2014-01-01

Epidermal growth factor receptor (EGFR) plays an important role in epithelial carcinogenesis and appears to be involved in STATs activation. In this study we investigated the possible interference of naturally occurring phenolic acids with EGFR, activator protein-1 (AP-1), and signal transducers and activators of transcription (STATs) pathways activated by topical application of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Balb/c mice epidermis. Pretreatment with tannic or chlorogenic acid resulted in a significant decrease in the phosphorylation of EGFR Y-1068 and Y-1173 tyrosine residues, which was accompanied by reduced activation of AP-1. Tannic acid decreased also the c-Jun AP-1 subunit level and binding to TPA response element (TRE) (3- and 2-fold in comparison with TPA-treated group respectively). Simultaneous reduction of JNK activity might be responsible for reduced activation of AP-1. In contrast to these more complex phenolics, protocatechuic acid increased the activity of JNK and was also the most efficient inhibitor of STATs activation. These results indicate that naturally occurring phenolic acids, by decreasing EGFR, AP-1, and STATs activation, may modulate other elements both upstream and downstream in these pathways and thus inhibit the tumor development. Although more complex phenolics affect mainly the EGFR/AP-1 pathway, STATs seem to be the most important targets for simple compounds, such as protocatechuic acid.

Approximate entropy of human respiratory movement during eye-closed waking and different sleep stages.

PubMed

Burioka, Naoto; Cornélissen, Germaine; Halberg, Franz; Kaplan, Daniel T; Suyama, Hisashi; Sako, Takanori; Shimizu, Eiji

2003-01-01

The breath-to-breath variability of respiratory parameters changes with sleep stage. This study investigates any alteration in the approximate entropy (ApEn) of respiratory movement as a gauge of complexity in respiration, by stage of consciousness, in the light of putative brain interactions. Eight healthy men, who were between the ages of 23 and 29 years, were investigated. The signals of chest wall movement and EEG were recorded from 10:30 PM to 6:00 AM. After analog-to-digital conversion, the ApEn of respiratory movement (3 min) and EEG (20 s) were computed. Surrogate data were tested for nonlinearity in the original time series. The most impressive reduction in the ApEn of respiratory movement was associated with stage IV sleep, when the ApEn of the EEG was also statistically significantly decreased. A statistically significant linear relation is found between the ApEn of both variables. Surrogate data indicated that respiratory movement had nonlinear properties during all stages of consciousness that were investigated. Respiratory movement and EEG signals are more regular during stage IV sleep than during other stages of consciousness. The change in complexity described by the ApEn of respiration depends in part on the ApEn of the EEG, suggesting the involvement of nonlinear dynamic processes in the coordination between brain and lungs.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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.more » 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.« less

Adaptor assembly for coupling turbine blades to rotor disks

DOEpatents

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.

Visualization of DNA and Protein-DNA Complexes with Atomic Force Microscopy

PubMed Central

Lyubchenko, Yuri L.; Gall, Alexander A.; Shlyakhtenko, Luda S.

2014-01-01

This article describes sample preparation techniques for AFM imaging of DNA and protein–DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purifi cation of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein–DNA complexes. PMID:24357372

Synthesis and properties of ApA analogues with shortened phosphonate internucleotide linkage.

PubMed

Králíková, Sárka; Buděšínský, Miloš; Barvík, Ivan; Masojídková, Milena; Točík, Zdeněk; Rosenberg, Ivan

2011-01-01

A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 ″ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 ″ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.

Kinetic studies on the oxidation of oxyhemoglobin by biologically active iron thiosemicarbazone complexes: relevance to iron-chelator-induced methemoglobinemia.

PubMed

Basha, Maram T; Rodríguez, Carlos; Richardson, Des R; Martínez, Manuel; Bernhardt, Paul V

2014-03-01

The oxidation of oxyhemoglobin to methemoglobin has been found to be facilitated by low molecular weight iron(III) thiosemicarbazone complexes. This deleterious reaction, which produces hemoglobin protein units unable to bind dioxygen and occurs during the administration of iron chelators such as the well-known 3-aminopyridine-2-pyridinecarbaldehyde thiosemicarbazone (3-AP; Triapine), has been observed in the reaction with Fe(III) complexes of some members of the 3-AP structurally-related thiosemicarbazone ligands derived from di-2-pyridyl ketone (HDpxxT series). We have studied the kinetics of this oxidation reaction in vitro using human hemoglobin and found that the reaction proceeds with two distinct time-resolved steps. These have been associated with sequential oxidation of the two different oxyheme cofactors in the α and β protein chains. Unexpected steric and hydrogen-bonding effects on the Fe(III) complexes appear to be the responsible for the observed differences in the reaction rate across the series of HDpxxT ligand complexes used in this study.

Mutation of CD2AP and SH3KBP1 Binding Motif in Alphavirus nsP3 Hypervariable Domain Results in Attenuated Virus.

PubMed

Mutso, Margit; Morro, Ainhoa Moliner; Smedberg, Cecilia; Kasvandik, Sergo; Aquilimeba, Muriel; Teppor, Mona; Tarve, Liisi; Lulla, Aleksei; Lulla, Valeria; Saul, Sirle; Thaa, Bastian; McInerney, Gerald M; Merits, Andres; Varjak, Margus

2018-04-27

Infection by Chikungunya virus (CHIKV) of the Old World alphaviruses (family Togaviridae) in humans can cause arthritis and arthralgia. The virus encodes four non-structural proteins (nsP) (nsP1, nsp2, nsP3 and nsP4) that act as subunits of the virus replicase. These proteins also interact with numerous host proteins and some crucial interactions are mediated by the unstructured C-terminal hypervariable domain (HVD) of nsP3. In this study, a human cell line expressing EGFP tagged with CHIKV nsP3 HVD was established. Using quantitative proteomics, it was found that CHIKV nsP3 HVD can bind cytoskeletal proteins, including CD2AP, SH3KBP1, CAPZA1, CAPZA2 and CAPZB. The interaction with CD2AP was found to be most evident; its binding site was mapped to the second SH3 ligand-like element in nsP3 HVD. Further assessment indicated that CD2AP can bind to nsP3 HVDs of many different New and Old World alphaviruses. Mutation of the short binding element hampered the ability of the virus to establish infection. The mutation also abolished ability of CD2AP to co-localise with nsP3 and replication complexes of CHIKV; the same was observed for Semliki Forest virus (SFV) harbouring a similar mutation. Similar to CD2AP, its homolog SH3KBP1 also bound the identified motif in CHIKV and SFV nsP3.

Cognitive Style, Creativity Framing and Effects

ERIC Educational Resources Information Center

Dew, Robert

2009-01-01

This study investigates how individuals with different cognitive styles respond to choices involving framing effects. The results suggest that cognitive style as defined by Kirton (1976) is far more complex than previous studies indicate. Kirton characterises "Innovators" as rule breakers and "Adaptors" as conformists. The most…

T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex.

PubMed

Breuning, Johannes; Brown, Marion H

2017-06-01

The cell surface receptor CD6 regulates T cell activation in both activating and inhibitory manners. The adaptor protein SLP-76 is recruited to the phosphorylated CD6 cytoplasmic Y662 residue during T cell activation, providing an activating signal to T cells. In this study, a biochemical approach identified the SH2 domain-containing adaptor protein GADS as the dominant interaction partner for the CD6 cytoplasmic Y629 residue. Functional experiments in human Jurkat and primary T cells showed that both mutations Y629F and Y662F abolished costimulation by CD6. In addition, a restraint on T cell activation by CD6 was revealed in primary T cells expressing CD6 mutated at Y629 and Y662. These data are consistent with a model in which bivalent recruitment of a GADS/SLP-76 complex is required for costimulation by CD6. Copyright © 2017 Breuning and Brown.

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics

PubMed Central

Bennett, Eric J.; Rush, John; Gygi, Steven P.; Harper, J. Wade

2010-01-01

Dynamic reorganization of signaling systems frequently accompany pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex Absolute Quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules while only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. PMID:21145461

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.

PubMed

Bennett, Eric J; Rush, John; Gygi, Steven P; Harper, J Wade

2010-12-10

Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization. Copyright © 2010 Elsevier Inc. All rights reserved.

RNF166 Determines Recruitment of Adaptor Proteins during Antibacterial Autophagy.

PubMed

Heath, Robert J; Goel, Gautam; Baxt, Leigh A; Rush, Jason S; Mohanan, Vishnu; Paulus, Geraldine L C; Jani, Vijay; Lassen, Kara G; Xavier, Ramnik J

2016-11-22

Xenophagy is a form of selective autophagy that involves the targeting and elimination of intracellular pathogens through several recognition, recruitment, and ubiquitination events. E3 ubiquitin ligases control substrate selectivity in the ubiquitination cascade; however, systematic approaches to map the role of E3 ligases in antibacterial autophagy have been lacking. We screened more than 600 putative human E3 ligases, identifying E3 ligases that are required for adaptor protein recruitment and LC3-bacteria colocalization, critical steps in antibacterial autophagy. An unbiased informatics approach pinpointed RNF166 as a key gene that interacts with the autophagy network and controls the recruitment of ubiquitin as well as the autophagy adaptors p62 and NDP52 to bacteria. Mechanistic studies demonstrated that RNF166 catalyzes K29- and K33-linked polyubiquitination of p62 at residues K91 and K189. Thus, our study expands the catalog of E3 ligases that mediate antibacterial autophagy and identifies a critical role for RNF166 in this process. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The adaptor protein SLP-76 regulates HIV-1 release and cell to cell transmission in T-cells

PubMed Central

Nagaraja, Tirumuru; Anand, Appakkudal R.; Zhao, Helong; Ganju, Ramesh K.

2014-01-01

HIV-1 infection in T-cells is regulated by T-cell receptor (TCR) activation. However, the cellular proteins of the TCR pathway that regulate HIV-1 infection are poorly characterized. Here, we elucidated the role of SLP-76, a key adaptor protein of the TCR signaling complex, in HIV-1 infection. We observed a significant reduction of HIV-1 virus production in SLP-76-deficient Jurkat T-cells compared to wild-type and SLP-76-reconstituted Jurkat T-cells. We further confirmed the role of SLP-76 in HIV-1 infection by siRNA-mediated knockdown in MT4 cells and PBMCs. Structural-functional analysis revealed that the amino-terminal domain of SLP-76 was important for regulating HIV-1 infection. Further mechanistic studies revealed that lack of SLP-76 impaired virus release, but did not affect viral entry, integration and transcription. We also showed that SLP-76 plays a critical role in cell-to-cell transmission of HIV-1. Signaling studies revealed that SLP-76 associated with viral Nef protein and multiple signaling molecules during HIV-1 infection. Furthermore, SLP-76 facilitated the association of Nef and F-actin, suggesting that SLP-76 mediates the formation of a signaling complex that may regulate viral release via cytoskeletal changes. Taken together, our studies demonstrate a novel role for the adaptor molecule, SLP-76 in regulating HIV-1 infection in T-cells with potential to develop innovative strategies against HIV-1. PMID:22323535

Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism.

PubMed

Ogi, Kazuhiro; Nakashima, Kenji; Chihara, Kazuyasu; Takeuchi, Kenji; Horiguchi, Tomoko; Fujieda, Shigeharu; Sada, Kiyonao

2011-09-01

Tyrosine phosphorylation of adaptor protein c-Abl-Src homology 3 (SH3) domain-binding protein-2 (3BP2, also referred to SH3BP2) positively regulates the B-cell antigen receptor (BCR)-mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B-cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2-P416R, corresponding to P418R in human protein) enhanced BCR-mediated activation of NFAT. 3BP2-P416R increased the signaling complex formation with Syk, phospholipase C-γ2 (PLC-γ2), and Vav1. In contrast, 3BP2-P416R could not change the association with the negative regulator 14-3-3. Loss of the association mutant that was incapable to associate with 14-3-3 could not mimic BCR-mediated NFAT activation in Syk-deficient cells. Moreover, BCR-mediated phosphorylation of extracellular signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Ubiquitin conjugating enzyme E2-N and sequestosome-1 (p62) are components of the ubiquitination process mediated by the malin-laforin E3-ubiquitin ligase complex.

PubMed

Sánchez-Martín, Pablo; Romá-Mateo, Carlos; Viana, Rosa; Sanz, Pascual

2015-12-01

Lafora disease (LD, OMIM254780, ORPHA501) is a rare neurodegenerative form of epilepsy related to mutations in two proteins: laforin, a dual specificity phosphatase, and malin, an E3-ubiquitin ligase. Both proteins form a functional complex, where laforin recruits specific substrates to be ubiquitinated by malin. However, little is known about the mechanism driving malin-laforin mediated ubiquitination of its substrates. In this work we present evidence indicating that the malin-laforin complex interacts physically and functionally with the ubiquitin conjugating enzyme E2-N (UBE2N). This binding determines the topology of the chains that the complex is able to promote in the corresponding substrates (mainly K63-linked polyubiquitin chains). In addition, we demonstrate that the malin-laforin complex interacts with the selective autophagy adaptor sequestosome-1 (p62). Binding of p62 to the malin-laforin complex allows its recognition by LC3, a component of the autophagosomal membrane. In addition, p62 enhances the ubiquitinating activity of the malin-laforin E3-ubiquitin ligase complex. These data enrich our knowledge on the mechanism of action of the malin-laforin complex as an E3-ubiquitin ligase and reinforces the role of this complex in targeting substrates toward the autophagy pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair.

PubMed

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-10-01

USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1.

The USP1-UAF1 complex interacts with RAD51AP1 to promote homologous recombination repair

PubMed Central

Cukras, Scott; Lee, Euiho; Palumbo, Emily; Benavidez, Pamela; Moldovan, George-Lucian; Kee, Younghoon

2016-01-01

ABSTRACT USP1 deubiquitinating enzyme and its stoichiometric binding partner UAF1 play an essential role in promoting DNA homologous recombination (HR) repair in response to various types of DNA damaging agents. Deubiquitination of FANCD2 may be attributed to the key role of USP1-UAF1 complex in regulating HR repair, however whether USP1-UAF1 promotes HR repair independently of FANCD2 deubiquitination is not known. Here we show evidence that the USP1-UAF1 complex has a FANCD2-independent function in promoting HR repair. Proteomic search of UAF1-interacting proteins revealed that UAF1 associates with RAD51AP1, a RAD51-interacting protein implicated in HR repair. We show that UAF1 mediates the interaction between USP1 and RAD51AP1, and that depletion of USP1 or UAF1 led to a decreased stability of RAD51AP1. Protein interaction mapping analysis identified some key residues within RAD51AP1 required for interacting with the USP1-UAF1 complex. Cells expressing the UAF1 interaction-deficient mutant of RAD51AP1 show increased chromosomal aberrations in response to Mitomycin C treatment. Moreover, similar to the RAD51AP1 depleted cells, the cells expressing UAF1-interaction deficient RAD51AP1 display persistent RAD51 foci following DNA damage exposure, indicating that these factors regulate a later step during the HR repair. These data altogether suggest that the USP1-UAF1 complex promotes HR repair via multiple mechanisms: through FANCD2 deubiquitination, as well as by interacting with RAD51AP1. PMID:27463890

Spiral biasing adaptor for use in Si drift detectors and Si drift detector arrays

DOEpatents

Li, Zheng; Chen, Wei

2016-07-05

A drift detector array, preferably a silicon drift detector (SDD) array, that uses a low current biasing adaptor is disclosed. The biasing adaptor is customizable for any desired geometry of the drift detector single cell with minimum drift time of carriers. The biasing adaptor has spiral shaped ion-implants that generate the desired voltage profile. The biasing adaptor can be processed on the same wafer as the drift detector array and only one biasing adaptor chip/side is needed for one drift detector array to generate the voltage profiles on the front side and back side of the detector array.

Insight into the architecture of the NuRD complex: structure of the RbAp48-MTA1 subcomplex.

PubMed

Alqarni, Saad S M; Murthy, Andal; Zhang, Wei; Przewloka, Marcin R; Silva, Ana P G; Watson, Aleksandra A; Lejon, Sara; Pei, Xue Y; Smits, Arne H; Kloet, Susan L; Wang, Hongxin; Shepherd, Nicholas E; Stokes, Philippa H; Blobel, Gerd A; Vermeulen, Michiel; Glover, David M; Mackay, Joel P; Laue, Ernest D

2014-08-08

The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

KCTD2, an adaptor of Cullin3 E3 ubiquitin ligase, suppresses gliomagenesis by destabilizing c-Myc

PubMed Central

Kim, Eun-Jung; Kim, Sung-Hak; Jin, Xiong; Jin, Xun; Kim, Hyunggee

2017-01-01

Cullin3 E3 ubiquitin ligase ubiquitinates a wide range of substrates through substrate-specific adaptors Bric-a-brac, Tramtrack, and Broad complex (BTB) domain proteins. These E3 ubiquitin ligase complexes are involved in diverse cellular functions. Our recent study demonstrated that decreased Cullin3 expression induces glioma initiation and correlates with poor prognosis of patients with malignant glioma. However, the substrate recognition mechanism associated with tumorigenesis is not completely understood. Through yeast two-hybrid screening, we identified potassium channel tetramerization domain-containing 2 (KCTD2) as a BTB domain protein that binds to Cullin3. The interaction of Cullin3 and KCTD2 was verified using immunoprecipitation and immunofluorescence. Of interest, KCTD2 expression was markedly decreased in patient-derived glioma stem cells (GSCs) compared with non-stem glioma cells. Depletion of KCTD2 using a KCTD2-specific short-hairpin RNA in U87MG glioma cells and primary Ink4a/Arf-deficient murine astrocytes markedly increased self-renewal activity in addition with an increased expression of stem cell markers, and mouse in vivo intracranial tumor growth. As an underlying mechanism for these KCTD2-mediated phenotypic changes, we demonstrated that KCTD2 interacts with c-Myc, which is a key stem cell factor, and causes c-Myc protein degradation by ubiquitination. As a result, KCTD2 depletion acquires GSC features and affects aerobic glycolysis via expression changes in glycolysis-associated genes through c-Myc protein regulation. Of clinical significance was our finding that patients having a profile of KCTD2 mRNA-low and c-Myc gene signature-high, but not KCTD2 mRNA-low and c-Myc mRNA-high, are strongly associated with poor prognosis. This study describes a novel regulatory mode of c-Myc protein in malignant gliomas and provides a potential framework for glioma therapy by targeting c-Myc function. PMID:28060381

The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3' kinase pathway.

PubMed

Sattler, M; Salgia, R; Okuda, K; Uemura, N; Durstin, M A; Pisick, E; Xu, G; Li, J L; Prasad, K V; Griffin, J D

1996-02-15

Chronic myelogenous leukemia (CML) and some acute lymphoblastic leukemias (ALL) are caused by the t(9;22) chromosome translocation, which produces the constitutively activated BCR/ABL tyrosine kinase. When introduced into factor dependent hematopoietic cell lines, BCR/ABL induces the tyrosine phosphorylation of many cellular proteins. One prominent BCR/ABL substrate is p120CBL, the cellular homolog of the v-Cbl oncoprotein. In an effort to understand the possible contribution of p120CBL to transformation by BCR/ABL, we looked for cellular proteins which associate with p120CBL in hematopoietic cell lines transformed by BCR/ABL. In addition to p210BCR/ABL and c-ABL, p120CBL coprecipitated with an 85 kDa phosphoprotein, which was identified as the p85 subunit of PI3K. Anti-p120CBL immunoprecipitates from BCR/ABL-transformed, but not from untransformed, cell lines contained PI3K lipid kinase activity. Interestingly, the adaptor proteins CRKL and c-CRK were also found in these complexes. In vitro binding studies indicated that the SH2 domains of CRKL and c-CRK bound directly to p120CBL, while the SH3 domains of c-CRK and CRKL bound to BCR/ABL and c-ABL. The N-terminal and the C-terminal SH2 and the SH3 domain of p85PI3K bound directly in vitro to p120CBL. The ABL-SH2, but not ABL-SH3, could also bind to p120CBL. These data suggest that BCR/ABL may induce the formation of multimeric complexes of signaling proteins which include p120CBL, PI3K, c-CRK or CRKL, c-ABL and BCR/ABL itself.

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,…

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 is...

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 is...

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 is...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages.

PubMed

den Hartigh, Andreas B; Fink, Susan L

2018-05-21

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1β and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis. In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

PubMed

Shin, John J; Aftab, Qurratulain; Austin, Pamela; McQueen, Jennifer A; Poon, Tak; Li, Shu Chen; Young, Barry P; Roskelley, Calvin D; Loewen, Christopher J R

2016-09-01

A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain complexes required for their proliferation. © 2016. Published by The Company of Biologists Ltd.

21 CFR 870.4290 - Cardiopulmonary bypass adaptor, stopcock, manifold, or fitting.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 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 diagnostic...

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 diagnostic...

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 it...

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 it...

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 it...

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 it...

Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction.

PubMed

Bule, Pedro; Pires, Virgínia M R; Alves, Victor D; Carvalho, Ana Luísa; Prates, José A M; Ferreira, Luís M A; Smith, Steven P; Gilbert, Harry J; Noach, Ilit; Bayer, Edward A; Najmudin, Shabir; Fontes, Carlos M G A

2018-05-03

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

Current opinion in Microbiology Roles of adaptor proteins in regulation of bacterial proteolysis

PubMed Central

Battesti, Aurelia; Gottesman, Susan

2013-01-01

Elimination of non-functional or unwanted proteins is critical for cell growth and regulation. In bacteria, ATP-dependent proteases target cytoplasmic proteins for degradation, contributing to both protein quality control and regulation of specific proteins, thus playing roles parallel to that of the proteasome in eukaryotic cells. Adaptor proteins provide a way to modulate the substrate specificity of the proteases and allow regulated proteolysis. Advances over the past few years have provided new insight into how adaptor proteins interact with both substrates and proteases and how adaptor functions are regulated. An important advance has come with the recognition of the critical roles of anti-adaptor proteins in regulating adaptor availability. PMID:23375660

Distribution of AMPA receptor subunits GluR1-4 in the dorsal vagal complex of the rat: a light and electron microscope immunocytochemical study.

PubMed

Kessler, J P; Baude, A

1999-10-01

The dorsal vagal complex, localized in the dorsomedial medulla, includes the nucleus tractus solitarii (NTS), the dorsal motor nucleus of the vagus nerve (DMN) and the area postrema (AP). The distribution of AMPA-preferring glutamate receptors (AMPA receptors) within this region was investigated using immunohistochemistry and antibodies recognizing either one (GluR1 or GluR4) or two (GluR2 and GluR3) AMPA receptors subunits. The distribution of GluR1 immunoreactivity showed high contrast of staining between strongly and lightly labeled areas. Labeling was intense in the AP and weak in the NTS, except for its medial and dorsalmost parts which exhibited moderate staining. Almost no GluR1 immunoreactivity was found in the DMN. GluR2/3 immunolabeling was present in the entire dorsal vagal complex. This labeling was strong in the AP, the DMN and the medial half of the NTS and moderate in the lateral half of the NTS, except for the interstitial subdivision which exhibited intense staining. Labeling induced by the GluR4 antibody was very weak throughout the dorsal vagal complex. Ultrastructural examination showed that GluR1 and GluR2/3 immunoreactivity was localized in neuronal cell bodies and dendrites. No labeled axon terminal or glial cell body was found. Immunoperoxidase staining in labeled cell bodies and dendrites was associated with intracellular organelles (microtubules, mitochondria, cisternae of the endoplasmic reticulum,.) and/or parts of the plasma membrane. Plasma membrane labeling was often associated with asymmetrical synaptic differentiations. No labeled symmetrical synapse was found using either GluR1 or GluR2/3 antibody. The present results show that AMPA receptors have a widespread distribution in neuronal perikarya and dendrites of the rat dorsal vagal complex. They suggest differences in subunit composition between AMPA receptors localized in the NTS, the DMN and the AP. Ultrastructural data are consistent with the fact that AMPA receptors associated with the plasma membrane are mostly synaptic receptors. However, they also suggest the existence of a large intracellular pool of receptor subunits in neuronal soma and dendrites. Copyright 1999 Wiley-Liss, Inc.

A Relationship Between Visual Complexity and Aesthetic Appraisal of Car Front Images: An Eye-Tracker Study.

PubMed

Chassy, Philippe; Lindell, Trym A E; Jones, Jessica A; Paramei, Galina V

2015-01-01

Image aesthetic pleasure (AP) is conjectured to be related to image visual complexity (VC). The aim of the present study was to investigate whether (a) two image attributes, AP and VC, are reflected in eye-movement parameters; and (b) subjective measures of AP and VC are related. Participants (N=26) explored car front images (M=50) while their eye movements were recorded. Following image exposure (10 seconds), its VC and AP were rated. Fixation count was found to positively correlate with the subjective VC and its objective proxy, JPEG compression size, suggesting that this eye-movement parameter can be considered an objective behavioral measure of VC. AP, in comparison, positively correlated with average dwelling time. Subjective measures of AP and VC were related too, following an inverted U-shape function best-fit by a quadratic equation. In addition, AP was found to be modulated by car prestige. Our findings reveal a close relationship between subjective and objective measures of complexity and aesthetic appraisal, which is interpreted within a prototype-based theory framework. © The Author(s) 2015.

Role of Adaptor Protein Toll-Like Interleukin Domain Containing Adaptor Inducing Interferon β in Toll-Like Receptor 3- and 4-Mediated Regulation of Hepatic Drug Metabolizing Enzyme and Transporter Genes.

PubMed

Shah, Pranav; Omoluabi, Ozozoma; Moorthy, Bhagavatula; Ghose, Romi

2016-01-01

The expressions and activities of hepatic drug-metabolizing enzymes and transporters (DMETs) are altered during infection and inflammation. Inflammatory responses in the liver are mediated primarily by Toll-like receptor (TLR)-signaling, which involves recruitment of Toll/interleukin (IL)-1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-β (TRIF) that eventually leads to induction of proinflammatory cytokines and mitogen-activated protein kinases (MAPKs). Lipopolysaccharide (LPS) activates the Gram-negative bacterial receptor TLR4 and polyinosinic:polycytidylic acid (polyI:C) activates the viral receptor TLR3. TLR4 signaling involves TIRAP and TRIF, whereas TRIF is the only adaptor protein involved in the TLR3 pathway. We have shown previously that LPS-mediated downregulation of DMETs is independent of TIRAP. To determine the role of TRIF, we treated TRIF(+/+) and TRIF(-/-) mice with LPS or polyI:C. LPS downregulated (∼40%-60%) Cyp3a11, Cyp2a4, Ugt1a1, Mrp2 mRNA levels, whereas polyI:C downregulated (∼30%-60%) Cyp3a11, Cyp2a4, Cyp1a2, Cyp2b10, Ugt1a1, Mrp2, and Mrp3 mRNA levels in TRIF(+/+) mice. This downregulation was not attenuated in TRIF(-/-) mice. Induction of cytokines by LPS was observed in both TRIF(+/+) and TRIF(-/-) mice. Cytokine induction was delayed in polyI:C-treated TRIF(-/-) mice, indicating that multiple mechanisms mediating polyI:C signaling exist. To assess the role of MAPKs, primary hepatocytes were pretreated with specific inhibitors before treatment with LPS/polyI:C. We found that only the c-jun-N-terminal kinase (JNK) inhibitor attenuated the down-regulation of DMETs. These results show that TRIF-independent pathways can be involved in the downregulation of DMETs through TLR4 and 3. JNK-dependent mechanisms likely mediate this downregulation. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

PubMed Central

Babinsky, Valerie N.; Head, Rosie A.; Cranston, Treena; Rust, Nigel; Hobbs, Maurine R.; Heath, Hunter; Thakker, Rajesh V.

2013-01-01

BACKGROUND Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein structures, and assessment on the basis of in vitro expression showed that familial hypocalciuric hypercalcemia type 2–associated mutations decreased the sensitivity of cells expressing calcium-sensing receptors to changes in extracellular calcium concentrations, whereas autosomal dominant hypocalcemia type 2–associated mutations increased cell sensitivity. CONCLUSIONS Gα11 mutants with loss of function cause familial hypocalciuric hypercalcemia type 2, and Gα11 mutants with gain of function cause a clinical disorder designated as autosomal dominant hypocalcemia type 2. (Funded by the United Kingdom Medical Research Council and others.) PMID:23802516

Transcriptome and Proteome Analyses of TNFAIP8 Knockdown Cancer Cells Reveal New Insights into Molecular Determinants of Cell Survival and Tumor Progression.

PubMed

Day, Timothy F; Mewani, Rajshree R; Starr, Joshua; Li, Xin; Chakravarty, Debyani; Ressom, Habtom; Zou, Xiaojun; Eidelman, Ofer; Pollard, Harvey B; Srivastava, Meera; Kasid, Usha N

2017-01-01

Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is the first discovered oncogenic and an anti-apoptotic member of a conserved TNFAIP8 or TIPE family of proteins. TNFAIP8 mRNA is induced by NF-kB, and overexpression of TNFAIP8 has been correlated with poor prognosis in many cancers. Downregulation of TNFAIP8 expression has been associated with decreased pulmonary colonization of human tumor cells, and enhanced sensitivities of tumor xenografts to radiation and docetaxel. Here we have investigated the effects of depletion of TNFAIP8 on the mRNA, microRNA and protein expression profiles in prostate and breast cancers and melanoma. Depending on the tumor cell type, knockdown of TNFAIP8 was found to be associated with increased mRNA expression of several antiproliferative and apoptotic genes (e.g., IL-24, FAT3, LPHN2, EPHA3) and fatty acid oxidation gene ACADL, and decreased mRNA levels of oncogenes (e.g., NFAT5, MALAT1, MET, FOXA1, KRAS, S100P, OSTF1) and glutamate transporter gene SLC1A1. TNFAIP8 knockdown cells also exhibited decreased expression of multiple onco-proteins (e.g., PIK3CA, SRC, EGFR, IL5, ABL1, GAP43), and increased expression of the orphan nuclear receptor NR4A1 and alpha 1 adaptin subunit of the adaptor-related protein complex 2 AP2 critical to clathrin-mediated endocytosis. TNFAIP8-centric molecules were found to be predominately implicated in the hypoxia-inducible factor-1α (HIF-1α) signaling pathway, and cancer and development signaling networks. Thus TNFAIP8 seems to regulate the cell survival and cancer progression processes in a multifaceted manner. Future validation of the molecules identified in this study is likely to lead to new subset of molecules and functional determinants of cancer cell survival and progression.

Multiple hydrogen bonding in excited states of aminopyrazine in methanol solution: time-dependent density functional theory study.

PubMed

Chai, Shuo; Yu, Jie; Han, Yong-Chang; Cong, Shu-Lin

2013-11-01

Aminopyrazine (AP) and AP-methanol complexes have been theoretically studied by using density functional theory (DFT) and time-dependent density functional theory (TDDFT). The excited-state hydrogen bonds are discussed in detail. In the ground state the intermolecular multiple hydrogen bonds can be formed between AP molecule and protic solvents. The AP monomer and hydrogen-bonded complex of AP with one methanol are photoexcited initially to the S2 state, and then transferred to the S1 state via internal conversion. However the complex of AP with two methanol molecules is directly excited to the S1 state. From the calculated electronic excited energies and simulated absorption spectra, we find that the intermolecular hydrogen bonds are strengthened in the electronic excited states. The strengthening is confirmed by the optimized excited-state geometries. The photochemical processes in the electronic excited states are significantly influenced by the excited-state hydrogen bond strengthening. Copyright © 2013 Elsevier B.V. All rights reserved.

Effect of the magnetism of impurities on their diffusion in metals: Bulk diffusion of iron, cobalt, and rhodium in iridium single crystals

NASA Astrophysics Data System (ADS)

Klotsman, S. M.; Tatarinova, G. N.

2008-12-01

The coefficients and parameters of the temperature dependences of the coefficients of bulk diffusion of Fe, Co, Rh, and Au atomic probes (APs) in iridium single crystals (mono-Ir) have been determined from the diffusion profiles obtained using secondary-ion mass spectrometry of the diffusion zones. The enthalpies of activation of diffusion of Fe, Co, and Rh APs are considerably lower than the enthalpy of activation of selfdiffusion in mono-Ir. This is caused by the negative contributions of the intraatomic exchange energy and energy of relaxation of the environment of the d transition APs to the enthalpy of interaction of magnetically active APs with the vacancies in the iridium lattice. The interaction energy of partners in such complexes and the relationships between the magnetic moments of d transition APs in complexes with vacancies have been estimated. The Rh APs in complexes with vacancies in iridium possess stable magnetic moments.

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.

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.

Contrasting styles of Mount Vesuvius activity in the period between the Avellino and Pompeii Plinian eruptions, and some implications for assessment of future hazards

NASA Astrophysics Data System (ADS)

Andronico, Daniele; Cioni, Raffaello

2002-09-01

Intense explosive activity occurred repeatedly at Vesuvius during the nearly 1,600-year period between the two Plinian eruptions of Avellino (3.5 ka) and Pompeii (79 A.D.). By correlating stratigraphic sections from more than 40 sites around the volcano, we identify the deposits of six main eruptions (AP1-AP6) and of some minor intervening events. Several deposits can be traced up to 20 km from the vent. Their stratigraphic and dispersal features suggest the prevalence of two main contrasting eruptive styles, each involving a complex relationship between magmatic and phreatomagmatic phases. The two main eruption styles are (1) sub-Plinian to phreato-Plinian events (AP1 and AP2 members), where deposits consist of pumice and scoria fall layers alternating with fine-grained, vesiculated, accretionary lapilli-bearing ashes; and (2) mixed, violent Strombolian to Vulcanian events (AP3-AP6 members), which deposited a complex sequence of fallout, massive to thinly stratified, scoria-bearing lapilli layers and fine ash beds. Morphology and density variations of the juvenile fragments confirm the important role played by magma-water interaction in the eruptive dynamics. The mean composition of the ejected material changes with time, and shows a strong correlation with vent position and eruption style. The ranges of intensity and magnitude of these events, derived by estimations of peak column height and volume of the ejecta, are significantly smaller than the values for the better known Plinian and sub-Plinian eruptions of Vesuvius, enlarging the spectrum of the possible eruptive scenarios at Vesuvius, useful in the assessment of its potential hazard.

Calcium Dynamics in Basal Dendrites of Layer 5A and 5B Pyramidal Neurons Is Tuned to the Cell-Type Specific Physiological Action Potential Discharge

PubMed Central

Krieger, Patrik; de Kock, Christiaan P. J.; Frick, Andreas

2017-01-01

Layer 5 (L5) is a major neocortical output layer containing L5A slender-tufted (L5A-st) and L5B thick-tufted (L5B-tt) pyramidal neurons. These neuron types differ in their in vivo firing patterns, connectivity and dendritic morphology amongst other features, reflecting their specific functional role within the neocortical circuits. Here, we asked whether the active properties of the basal dendrites that receive the great majority of synaptic inputs within L5 differ between these two pyramidal neuron classes. To quantify their active properties, we measured the efficacy with which action potential (AP) firing patterns backpropagate along the basal dendrites by measuring the accompanying calcium transients using two-photon laser scanning microscopy in rat somatosensory cortex slices. For these measurements we used both “artificial” three-AP patterns and more complex physiological AP patterns that were previously recorded in anesthetized rats in L5A-st and L5B-tt neurons in response to whisker stimulation. We show that AP patterns with relatively few APs (3APs) evoke a calcium response in L5B-tt, but not L5A-st, that is dependent on the temporal pattern of the three APs. With more complex in vivo recorded AP patterns, the average calcium response was similar in the proximal dendrites but with a decay along dendrites (measured up to 100 μm) of L5B-tt but not L5A-st neurons. Interestingly however, the whisker evoked AP patterns—although very different for the two cell types—evoke similar calcium responses. In conclusion, although the effectiveness with which different AP patterns evoke calcium transients vary between L5A-st and L5B-tt cell, the calcium influx appears to be tuned such that whisker-evoked calcium transients are within the same dynamic range for both cell types. PMID:28744201

Structure of an Arrestin2-clathrin Complex Reveals a Novel Clathrin Binding Domain that Modulates Receptor Trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, D.; Kern, R; Puthenveedu, M

2009-01-01

Non-visual arrestins play a pivotal role as adaptor proteins in regulating the signaling and trafficking of multiple classes of receptors. Although arrestin interaction with clathrin, AP-2, and phosphoinositides contributes to receptor trafficking, little is known about the configuration and dynamics of these interactions. Here, we identify a novel interface between arrestin2 and clathrin through x-ray diffraction analysis. The intrinsically disordered clathrin binding box of arrestin2 interacts with a groove between blades 1 and 2 in the clathrin {beta}-propeller domain, whereas an 8-amino acid splice loop found solely in the long isoform of arrestin2 (arrestin2L) interacts with a binding pocket formedmore » by blades 4 and 5 in clathrin. The apposition of the two binding sites in arrestin2L suggests that they are exclusive and may function in higher order macromolecular structures. Biochemical analysis demonstrates direct binding of clathrin to the splice loop in arrestin2L, whereas functional analysis reveals that both binding domains contribute to the receptor-dependent redistribution of arrestin2L to clathrin-coated pits. Mutagenesis studies reveal that the clathrin binding motif in the splice loop is (L/I){sub 2}GXL. Taken together, these data provide a framework for understanding the dynamic interactions between arrestin2 and clathrin and reveal an essential role for this interaction in arrestin-mediated endocytosis.« less

Macromolecular assembly of the adaptor SLP-65 at intracellular vesicles in resting B cells.

PubMed

Engelke, Michael; Pirkuliyeva, Sona; Kühn, Julius; Wong, Leo; Boyken, Janina; Herrmann, Nadine; Becker, Stefan; Griesinger, Christian; Wienands, Jürgen

2014-08-19

The traditional view of how intracellular effector proteins are recruited to the B cell antigen receptor (BCR) complex at the plasma membrane is based on the occurrence of direct protein-protein interactions, as exemplified by the recruitment of the tyrosine kinase Syk (spleen tyrosine kinase) to phosphorylated motifs in BCR signaling subunits. By contrast, the subcellular targeting of the cytosolic adaptor protein SLP-65 (Src homology 2 domain-containing leukocyte adaptor protein of 65 kD), which serves as a proximal Syk substrate, is unclear. We showed that SLP-65 activation required its association at vesicular compartments in resting B cells. A module of ~50 amino acid residues located at the amino terminus of SLP-65 anchored SLP-65 to the vesicles. Nuclear magnetic resonance spectroscopy showed that the SLP-65 amino terminus was structurally disordered in solution but could bind in a structured manner to noncharged lipid components of cellular membranes. Our finding that preformed vesicular signaling scaffolds are required for B cell activation indicates that vesicles may deliver preassembled signaling cargo to sites of BCR activation. Copyright © 2014, American Association for the Advancement of Science.

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

PubMed Central

Scott, R. H.; Sweeney, M. I.; Kobrinsky, E. M.; Pearson, H. A.; Timms, G. H.; Pullar, I. A.; Wedley, S.; Dolphin, A. C.

1992-01-01

1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1380382

Structural insights into the p97-Ufd1-Npl4 complex

PubMed Central

Pye, Valerie E.; Beuron, Fabienne; Keetch, Catherine A.; McKeown, Ciaran; Robinson, Carol V.; Meyer, Hemmo H.; Zhang, Xiaodong; Freemont, Paul S.

2007-01-01

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is ≈80 × 30 Å in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways. PMID:17202270

The signaling adapter Gab1 regulates cell polarity by acting as a PAR protein scaffold

PubMed Central

Yang, Ziqiang; Xue, Bin; Umitsu, Masataka; Ikura, Mitsuhiko; Muthuswamy, Senthil K.; Neel, Benjamin G.

2012-01-01

Summary Cell polarity plays a key role in development and is disrupted in tumors, yet the molecules and mechanisms that regulate polarity remain poorly defined. We found that the scaffolding adaptor GAB1 interacts with two polarity proteins, PAR1 and PAR3. GAB1 binds PAR1 and enhances its kinase activity. GAB1 brings PAR1 and PAR3 into a transient complex, stimulating PAR3 phosphorylation by PAR1. GAB1 and PAR6 bind the PAR3 PDZ1 domain and thereby compete for PAR3 binding. Consequently, GAB1 depletion causes PAR3 hypo-phosphorylation and increases PAR3/PAR6 complex formation, resulting in accelerated and enhanced tight junction formation, increased trans-epithelial resistance and lateral domain shortening. Conversely, GAB1 over-expression, in a PAR1/PAR3-dependent manner, disrupts epithelial apical-basal polarity, promotes multi-lumen cyst formation, and enhances growth factor-induced epithelial cell scattering. Our results identify GAB1 as a novel negative regulator of epithelial cell polarity that functions as a scaffold for modulating PAR protein complexes on the lateral membrane. PMID:22883624

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

PubMed Central

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

2017-01-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

Structure-based design of an osteoclast-selective, nonpeptide Src homology 2 inhibitor with in vivo antiresorptive activity

PubMed Central

Shakespeare, William; Yang, Michael; Bohacek, Regine; Cerasoli, Franklin; Stebbins, Karin; Sundaramoorthi, Raji; Azimioara, Mihai; Vu, Chi; Pradeepan, Selvi; Metcalf, Chester; Haraldson, Chad; Merry, Taylor; Dalgarno, David; Narula, Surinder; Hatada, Marcos; Lu, Xiaode; van Schravendijk, Marie Rose; Adams, Susan; Violette, Shelia; Smith, Jeremy; Guan, Wei; Bartlett, Catherine; Herson, Jay; Iuliucci, John; Weigele, Manfred; Sawyer, Tomi

2000-01-01

Targeted disruption of the pp60src (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3′,4′-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC50 = 0.30 μM for AP22408 and 5.5 μM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model. PMID:10944210

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO.

PubMed

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E; Stenmark, Harald; Platta, Harald W

2014-12-23

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes-autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept.

Regulation of the Tumor-Suppressor Function of the Class III Phosphatidylinositol 3-Kinase Complex by Ubiquitin and SUMO

PubMed Central

Reidick, Christina; El Magraoui, Fouzi; Meyer, Helmut E.; Stenmark, Harald; Platta, Harald W.

2014-01-01

The occurrence of cancer is often associated with a dysfunction in one of the three central membrane-involution processes—autophagy, endocytosis or cytokinesis. Interestingly, all three pathways are controlled by the same central signaling module: the class III phosphatidylinositol 3-kinase (PI3K-III) complex and its catalytic product, the phosphorylated lipid phosphatidylinositol 3-phosphate (PtdIns3P). The activity of the catalytic subunit of the PI3K-III complex, the lipid-kinase VPS34, requires the presence of the membrane-targeting factor VPS15 as well as the adaptor protein Beclin 1. Furthermore, a growing list of regulatory proteins associates with VPS34 via Beclin 1. These accessory factors define distinct subunit compositions and thereby guide the PI3K-III complex to its different cellular and physiological roles. Here we discuss the regulation of the PI3K-III complex components by ubiquitination and SUMOylation. Especially Beclin 1 has emerged as a highly regulated protein, which can be modified with Lys11-, Lys48- or Lys63-linked polyubiquitin chains catalyzed by distinct E3 ligases from the RING-, HECT-, RBR- or Cullin-type. We also point out other cross-links of these ligases with autophagy in order to discuss how these data might be merged into a general concept. PMID:25545884

Gallium(III) complexes of NOTA-bis (phosphonate) conjugates as PET radiotracers for bone imaging.

PubMed

Holub, Jan; Meckel, Marian; Kubíček, Vojtěch; Rösch, Frank; Hermann, Petr

2015-01-01

Ligands with geminal bis(phosphonic acid) appended to 1,4,7-triazacyclonone-1,4-diacetic acid fragment through acetamide (NOTAM(BP) ) or methylenephosphinate (NO2AP(BP) ) spacers designed for (68) Ga were prepared. Ga(III) complexation is much faster for ligand with methylenephosphinate spacer than that with acetamide one, in both chemical (high reactant concentrations) and radiolabeling studies with no-carrier-added (68) Ga. For both ligands, formation of Ga(III) complex was slower than that with NOTA owing to the strong out-of-cage binding of bis(phosphonate) group. Radiolabeling was efficient and fast only above 60 °C and in a narrow acidity region (pH ~3). At higher temperature, hydrolysis of amide bond of the carboxamide-bis(phosphonate) conjugate was observed during complexation reaction leading to Ga-NOTA complex. In vitro sorption studies confirmed effective binding of the (68) Ga complexes to hydroxyapatite being comparable with that found for common bis(phosphonate) drugs such as pamindronate. Selective bone uptake was confirmed in healthy rats by biodistribution studies ex vivo and by positron emission tomography imaging in vivo. Bone uptake was very high, with SUV (standardized uptake value) of 6.19 ± 1.27 for [(68) Ga]NO2AP(BP) ) at 60 min p.i., which is superior to uptake of (68) Ga-DOTA-based bis(phosphonates) and [(18) F]NaF reported earlier (SUV of 4.63 ± 0.38 and SUV of 4.87 ± 0.32 for [(68) Ga]DO3AP(BP) and [(18) F]NaF, respectively, at 60 min p.i.). Coincidently, accumulation in soft tissue is generally low (e.g. for kidneys SUV of 0.26 ± 0.09 for [(68) Ga]NO2AP(BP) at 60 min p.i.), revealing the new (68) Ga complexes as ideal tracers for noninvasive, fast and quantitative imaging of calcified tissue and for metastatic lesions using PET or PET/CT. Copyright © 2014 John Wiley & Sons, Ltd.

A variation in NOS1AP gene is associated with repaglinide efficacy on insulin resistance in type 2 diabetes of Chinese

PubMed Central

Qin, Wen; Zhang, Rong; Hu, Cheng; Wang, Cong-rong; Lu, Jing-yi; Yu, Wei-hui; Bao, Yu-qian; Xiang, Kun-san; Jia, Wei-ping

2010-01-01

Aim: To investigate a potential association between SNP rs10494366 in the neural nitric oxide synthase adaptor protein (NOS1AP) and efficacy of repaglinide (an insulin secretagogue) in newly diagnosed Shanghai Chinese type 2 diabetes patients. Methods: A total of 104 newly diagnosed type 2 diabetes patients (69 men, 35 women) were recruited and treated with repaglinide for 24 weeks. Anthropometric measurements, clinical laboratory tests were obtained at baseline and after 24-week treatment. Genotyping was performed by sequencing. Results: The baseline value of BMI, HOMA-IR, HOMA-B, and fasting insulin level were significantly different between GG, GT, and TT genotypes (P=0.024, 0.030, 0.005, and 0.007, respectively). Carriers of TT genotype were in significant insulin resistance at baseline. After 24-week repaglinide monotherapy, the Δ value of fasting insulin (P=0.019) and HOMA-IR (P=0.011) were significantly different. TT carriers had the least insulin resistance after treatment. The mixed model analysis showed that the variation had an interaction effect with repaglinide treatment only on HOMA-IR (P=0.013). Conclusion: A common variant in rs10494366 is associated with repaglinide monotherapy efficacy on insulin resistance in newly diagnosed Shanghai Chinese type 2 diabetes patients. PMID:20305679

A variation in NOS1AP gene is associated with repaglinide efficacy on insulin resistance in type 2 diabetes of Chinese.

PubMed

Qin, Wen; Zhang, Rong; Hu, Cheng; Wang, Cong-rong; Lu, Jing-yi; Yu, Wei-hui; Bao, Yu-qian; Xiang, Kun-san; Jia, Wei-ping

2010-04-01

To investigate a potential association between SNP rs10494366 in the neural nitric oxide synthase adaptor protein (NOS1AP) and efficacy of repaglinide (an insulin secretagogue) in newly diagnosed Shanghai Chinese type 2 diabetes patients. A total of 104 newly diagnosed type 2 diabetes patients (69 men, 35 women) were recruited and treated with repaglinide for 24 weeks. Anthropometric measurements, clinical laboratory tests were obtained at baseline and after 24-week treatment. Genotyping was performed by sequencing. The baseline value of BMI, HOMA-IR, HOMA-B, and fasting insulin level were significantly different between GG, GT, and TT genotypes (P=0.024, 0.030, 0.005, and 0.007, respectively). Carriers of TT genotype were in significant insulin resistance at baseline. After 24-week repaglinide monotherapy, the Delta value of fasting insulin (P=0.019) and HOMA-IR (P=0.011) were significantly different. TT carriers had the least insulin resistance after treatment. The mixed model analysis showed that the variation had an interaction effect with repaglinide treatment only on HOMA-IR (P=0.013). A common variant in rs10494366 is associated with repaglinide monotherapy efficacy on insulin resistance in newly diagnosed Shanghai Chinese type 2 diabetes patients.

Secondary coordination sphere interactions within the biomimetic iron azadithiolate complexes related to Fe-only hydrogenase: dynamic measure of electron density about the Fe sites.

PubMed

Liu, Yu-Chiao; Tu, Ling-Kuang; Yen, Tao-Hung; Lee, Gene-Hsiang; Yang, Shu-Ting; Chiang, Ming-Hsi

2010-07-19

A series of iron azadithiolate complexes possessing an intramolecular secondary coordination sphere interaction and an ability to reduce HOAc at the potential near the first electron-transfer process are reported. A unique structural feature in which the aza nitrogen has its lone pair point toward the apical carbonyl carbon is observed in [Fe(2)(mu-S(CH(2))(2)NR(CH(2))(2)S)(CO)(6-x)L(x)](2) (R = (n)Pr, x = 0, 1a; R = (i)Pr, x = 0, 1b; R = (n)Pr, L = PPh(3), x = 1, 2; R = (n)Pr, L = P(n)Bu(3), x = 1, 3) as biomimetic models of the active site of Fe-only hydrogenase. The presence of this weak N...C(CO(ap)) interaction provides electronic perturbation at the Fe center. The distance of the N...C(CO(ap)) contact is 3.497 A in 1a. It increases by 0.455 A in 2 when electronic density of the Fe site is slightly enriched by a weak sigma-donating ligand, PPh(3). A longer distance (4.040 A) is observed for the P(n)Bu(3) derivative, 3. This N...C(CO(ap)) distance is thus a dynamic measure of electronic nature of the Fe(2) core. Variation of electronic richness within the Fe(2) moiety among the complexes reflects on their electrochemical response. Reduction of 2 is recorded at the potential of -2.17 V, which is 270 mV more negative than that of 1. Complex 3 requires additional 150 mV for the same reduction. Such cathodic shift results from CO substitution by phosphines. Electrocatalytic hydrogen production from HOAc by both kinds of complexes (all-CO and phosphine-substituted species) requires the potential close to that for reduction of the parent molecules in the absence of acids. The catalytic mechanism of 1a is proposed to involve proton uptake at the Fe(0)Fe(I) redox level instead of the Fe(0)Fe(0) level. This result is the first observation among the all-CO complexes with respect to electrocatalysis of HOAc.

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study.

PubMed

Wszelaka-Rylik, Małgorzata; Witkiewicz-Kucharczyk, Aleksandra; Wójcik, Jacek; Bal, Wojciech

2007-05-01

Diadenosine 5',5''-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.

Complexity measures of the central respiratory networks during wakefulness and sleep

NASA Astrophysics Data System (ADS)

Dragomir, Andrei; Akay, Yasemin; Curran, Aidan K.; Akay, Metin

2008-06-01

Since sleep is known to influence respiratory activity we studied whether the sleep state would affect the complexity value of the respiratory network output. Specifically, we tested the hypothesis that the complexity values of the diaphragm EMG (EMGdia) activity would be lower during REM compared to NREM. Furthermore, since REM is primarily generated by a homogeneous population of neurons in the medulla, the possibility that REM-related respiratory output would be less complex than that of the awake state was also considered. Additionally, in order to examine the influence of neuron vulnerabilities within the rostral ventral medulla (RVM) on the complexity of the respiratory network output, we inhibited respiratory neurons in the RVM by microdialysis of GABAA receptor agonist muscimol. Diaphragm EMG, nuchal EMG, EEG, EOG as well as other physiological signals (tracheal pressure, blood pressure and respiratory volume) were recorded from five unanesthetized chronically instrumented intact piglets (3-10 days old). Complexity of the diaphragm EMG (EMGdia) signal during wakefulness, NREM and REM was evaluated using the approximate entropy method (ApEn). ApEn values of the EMGdia during NREM and REM sleep were found significantly (p < 0.05 and p < 0.001, respectively) lower than those of awake EMGdia after muscimol inhibition. In the absence of muscimol, only the differences between REM and wakefulness ApEn values were found to be significantly different.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium

PubMed Central

Palanisamy, Arun P.; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D.; Kuppuswamy, Dhandapani

2017-01-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src’s adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24–48 h PO myocardium. Our studies indicate that c-Src’s adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. PMID:25976166

IKKα contributes to UVB-induced VEGF expression by regulating AP-1 transactivation

PubMed Central

Dong, Wen; Li, Yi; Gao, Ming; Hu, Meiru; Li, Xiaoguang; Mai, Sanyue; Guo, Ning; Yuan, Shengtao; Song, Lun

2012-01-01

Exposure to ultraviolet B (UVB) irradiation from sunlight induces the upregulation of VEGF, a potent angiogenic factor that is critical for mediating angiogenesis-associated photodamage. However, the molecular mechanisms related to UVB-induced VEGF expression have not been fully defined. Here, we demonstrate that one of the catalytic subunits of the IκB kinase complex (IKK), IKKα, plays a critical role in mediating UVB-induced VEGF expression in mouse embryonic fibroblasts (MEFs), which requires IKKα kinase activity but is independent of IKKβ, IKKγ and the transactivation of NF-κB. We further show that the transcriptional factor AP-1 functions as the downstream target of IKKα that is responsible for VEGF induction under UVB exposure. Both the accumulation of AP-1 component, c-Fos and the transactivation of AP-1 by UVB require the activated IKKα located within the nucleus. Moreover, nuclear IKKα can associate with c-Fos and recruit to the vegf promoter regions containing AP-1-responsive element and then trigger phosphorylation of the promoter-bound histone H3. Thus, our results have revealed a novel independent role for IKKα in controlling VEGF expression during the cellular UVB response by regulating the induction of the AP-1 component and phosphorylating histone H3 to facilitate AP-1 transactivation. Targeting IKKα shows promise for the prevention of UVB-induced angiogenesis and the associated photodamage. PMID:22169952

Direct 3D Printing of Catalytically Active Structures

DOE PAGES

Manzano, J. Sebastian; Weinstein, Zachary B.; Sadow, Aaron D.; ...

2017-09-22

3D printing of materials with active functional groups can provide custom-designed structures that promote chemical conversions. Catalytically active architectures were produced by photopolymerizing bifunctional molecules using a commercial stereolithographic 3D printer. Functionalities in the monomers included a polymerizable vinyl group to assemble the 3D structures and a secondary group to provide them with active sites. The 3D-printed architectures containing accessible carboxylic acid, amine, and copper carboxylate functionalities were catalytically active for the Mannich, aldol, and Huisgen cycloaddition reactions, respectively. The functional groups in the 3D-printed structures were also amenable to post-printing chemical modification. And as proof of principle, chemically activemore » cuvette adaptors were 3D printed and used to measure in situ the kinetics of a heterogeneously catalyzed Mannich reaction in a conventional solution spectrophotometer. In addition, 3D-printed millifluidic devices with catalytically active copper carboxylate complexes were used to promote azide-alkyne cycloaddition under flow conditions. The importance of controlling the 3D architecture of the millifluidic devices was evidenced by enhancing reaction conversion upon increasing the complexity of the 3D prints.« less

Direct 3D Printing of Catalytically Active Structures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Manzano, J. Sebastian; Weinstein, Zachary B.; Sadow, Aaron D.

3D printing of materials with active functional groups can provide custom-designed structures that promote chemical conversions. Catalytically active architectures were produced by photopolymerizing bifunctional molecules using a commercial stereolithographic 3D printer. Functionalities in the monomers included a polymerizable vinyl group to assemble the 3D structures and a secondary group to provide them with active sites. The 3D-printed architectures containing accessible carboxylic acid, amine, and copper carboxylate functionalities were catalytically active for the Mannich, aldol, and Huisgen cycloaddition reactions, respectively. The functional groups in the 3D-printed structures were also amenable to post-printing chemical modification. And as proof of principle, chemically activemore » cuvette adaptors were 3D printed and used to measure in situ the kinetics of a heterogeneously catalyzed Mannich reaction in a conventional solution spectrophotometer. In addition, 3D-printed millifluidic devices with catalytically active copper carboxylate complexes were used to promote azide-alkyne cycloaddition under flow conditions. The importance of controlling the 3D architecture of the millifluidic devices was evidenced by enhancing reaction conversion upon increasing the complexity of the 3D prints.« less

TRAF3IP2 Mediates Aldosterone/Salt-Induced Cardiac Hypertrophy and Fibrosis

PubMed Central

Sakamuri, Siva S.V.P; Valente, Anthony J.; Siddesha, Jalahalli M.; Delafontaine, Patrice; Siebenlist, Ulrich; Gardner, Jason D.; Chandrasekar, Bysani

2016-01-01

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3-interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. Importantly, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen 3α1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) gene expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease. PMID:27040306

Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump.

PubMed

Hinchliffe, Philip; Greene, Nicholas P; Paterson, Neil G; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2014-08-25

Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Integration of Synaptic Vesicle Cargo Retrieval with Endocytosis at Central Nerve Terminals

PubMed Central

Cousin, Michael A.

2017-01-01

Central nerve terminals contain a limited number of synaptic vesicles (SVs) which mediate the essential process of neurotransmitter release during their activity-dependent fusion. The rapid and accurate formation of new SVs with the appropriate cargo is essential to maintain neurotransmission in mammalian brain. Generating SVs containing the correct SV cargo with the appropriate stoichiometry is a significant challenge, especially when multiple modes of endocytosis exist in central nerve terminals, which occur at different locations within the nerve terminals. These endocytosis modes include ultrafast endocytosis, clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE) which are triggered by specific patterns of neuronal activity. This review article will assess the evidence for the role of classical adaptor protein complexes in SV retrieval, discuss the role of monomeric adaptors and how interactions between specific SV cargoes can facilitate retrieval. In addition it will consider the evidence for preassembled plasma membrane cargo complexes and their role in facilitating these endocytosis modes. Finally it will present a unifying model for cargo retrieval at the presynapse, which integrates endocytosis modes in time and space. PMID:28824381

T cell receptor for antigen induces linker for activation of T cell–dependent activation of a negative signaling complex involving Dok-2, SHIP-1, and Grb-2

PubMed Central

Dong, Shen; Corre, Béatrice; Foulon, Eliane; Dufour, Evelyne; Veillette, André; Acuto, Oreste; Michel, Frédérique

2006-01-01

Adaptor proteins positively or negatively regulate the T cell receptor for antigen (TCR) signaling cascade. We report that after TCR stimulation, the inhibitory adaptor downstream of kinase (Dok)-2 and its homologue Dok-1 are involved in a multimolecular complex including the lipid phosphatase Src homology 2 domain–containing inositol polyphosphate 5′-phosphatase (SHIP)-1 and Grb-2 which interacts with the membrane signaling scaffold linker for activation of T cells (LAT). Knockdown of LAT and SHIP-1 expression indicated that SHIP-1 favored recruitment of Dok-2 to LAT. Knockdown of Dok-2 and Dok-1 revealed their negative control on Akt and, unexpectedly, on Zap-70 activation. Our findings support the view that Dok-1 and -2 are critical elements of a LAT-dependent negative feedback loop that attenuates early TCR signal. Dok-1 and -2 may therefore exert a critical role in shaping the immune response and as gatekeepers for T cell tolerance. PMID:17043143

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

PubMed

Skrott, Zdenek; Mistrik, Martin; Andersen, Klaus Kaae; Friis, Søren; Majera, Dusana; Gursky, Jan; Ozdian, Tomas; Bartkova, Jirina; Turi, Zsofia; Moudry, Pavel; Kraus, Marianne; Michalova, Martina; Vaclavkova, Jana; Dzubak, Petr; Vrobel, Ivo; Pouckova, Pavla; Sedlacek, Jindrich; Miklovicova, Andrea; Kutt, Anne; Li, Jing; Mattova, Jana; Driessen, Christoph; Dou, Q Ping; Olsen, Jørgen; Hajduch, Marian; Cvek, Boris; Deshaies, Raymond J; Bartek, Jiri

2017-12-14

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.

TRAF3IP2 mediates aldosterone/salt-induced cardiac hypertrophy and fibrosis.

PubMed

Sakamuri, Siva S V P; Valente, Anthony J; Siddesha, Jalahalli M; Delafontaine, Patrice; Siebenlist, Ulrich; Gardner, Jason D; Bysani, Chandrasekar

2016-07-05

Aberrant activation of the renin-angiotensin-aldosterone system (RAAS) contributes to adverse cardiac remodeling and eventual failure. Here we investigated whether TRAF3 Interacting Protein 2 (TRAF3IP2), a redox-sensitive cytoplasmic adaptor molecule and an upstream regulator of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), mediates aldosterone-induced cardiac hypertrophy and fibrosis. Wild type (WT) and TRAF3IP2-null mice were infused with aldosterone (0.2 mg/kg/day) for 4 weeks along with 1%NaCl in drinking water. Aldosterone/salt, but not salt alone, upregulated TRAF3IP2 expression in WT mouse hearts. Further, aldosterone elevated blood pressure to a similar extent in both WT and TRAF3IP2-null groups. However, TRAF3IP2 gene deletion attenuated aldosterone/salt-induced (i) p65 and c-Jun activation, (ii) extracellular matrix (collagen Iα1 and collagen IIIα1), matrix metalloproteinase (MMP2), lysyl oxidase (LOX), inflammatory cytokine (IL-6 and IL-18), chemokine (CXCL1 and CXCL2), and adhesion molecule (ICAM1) mRNA expression in hearts, (iii) IL-6, IL-18, and MMP2 protein levels, (iv) systemic IL-6 and IL-18 levels, and (iv) cardiac hypertrophy and fibrosis. These results indicate that TRAF3IP2 is a critical signaling intermediate in aldosterone/salt-induced myocardial hypertrophy and fibrosis, and thus a potential therapeutic target in hypertensive heart disease. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Murine natural killer immunoreceptors use distinct proximal signaling complexes to direct cell function

PubMed Central

May, Rebecca M.; Okumura, Mariko; Hsu, Chin-Jung; Bassiri, Hamid; Yang, Enjun; Rak, Gregory; Mace, Emily M.; Philip, Naomi H.; Zhang, Weiguo; Baumgart, Tobias; Orange, Jordan S.; Nichols, Kim E.

2013-01-01

Signaling pathways leading to natural killer (NK)–cell effector function are complex and incompletely understood. Here, we investigated the proximal signaling pathways downstream of the immunotyrosine-based activation motif (ITAM) bearing activating receptors. We found that the adaptor molecule SH2 domain-containing leukocyte protein of 76 kD (SLP-76) is recruited to microclusters at the plasma membrane in activated NK cells and that this is required for initiation of downstream signaling and multiple NK-cell effector functions in vitro and in vivo. Surprisingly, we found that 2 types of proximal signaling complexes involving SLP-76 were formed. In addition to the canonical membrane complex formed between SLP-76 and linker for activation of T cells (LAT) family members, a novel LAT family–independent SLP-76–dependent signaling pathway was identified. The LAT family–independent pathway involved the SH2 domain of SLP-76 and adhesion and degranulation-promoting adaptor protein (ADAP). Both the LAT family–dependent and ADAP-dependent pathway contributed to interferon-gamma production and cytotoxicity; however, they were not essential for other SLP-76–dependent events, including phosphorylation of AKT and extracellular signal–related kinase and cellular proliferation. These results demonstrate that NK cells possess an unexpected bifurcation of proximal ITAM-mediated signaling, each involving SLP-76 and contributing to optimal NK-cell function. PMID:23407547

The Legionella pneumophila IcmS-LvgA protein complex is important for Dot/Icm-dependent intracellular growth.

PubMed

Vincent, Carr D; Vogel, Joseph P

2006-08-01

Many bacterial pathogens require a functional type IV secretion system (T4SS) for virulence. Legionella pneumophila, the causative agent of Legionnaires' disease, employs the Dot/Icm T4SS to inject a large number of protein substrates into its host, thereby altering phagosome trafficking. The L. pneumophila T4SS substrate SdeA has been shown to require the accessory factor IcmS for its export. IcmS, defined as a type IV adaptor, exists as a heterodimer with IcmW and this complex functions in a manner similar to a type III secretion chaperone. Here we report an interaction between IcmS and the previously identified virulence factor LvgA. Similar to the icmS mutant, the lvgA mutant appears to assemble a fully functional Dot/Icm complex. Both LvgA and IcmS are small, acidic proteins localized to the cytoplasm and are not exported by the Dot/Icm system, suggesting they form a novel type IV adaptor complex. Inactivation of lvgA causes a minimal defect in growth in the human monocytic cell line U937 and the environmental host Acanthamoeba castellanii. However, the lvgA mutant was severely attenuated for intracellular growth of L. pneumophila in mouse macrophages, suggesting LvgA may be a critical factor that confers host specificity.

Tuning of "antenna effect" of Eu(III) in ternary systems in aqueous medium through binding with protein.

PubMed

Ghorai, Shyamal Kr; Samanta, Swarna Kamal; Mukherjee, Manini; Saha Sardar, Pinki; Ghosh, Sanjib

2013-02-04

A simple ternary system containing a protein [human serum albumin (HSA)/bovine serum albumin (BSA)], tetracycline hydrochloride (TC), and Eu(III) in suitable aqueous buffer medium at physiological pH (= 7.2) has been shown to exhibit highly efficient "antenna effect" compared to the binary complex of TC with Eu(III) (Eu(3)TC). The ternary system containing E. coli alkaline phosphatase (AP), TC, and Eu(III), however, shows a slight enhancement of Eu(III) emission, although the binding constant of AP with TC is 2 orders of magnitude greater than with BSA/HSA. The enhanced emission of bound TC in the binary systems containing proteins and TC gets quenched in the ternary systems containing HSA/BSA, showing the efficient energy transfer (ET) from TC to Eu(III). Steady state and time-resolved emission studies of each component in all the ternary systems in H(2)O and in D(2)O medium reveal that Eu(III) is very well protected from the O-H oscillator in the ternary system containing HSA/BSA compared to that containing AP. The docking studies locating the binding site of TC in the proteins suggest that TC binds near the surface of AP. In the case of HSA/BSA, TC resides in the interior of the protein resulting in a large shielding effect of Eu(III). The rotational correlation time (θ(c)) determined from the anisotropy decay of bound TC in the complexes and the accessible surface area (ASA) of the ligand in the complexes obtained from the docking studies also support the contention that Eu(3)TC is more exposed to solvent in the case of the ternary system consisting of AP, TC, and Eu(III). The calculated radiative lifetime and the sensitization efficiency ratio of Eu(III) in all the systems clearly demonstrate the protein mediated tuning of "antenna effect" in Eu(III).

Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

PubMed

Le, N; Simon, M A

1998-08-01

DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

Frequency and correlates of DSM-5 attenuated psychosis syndrome in a sample of adolescent inpatients with nonpsychotic psychiatric disorders.

PubMed

Gerstenberg, Miriam; Hauser, Marta; Al-Jadiri, Aseel; Sheridan, Eva M; Kishimoto, Taishiro; Borenstein, Yehonatan; Vernal, Ditte L; David, Lisa; Saito, Ema; Landers, Sara E; Carella, Morgan; Singh, Sukhbir; Carbon, Maren; Jiménez-Fernández, Sara; Birnbaum, Michael L; Auther, Andrea; Carrión, Ricardo E; Cornblatt, Barbara A; Kane, John M; Walitza, Susanne; Correll, Christoph U

2015-11-01

DSM-5 conceptualized attenuated psychosis syndrome (APS) as self-contained rather than as a risk syndrome, including it under "Conditions for Further Study," but also as a codable/billable condition in the main section. Since many major mental disorders emerge during adolescence, we assessed the frequency and characteristics of APS in adolescent psychiatric inpatients. Consecutively recruited adolescents hospitalized for nonpsychotic disorders (September 2009-May 2013) were divided into APS youth versus non-APS youth, based on the Structured Interview of Prodromal Syndromes (SIPS) and according to DSM-5 criteria, and compared across multiple characteristics. Of 89 adolescents (mean ± SD age = 15.1 ± 1.6 years), 21 (23.6%) had APS. Compared to non-APS, APS was associated with more comorbid disorders (2.7 ± 1.0 vs 2.2 ± 1.3), major depressive disorder (61.9% vs 27.9%), oppositional defiant disorder/conduct disorder (52.4% vs 25.0%), and personality disorder traits (57.1% vs 7.4%, the only diagnostic category surviving Bonferroni correction). APS youth were more severely ill, having higher SIPS total positive, negative, and general symptoms; Brief Psychiatric Rating Scale total and positive scores; depression and global illness ratings; and lower Global Assessment of Functioning (GAF). Conversely, Young Mania Rating Scale scores, suicidal behavior, prescribed psychotropic medications, and mental disorder awareness were similar between APS and non-APS groups. In multivariable analysis, lowest GAF score in the past year (odds ratio [OR] = 51.15; 95% confidence interval [CI], 2.46-2,439.0) and social isolation (OR = 27.52; 95% CI, 3.36-313.87) were independently associated with APS (r(2) = 0.302, P < .0001). Although psychotic disorders were excluded, 65.2% (APS = 57.1%, non-APS = 67.7%, P = .38) received antipsychotics. One in 4 nonpsychotic adolescent inpatients met DSM-5 criteria for APS. APS youth were more impaired, showing a complex entanglement with a broad range of psychiatric symptoms and disorders, including depression, impulse-control, and, especially, emerging personality disorders. ClinicalTrials.gov identifier: NCT01383915. © Copyright 2015 Physicians Postgraduate Press, Inc.

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

PubMed

Scott, R H; Sweeney, M I; Kobrinsky, E M; Pearson, H A; Timms, G H; Pullar, I A; Wedley, S; Dolphin, A C

1992-05-01

1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents. However, at higher concentrations 1-10 microM AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.

AP39, a mitochondria‐targeting hydrogen sulfide (H2S) donor, protects against myocardial reperfusion injury independently of salvage kinase signalling

PubMed Central

Karwi, Qutuba G; Bornbaum, Julia; Boengler, Kerstin; Torregrossa, Roberta; Whiteman, Matthew; Wood, Mark E; Schulz, Rainer

2017-01-01

Background and Purpose H2S protects myocardium against ischaemia/reperfusion injury. This protection may involve the cytosolic reperfusion injury salvage kinase (RISK) pathway, but direct effects on mitochondrial function are possible. Here, we investigated the potential cardioprotective effect of a mitochondria‐specific H2S donor, AP39, at reperfusion against ischaemia/reperfusion injury. Experimental Approach Anaesthetized rats underwent myocardial ischaemia (30 min)/reperfusion (120 min) with randomization to receive interventions before reperfusion: vehicle, AP39 (0.01, 0.1, 1 μmol·kg−1), or control compounds AP219 and ADT‐OH (1 μmol·kg−1). LY294002, L‐NAME or ODQ were used to investigate the involvement of the RISK pathway. Myocardial samples harvested 5 min after reperfusion were analysed for RISK protein phosphorylation and isolated cardiac mitochondria were used to examine the direct mitochondrial effects of AP39. Key Results AP39, dose‐dependently, reduced infarct size. Inhibition of either PI3K/Akt, eNOS or sGC did not affect this effect of AP39. Western blot analysis confirmed that AP39 did not induce phosphorylation of Akt, eNOS, GSK‐3β or ERK1/2. In isolated subsarcolemmal and interfibrillar mitochondria, AP39 significantly attenuated mitochondrial ROS generation without affecting respiratory complexes I or II. Furthermore, AP39 inhibited mitochondrial permeability transition pore (PTP) opening and co‐incubation of mitochondria with AP39 and cyclosporine A induced an additive inhibitory effect on the PTP. Conclusion and Implications AP39 protects against reperfusion injury independently of the cytosolic RISK pathway. This cardioprotective effect could be mediated by inhibiting PTP via a cyclophilin D‐independent mechanism. Thus, selective delivery of H2S to mitochondria may be therapeutically applicable for employing the cardioprotective utility of H2S. PMID:27930802

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.

Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partnermore » and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.« less

Membrane attachment is key to protecting transducin GTPase-activating complex from intracellular proteolysis in photoreceptors.

PubMed

Gospe, Sidney M; Baker, Sheila A; Kessler, Christopher; Brucato, Martha F; Winter, Joan R; Burns, Marie E; Arshavsky, Vadim Y

2011-10-12

The members of the R7 regulator of G-protein signaling (RGS) protein subfamily are versatile regulators of G-protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins that serve as versatile modulators of their activity, intracellular targeting, and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1 · Gβ5 GTPase-activating complex responsible for the rapid inactivation of the G-protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gβ5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1 · Gβ5 to cellular membranes. Furthermore, membrane attachment of RGS9-1 · Gβ5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1 · Gβ5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP · RGS9-1 · Gβ5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

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

PubMed

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

2009-08-01

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.

Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion.

PubMed

Bänfer, Sebastian; Schneider, Dominik; Dewes, Jenny; Strauss, Maximilian T; Freibert, Sven-A; Heimerl, Thomas; Maier, Uwe G; Elsässer, Hans-Peter; Jungmann, Ralf; Jacob, Ralf

2018-05-08

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4a E228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

Retriever, a multiprotein complex for retromer-independent endosomal cargo recycling

PubMed Central

McNally, Kerrie E.; Faulkner, Rebecca; Steinberg, Florian; Gallon, Matthew; Ghai, Rajesh; Pim, David; Langton, Paul; Pearson, Neil; Danson, Chris M.; Nägele, Heike; Morris, Lindsey M; Singla, Arnika; Overlee, Brittany L; Heesom, Kate J.; Sessions, Richard; Banks, Lawrence; Collins, Brett M; Berger, Imre; Billadeau, Daniel D.; Burstein, Ezra; Cullen, Peter J.

2018-01-01

Following endocytosis and entry into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are alternatively retrieved and recycled back to the cell surface. Here we describe the discovery of an ancient and conserved multi-protein complex which orchestrates cargo retrieval and recycling and importantly, is biochemically and functionally distinct to the established retromer pathway. Composed of a heterotrimer of DSCR3, C16orf62 and VPS29, and bearing striking similarity with retromer, we have called this complex ‘retriever’. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to the CCC and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α5β1-integrin. Through quantitative proteomic analysis we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, which require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major new endosomal retrieval and recycling pathway. PMID:28892079

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.

Replication of Caucasian loci associated with bone mineral density in Koreans.

PubMed

Kim, Y A; Choi, H J; Lee, J Y; Han, B G; Shin, C S; Cho, N H

2013-10-01

Most bone mineral density (BMD) loci were reported in Caucasian genome-wide association studies (GWAS). This study investigated the association between 59 known BMD loci (+200 suggestive SNPs) and DXA-derived BMD in East Asian population with respect to sex and site specificity. We also identified four novel BMD candidate loci from the suggestive SNPs. Most GWAS have reported BMD-related variations in Caucasian populations. This study investigates whether the BMD loci discovered in Caucasian GWAS are also associated with BMD in East Asian ethnic samples. A total of 2,729 unrelated Korean individuals from a population-based cohort were analyzed. We selected 747 single-nucleotide polymorphisms (SNPs). These markers included 547 SNPs from 59 loci with genome-wide significance (GWS, p value less than 5 × 10(-8)) levels and 200 suggestive SNPs that showed weaker BMD association with p value less than 5 × 10(-5). After quality control, 535 GWS SNPs and 182 suggestive SNPs were included in the replication analysis. Of the 535 GWS SNPs, 276 from 25 loci were replicated (p < 0.05) in the Korean population with 51.6 % replication rate. Of the 182 suggestive variants, 16 were replicated (p < 0.05, 8.8 % of replication rate), and five reached a significant combined p value (less than 7.0 × 10(-5), 0.05/717 SNPs, corrected for multiple testing). Two markers (rs11711157, rs3732477) are for the same signal near the gene CPN2 (carboxypeptidase N, polypeptide 2). The other variants, rs6436440 and rs2291296, were located in the genes AP1S3 (adaptor-related protein complex 1, sigma 3 subunit) and RARB (retinoic acid receptor, beta). Our results illustrate ethnic differences in BMD susceptibility genes and underscore the need for further genetic studies in each ethnic group. We were also able to replicate some SNPs with suggestive associations. These SNPs may be BMD-related genetic markers and should be further investigated.

Inhibition of Mycobacterium tuberculosis Methionine Aminopeptidases by Bengamide Derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Jing-Ping; Yuan, Xiu-Hua; Yuan, Hai

Methionine aminopeptidase (MetAP) carries out an essential function of protein N-terminal processing in many bacteria and is a promising target for the development of novel antitubercular agents. Natural bengamides potently inhibit the proliferation of mammalian cells by targeting MetAP enzymes, and the X-ray crystal structure of human type 2 MetAP in complex with a bengamide derivative reveals the key interactions at the active site. By preserving the interactions with the conserved residues inside the binding pocket while exploring the differences between bacterial and human MetAPs around the binding pocket, seven bengamide derivatives were synthesized and evaluated for inhibition of MtMetAP1amore » and MtMetAP1c in different metalloforms, inhibition of M. tuberculosis growth in replicating and non-replicating states, and inhibition of human K562 cell growth. Potent inhibition of MtMetAP1a and MtMetAP1c and modest growth inhibition of M. tuberculosis were observed for some of these derivatives. Crystal structures of MtMetAP1c in complex with two of the derivatives provided valuable structural information for improvement of these inhibitors for potency and selectivity.« less

UV resonance Raman finds peptide bond-Arg side chain electronic interactions.

PubMed

Sharma, Bhavya; Asher, Sanford A

2011-05-12

We measured the UV resonance Raman excitation profiles and Raman depolarization ratios of the arginine (Arg) vibrations of the amino acid monomer as well as Arg in the 21-residue predominantly alanine peptide AAAAA(AAARA)(3)A (AP) between 194 and 218 nm. Excitation within the π → π* peptide bond electronic transitions result in UVRR spectra dominated by amide peptide bond vibrations. The Raman cross sections and excitation profiles indicate that the Arg side chain electronic transitions mix with the AP peptide bond electronic transitions. The Arg Raman bands in AP exhibit Raman excitation profiles similar to those of the amide bands in AP which are conformation specific. These Arg excitation profiles distinctly differ from the Arg monomer. The Raman depolarization ratios of Arg in monomeric solution are quite simple with ρ = 0.33 indicating enhancement by a single electronic transition. In contrast, we see very complex depolarization ratios of Arg in AP that indicate that the Arg residues are resonance enhanced by multiple electronic transitions.

Human SLP-65 isoforms contribute differently to activation and apoptosis of B lymphocytes.

PubMed

Grabbe, Annika; Wienands, Jürgen

2006-12-01

The SH2 domain-containing leukocyte adaptor protein of 65 kDa (SLP-65) is the key effector for signaling downstream of the B-cell antigen receptor (BCR). SLP-65 controls not only B lymphopoiesis and humoral immunity but also possesses a yet poorly defined tumor suppressor activity that is lost in many cases of acute lymphoblastic leukemia. We found that the 2 isoforms of human SLP-65 are differentially involved in positive and negative B-cell signaling. Reconstitution experiments revealed that an atypical SH3 domain-binding motif, which is present in the long but not in the short SLP-65 isoform, mediates association to Grb2 and suppresses activation of mitogen-activated protein kinases p38 and JNK as well as up-regulation of c-Fos expression. In turn, the short isoform activates not only AP1-driven but also NF-kappaB-driven gene transcription more potently than the long isoform. Conversely, the long rather than the short SLP-65 isoform promotes BCR-induced B-cell apoptosis. Our data further delineate the structural requirements of positive and negative SLP-65 signal transduction in normal and neoplastic cells.

Crystal structures of the CBS and DRTGG domains of the regulatory region of Clostridiumperfringens pyrophosphatase complexed with the inhibitor, AMP, and activator, diadenosine tetraphosphate.

PubMed

Tuominen, H; Salminen, A; Oksanen, E; Jämsen, J; Heikkilä, O; Lehtiö, L; Magretova, N N; Goldman, A; Baykov, A A; Lahti, R

2010-05-07

Nucleotide-binding cystathionine beta-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be established. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5',5-P1,P4-tetraphosphate (AP(4)A). The structures of the AMP and AP(4)A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 A resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP(4)A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides. Copyright 2010 Elsevier Ltd. All rights reserved.

Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models.

PubMed

Li, Li-Li; Melero-Fernandez de Mera, Raquel M; Chen, Jia; Ba, Wei; Kasri, Nael Nadif; Zhang, Mingjie; Courtney, Michael J

2015-05-13

The protein NOS1AP/CAPON mediates signaling from a protein complex of NMDA receptor, PSD95 and nNOS. The only stroke trial for neuroprotectants that showed benefit to patients targeted this ternary complex. NOS1AP/nNOS interaction regulates small GTPases, iron transport, p38MAPK-linked excitotoxicity, and anxiety. Moreover, the nos1ap gene is linked to disorders from schizophrenia, post-traumatic stress disorder, and autism to cardiovascular disorders and breast cancer. Understanding protein interactions required for NOS1AP function, therefore, has broad implications for numerous diseases. Here we show that the interaction of NOS1AP with nNOS differs radically from the classical PDZ docking assumed to be responsible. The NOS1AP PDZ motif does not bind nNOS as measured by multiple methods. In contrast, full-length NOS1AP forms an unusually stable interaction with nNOS. We mapped the discrepancy between full-length and C-terminal PDZ motif to a novel internal region we call the ExF motif. The C-terminal PDZ motif, although neither sufficient nor necessary for binding, nevertheless promotes the stability of the complex. It therefore potentially affects signal transduction and suggests that functional interaction of nNOS with NOS1AP might be targetable at two distinct sites. We demonstrate that excitotoxic pathways can be regulated, in cortical neuron and organotypic hippocampal slice cultures from rat, either by the previously described PDZ ligand TAT-GESV or by the ExF motif-bearing region of NOS1AP, even when lacking the critical PDZ residues as long as the ExF motif is intact and not mutated. This previously unrecognized heterodivalent interaction of nNOS with NOS1AP may therefore provide distinct opportunities for pharmacological intervention in NOS1AP-dependent signaling and excitotoxicity. Copyright © 2015 the authors 0270-6474/15/357349-16$15.00/0.

A comparative study of prebiotic and present day translational models

NASA Technical Reports Server (NTRS)

Rein, R.; Raghunathan, G.; Mcdonald, J.; Shibata, M.; Srinivasan, S.

1986-01-01

It is generally recognized that the understanding of the molecular basis of primitive translation is a fundamental step in developing a theory of the origin of life. However, even in modern molecular biology, the mechanism for the decoding of messenger RNA triplet codons into an amino acid sequence of a protein on the ribosome is understood incompletely. Most of the proposed models for prebiotic translation lack, not only experimental support, but also a careful theoretical scrutiny of their compatibility with well understood stereochemical and energetic principles of nucleic acid structure, molecular recognition principles, and the chemistry of peptide bond formation. Present studies are concerned with comparative structural modelling and mechanistic simulation of the decoding apparatus ranging from those proposed for prebiotic conditions to the ones involved in modern biology. Any primitive decoding machinery based on nucleic acids and proteins, and most likely the modern day system, has to satisfy certain geometrical constraints. The charged amino acyl and the peptidyl termini of successive adaptors have to be adjacent in space in order to satisfy the stereochemical requirements for amide bond formation. Simultaneously, the same adaptors have to recognize successive codons on the messenger. This translational complex has to be realized by components that obey nucleic acid conformational principles, stabilities, and specificities. This generalized condition greatly restricts the number of acceptable adaptor structures.

A neuromorphic implementation of multiple spike-timing synaptic plasticity rules for large-scale neural networks

PubMed Central

Wang, Runchun M.; Hamilton, Tara J.; Tapson, Jonathan C.; van Schaik, André

2015-01-01

We present a neuromorphic implementation of multiple synaptic plasticity learning rules, which include both Spike Timing Dependent Plasticity (STDP) and Spike Timing Dependent Delay Plasticity (STDDP). We present a fully digital implementation as well as a mixed-signal implementation, both of which use a novel dynamic-assignment time-multiplexing approach and support up to 226 (64M) synaptic plasticity elements. Rather than implementing dedicated synapses for particular types of synaptic plasticity, we implemented a more generic synaptic plasticity adaptor array that is separate from the neurons in the neural network. Each adaptor performs synaptic plasticity according to the arrival times of the pre- and post-synaptic spikes assigned to it, and sends out a weighted or delayed pre-synaptic spike to the post-synaptic neuron in the neural network. This strategy provides great flexibility for building complex large-scale neural networks, as a neural network can be configured for multiple synaptic plasticity rules without changing its structure. We validate the proposed neuromorphic implementations with measurement results and illustrate that the circuits are capable of performing both STDP and STDDP. We argue that it is practical to scale the work presented here up to 236 (64G) synaptic adaptors on a current high-end FPGA platform. PMID:26041985

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism

PubMed Central

Rammohan, Jayan; Ruiz Manzano, Ana; Garner, Ashley L.; Stallings, Christina L.; Galburt, Eric A.

2015-01-01

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera. PMID:25697505

Production and flux of carbohydrate species in the Gulf of Mexico

NASA Astrophysics Data System (ADS)

Hung, Chin-Chang; Guo, Laodong; Schultz, Gary E.; Pinckney, James L.; Santschi, Peter H.

2003-06-01

Carbohydrates are an important organic compound class in seawater and play an active role in the biogeochemical cycling of organic carbon and trace elements in the ocean, but are poorly characterized. To better understand the sources and role of carbohydrate species in marine environments, the concentrations and fluxes of particulate carbohydrates (CHO), total acid polysaccharides (APS), uronic acids (URA), phytoplankton composition and bacterial production were measured in the Gulf of Mexico in 2000 and 2001. A strong positive correlation between APS concentration and cyanobacteria abundance was found in 2000. In 2001, prymnesiophyte abundance correlated well with both concentrations of APS and URA. Bacterial production data, measured simultaneously in 2001, showed significant positive relationships with particulate organic carbon (POC), CHO, APS and URA concentrations, respectively. The average fluxes out of the euphotic zone of CHO, APS and URA in 2000 were 8.1, 1.3, and 0.7 mg C m-2 d-1, respectively. In 2001, the average fluxes of CHO, APS and URA were about 3 times higher than those in 2000, which was a time of lower nutrient concentrations, indicating that the fluxes of carbohydrate species are related to the nutrient status and phytoplankton composition. The results suggest that APS in the upper water column can be produced by cyanobacteria, prymnesiophytes, and heterotrophic bacteria. Most importantly, our data indicate that APS and CHO compounds are more resistant to biological degradation than other organic compounds, suggesting that the role of CHO compounds in carbon cycling in the ocean is more complex than previously thought.

Formal Verification of Complex Systems based on SysML Functional Requirements

DTIC Science & Technology

2014-12-23

Formal Verification of Complex Systems based on SysML Functional Requirements Hoda Mehrpouyan1, Irem Y. Tumer2, Chris Hoyle2, Dimitra Giannakopoulou3...requirements for design of complex engineered systems. The proposed ap- proach combines a SysML modeling approach to document and structure safety requirements...methods and tools to support the integration of safety into the design solution. 2.1. SysML for Complex Engineered Systems Traditional methods and tools

Four unprecedented 2D trinuclear Mn(II)-complexes with adenine nucleobase controlled by solvent or co-ligand: Hydrothermal synthesis, crystal structure and magnetic behaviour

NASA Astrophysics Data System (ADS)

Zhao, Hongkun; He, Hongming; Wang, Xiuguang; Liu, Zhongyi; Ding, Bo; Yang, Hanwen

2018-03-01

Four unique infinite 2D Mn(II) aggregates, [Mn3(μ3-ade)2(OAc)4X]n (X = DMF for 1, DMA for 2 and C2H5O- for 3), [Mn3(μ3-ade)2(ap)2DMF]n (4) (Hade = adenine; DMF = N,N-dimethylformamide; DMA = N,N-dimethylacetamide, OAc- = acetate ion, H2ap = adipic acid) with trinuclear Mn(II) as secondary building units (SBUs), have been successfully synthesized by the assembly of Hade nucleobase and manganese acetate under solvothermal conditions. The resultant complexes can be applied to explore the influence of solvent or co-ligands on the self-assembly and properties of metal complexes based on adenine. The Hade represent tridentate μ3-N3, N7, N9 bridging coordination modes. The acetate anions exhibit μ2-η1:η1 bidentate, μ2-η1:η2 tridentate mode, and μ2-η0:η2 bidentate mode. The adipate anions in complex 4 adopt two coordination modes: one is μ4-η2:η1:η1:η1 pentadentate mode, the other one is μ3-η1:η2:η2:η1 hexadentate mode. Their magnetic behaviors exhibit interesting variations, in which the local net magnetization at low temperature increases from 1 to 3. The MnII3 SBUs in 1-3 are symmetric with an inversion center, whereas that in 4 has three crystallographically independent MnII atoms. Thus, the magnetic behaviors of 4 are different from complex 1-3.

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

DOE PAGES

Zhao, Baoyu; Shu, Chang; Gao, Xinsheng; ...

2016-06-02

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 in this paper, we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF bindsmore » 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.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Baoyu; Shu, Chang; Gao, Xinsheng

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 in this paper, we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF bindsmore » 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.« less

Factors Associated With Length of Stay and 30-Day Revisits in Pediatric Acute Pancreatitis.

PubMed

Gay, Anna C; Barreto, Nicolas; Schrager, Sheree M; Russell, Christopher J

2018-05-30

Identify factors associated with length of stay (LOS) and 30-day hospital revisit for patients hospitalized with acute pancreatitis (AP). Multicenter, retrospective cohort study using the Pediatric Health Information System database. Multilevel linear and logistic regression was used to identify factors independently associated with the primary outcome variables of LOS and 30-day hospital revisit in children aged 1-18 years discharged with a primary discharge diagnosis of AP from participating hospitals between 2008 and 2013. For the 7693 discharges, median LOS was 4 days (interquartile range 3-7 days) and 30-day revisit rate 17.6% (n = 1356). Discharges were primarily female (55%), Caucasian (46%), and six years old or older (85%). On multilevel regression, factors independently associated with both longer LOS and higher revisit odds included malignant and gastrointestinal complex chronic conditions and total parenteral nutrition (TPN) use while hospitalized. Male gender was associated with both lower LOS (aLOS = -0.6 days, 95% CI = -0.8, -0.4) and decreased revisit odds (aOR 0.85; 95% CI = 0.74, 0.97). Hispanic ethnicity was associated with increased LOS (aLOS = +0.8 days, 95% CI = +0.5, +1.1) but no change in revisit odds. Certain demographic and clinical factors, including gender, ethnicity, and type of complex chronic condition, were independently associated with LOS and risk of 30-day hospital revisit for pediatric AP. Children with malignant and gastrointestinal complex chronic conditions who require TPN are at highest risk for both longer LOS and hospital revisit when admitted with AP. These patient populations may benefit from intensive care coordination when hospitalized for AP.

Teaching Materials and Strategies for the AP Music Theory Exam

ERIC Educational Resources Information Center

Lively, Michael T.

2017-01-01

Each year, many students take the Advanced Placement (AP) Music Theory Exam, and the majority of these students enroll in specialized AP music theory classes as part of the preparation process. For the teachers of these AP music theory classes, a number of challenges are presented by the difficulty and complexity of the exam subject material as…

Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

PubMed Central

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

2011-01-01

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis. PMID:21437209

Immunoglobulin class switch DNA recombination: induction, targeting and beyond

PubMed Central

Xu, Zhenming; Zan, Hong; Pone, Egest J.; Mai, Thach; Casali, Paolo

2012-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus is central to the maturation of the antibody response and critically requires the AID cytidine deaminase. CSR entails changes of the chromatin state and transcriptional activation of the IgH locus upstream and downstream switch (S) regions that are to undergo S-S DNA recombination, induction of AID, and targeting of CSR factors to S regions by 14-3-3 adaptors and as enabled by the transcription machinery and histone modifications. In this Review, we focus on recent advances in CSR induction and targeting. We also outline an integrated model of the assembly of macromolecular complexes that transduce critical epigenetic information to enzymatic effectors of the CSR machinery. PMID:22728528

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…

A novel motif in the yeast mitochondrial dynamin Dnm1 is essential for adaptor binding and membrane recruitment

PubMed Central

Bui, Huyen T.; Karren, Mary A.; Bhar, Debjani

2012-01-01

To initiate mitochondrial fission, dynamin-related proteins (DRPs) must bind specific adaptors on the outer mitochondrial membrane. The structural features underlying this interaction are poorly understood. Using yeast as a model, we show that the Insert B domain of the Dnm1 guanosine triphosphatase (a DRP) contains a novel motif required for association with the mitochondrial adaptor Mdv1. Mutation of this conserved motif specifically disrupted Dnm1–Mdv1 interactions, blocking Dnm1 recruitment and mitochondrial fission. Suppressor mutations in Mdv1 that restored Dnm1–Mdv1 interactions and fission identified potential protein-binding interfaces on the Mdv1 β-propeller domain. These results define the first known function for Insert B in DRP–adaptor interactions. Based on the variability of Insert B sequences and adaptor proteins, we propose that Insert B domains and mitochondrial adaptors have coevolved to meet the unique requirements for mitochondrial fission of different organisms. PMID:23148233

Nuclear pore complex tethers to the cytoskeleton.

PubMed

Goldberg, Martin W

2017-08-01

The nuclear envelope is tethered to the cytoskeleton. The best known attachments of all elements of the cytoskeleton are via the so-called LINC complex. However, the nuclear pore complexes, which mediate the transport of soluble and membrane bound molecules, are also linked to the microtubule network, primarily via motor proteins (dynein and kinesins) which are linked, most importantly, to the cytoplasmic filament protein of the nuclear pore complex, Nup358, by the adaptor BicD2. The evidence for such linkages and possible roles in nuclear migration, cell cycle control, nuclear transport and cell architecture are discussed. Copyright © 2017. Published by Elsevier Ltd.

Specificity of binding of clathrin adaptors to signals on the mannose-6-phosphate/insulin-like growth factor II receptor.

PubMed Central

Glickman, J N; Conibear, E; Pearse, B M

1989-01-01

Adaptors mediate the interaction of clathrin with select groups of receptors. Two distinct types of adaptors, the HA-II adaptors (found in plasma membrane coated pits) and the HA-I adaptors (localized to Golgi coated pits) bind to the cytoplasmic portion of the 270 kd mannose 6-phosphate (M6P) receptor-a receptor which is concentrated in coated pits on both the plasma membrane and in the trans-Golgi network. Neither type of adaptor appears to compete with the other for binding, suggesting that each type recognizes a distinct site on the M6P receptor tail. Mutation of the two tyrosines in the tail essentially eliminates the interaction with the HA-II plasma membrane adaptor, which recognizes a 'tyrosine' signal on other endocytosed receptors (for example, the LDL receptor and the poly Ig receptor). In contrast, the wild type and the mutant M6P receptor tail (lacking tyrosines) are equally effective at binding HA-I adaptors. This suggests that there is an HA-I recognition signal in another region of the M6P receptor tail, C-terminal to the tyrosine residues, which remains intact in the mutant. This signal is presumably responsible for the concentration of the M6P receptor, with bound lysosomal enzymes, into coated pits which bud from the trans-Golgi network, thus mediating efficient transfer of these enzymes to lysosomes. Images PMID:2545438

VizieR Online Data Catalog: Candidate X-ray OB stars in MYStIX regions (Povich+, 2017)

NASA Astrophysics Data System (ADS)

Povich, M. S.; Busk, H. A.; Feigelson, E. D.; Townsley, L. K.; Kuhn, M. A.

2017-10-01

X-ray point source catalogs for the 18 Massive Young Star-forming Complex Study in Infrared and X-Rays (MYStIX) regions studied here were produced by Kuhn+ (2010, J/ApJ/725/2485 and 2013, J/ApJS/209/27) and Townsley (2014+, J/ApJS/213/1) from archival Chandra Advanced CCD Imaging Camera (ACIS) observations. MYStIX JHKs NIR photometry was obtained from images taken with the United Kingdom Infrared Telescope (UKIRT) Wide-field Camera or from the Two-Micron All-Sky Survey (2MASS). See section 2 for further details. Spitzer MIR photometry at 3.6, 4.5, 5.8, and 8.0um was provided either by the Galactic Legacy Mid-Plane Survey Extraordinaire (GLIMPSE; Benjamin+ 2003PASP..115..953B) or by Kuhn+ (2013, J/ApJS/209/29). (4 data files).

Spectroscopic, electrochemical DNA binding and in vivo anti-inflammatory studies on newly synthesized Schiff bases of 4-aminophenazone.

PubMed

Arshad, Nasima; Ahmad, Mukhtar; Ashraf, Muhammad Zaman; Nadeem, Humaira

2014-09-05

4-Aminophenazone (Ap-1) Schiff bases i.e., 4-{(3,4,5-trimethoxybenzylidine) amino}phenazone (Ap-2), 4-{(2-chlorobenzylidine) amino}phenazone (Ap-3) and 4-{(4-chlorobenzylidine)amino} phenazone (Ap-4) were synthesized and characterized by different spectroscopic techniques. Interaction of these compounds with ds.DNA was investigated through UV-Visible spectroscopy, fluorescence spectroscopy and cyclic voltammetry at stomach (4.7) and blood (7.4) pH under 37 °C (human body temperature). Instrumental findings were further quantified both kinetically and thermodynamically. Results obtained through these techniques inferred intercalative mode of binding of all the compounds with DNA. The binding constant data, "Kb", and free energy change, ΔG, indicated comparatively greater binding affinity and more spontaneity of binding of compounds with DNA at stomach pH (4.7), respectively. However, among these compounds, Ap-4 showed comparatively greater binding at both the pH. Formation of compound-DNA complex was further confirmed through the decrease in diffusion rates after the addition of DNA. The in vivo anti-inflammatory activity of the compounds was evaluated using the carrageenan-induced hind paw edema method. The results revealed that among all the compounds, Ap-4 showed greater percentage of edema inhibition compared to standard drug. Copyright © 2014 Elsevier B.V. All rights reserved.

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

PubMed

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF andmore » 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.« less

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 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor...

A Complex Organic Slushy Bathing Low-Mass Protostars

NASA Astrophysics Data System (ADS)

Drozdovskaya, Maria; Walsh, Catherine; Visser, Ruud; Harsono, Daniel; van Dishoeck, Ewine

2015-08-01

Complex organic molecules are ubiquitous companions of young forming stars. They were first observed in hot cores surrounding high-mass protostars [e.g., 1], but have since also been detected in the environs of several low-mass counterparts [e.g., 2]. Recent studies have shown that colder envelopes and positions with impinging outflows may also glow with emission from complex organic species [e.g., 3, 4]. For this meeting, I would like to present physicochemical modeling results on the synthesis of complex organics in an envelope-cavity system that is subject to non-thermal processing. This includes wavelength-dependent radiative transfer calculations with RADMC [5] and a comprehensive gas-grain chemical network [6]. The results show that the morphology of such a system delineates three distinct regions: the cavity wall layer with time-dependent and species-variant enhancements; a torus rich in complex organic ices, but not reflected in gas-phase abundances; and the remaining outer envelope abundant in simpler solid and gaseous molecules. Within the adopted paradigm, complex organic molecules are demonstrated to have unique lifetimes and be grouped into early and late species [7]. Key chemical processes for forming and destroying complex organic molecules will be discussed. In addition, the results of adding newly experimentally verified routes [8] into the existing chemical networks will be shown.[1] Blake G. A., Sutton E. C., Masson C. R., Phillips T. G., 1987, ApJ, 315, 621[2] Jørgensen J. K., Favre C., Bisschop S. E., Bourke T. L., van Dishoeck E. F., Schmalzl M., 2012, ApJ, 757, L4[3] Arce H. G., Santiago-García J., Jørgensen J. K., Tafalla M., Bachiller R., 2008, ApJ, 681, L21[4] Öberg K. I., Bottinelli S., Jørgensen J. K., van Dishoeck E. F., 2010, ApJ, 716, 825[5] Dullemond C. P., Dominik C., 2004, A&A, 417, 159[6] Walsh C., Millar T. J., Nomura H., Herbst E., Widicus Weaver S., Aikawa Y., Laas J. C., Vasyunin A. I., 2014, A&A, 563, A33[7] Drozdovskaya M. N., Walsh C., Visser R., Harsono D., van Dishoeck E. F., MNRAS, subm.[8] Fedoseev G., Cuppen H. M., Ioppolo S., Lamberts T., Linnartz H., 2015, MNRAS, 448, 128

AMF/PGI transactivates the MMP-3 gene through the activation of Src-RhoA-phosphatidylinositol 3-kinase signaling to induce hepatoma cell migration.

PubMed

Shih, Wen-Ling; Liao, Ming-Huei; Yu, Feng-Ling; Lin, Ping-Yuan; Hsu, Hsue-Yin; Chiu, Shu-Jun

2008-11-08

We have previously shown that AMF/PGI induces hepatoma cell migration through the induction of MMP-3. This work investigates how AMF/PGI activates the MMP-3 gene. We demonstrated that AMF/PGI transactivates the MMP-3 gene promoter through AP-1. The transactivation and induction of cell migration effect of AMF/PGI directly correlates with its enzymatic activity. Various analyses showed that AMF/PGI stimulated the Src-RhoA-PI3-kinase signaling pathway, and these three signaling molecules could form a complex. Our results demonstrate a new mechanism of AMF/PGI-induced cell migration and a link between Src-RhoA-PI3-kinase, AP-1, MMP-3 and hepatoma cell migration.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

PubMed

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

2017-06-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1 -knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Anatomic Mesenchymal Stem Cell-Based Engineered Cartilage Constructs for Biologic Total Joint Replacement

PubMed Central

Saxena, Vishal; Kim, Minwook; Keah, Niobra M.; Neuwirth, Alexander L.; Stoeckl, Brendan D.; Bickard, Kevin; Restle, David J.; Salowe, Rebecca; Wang, Margaret Ye; Steinberg, David R.

2016-01-01

Cartilage has a poor healing response, and few viable options exist for repair of extensive damage. Hyaluronic acid (HA) hydrogels seeded with mesenchymal stem cells (MSCs) polymerized through UV crosslinking can generate functional tissue, but this crosslinking is not compatible with indirect rapid prototyping utilizing opaque anatomic molds. Methacrylate-modified polymers can also be chemically crosslinked in a cytocompatible manner using ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). The objectives of this study were to (1) compare APS/TEMED crosslinking with UV crosslinking in terms of functional maturation of MSC-seeded HA hydrogels; (2) generate an anatomic mold of a complex joint surface through rapid prototyping; and (3) grow anatomic MSC-seeded HA hydrogel constructs using this alternative crosslinking method. Juvenile bovine MSCs were suspended in methacrylated HA (MeHA) and crosslinked either through UV polymerization or chemically with APS/TEMED to generate cylindrical constructs. Minipig porcine femoral heads were imaged using microCT, and anatomic negative molds were generated by three-dimensional printing using fused deposition modeling. Molded HA constructs were produced using the APS/TEMED method. All constructs were cultured for up to 12 weeks in a chemically defined medium supplemented with TGF-β3 and characterized by mechanical testing, biochemical assays, and histologic analysis. Both UV- and APS/TEMED-polymerized constructs showed increasing mechanical properties and robust proteoglycan and collagen deposition over time. At 12 weeks, APS/TEMED-polymerized constructs had higher equilibrium and dynamic moduli than UV-polymerized constructs, with no differences in proteoglycan or collagen content. Molded HA constructs retained their hemispherical shape in culture and demonstrated increasing mechanical properties and proteoglycan and collagen deposition, especially at the edges compared to the center of these larger constructs. Immunohistochemistry showed abundant collagen type II staining and little collagen type I staining. APS/TEMED crosslinking can be used to produce MSC-seeded HA-based neocartilage and can be used in combination with rapid prototyping techniques to generate anatomic MSC-seeded HA constructs for use in filling large and anatomically complex chondral defects or for biologic joint replacement. PMID:26871863

ROLES OF ADIPOCYTES AND FIBROBLASTS IN ACTIVATION OF THE ALTERNATIVE PATHWAY OF COMPLEMENT IN INFLAMMATORY ARTHRITIS IN MICE

PubMed Central

Arend, William P.; Mehta, Gaurav; Antonioli, Alexandra H.; Takahashi, Minoru; Takahashi, Kazue; Stahl, Gregory L.; Holers, V. Michael; Banda, Nirmal K.

2013-01-01

The complement system is involved in mediation of joint damage in rheumatoid arthritis, with evidence suggesting activation of both the classical and alternative pathways (AP). The AP is both necessary and sufficient to mediate collagen antibody-induced arthritis (CAIA), an experimental animal model of immune complex (IC)-induced joint disease. The AP in mice is dependent on MASP-1/3 cleavage of pro-factor D (pro-FD) into mature FD. The objectives of the present study were to determine the cells synthesizing MASP-1/3 and pro-FD in synovial tissue. CAIA was studied in wild-type C57BL/6 mice, and the localization of mRNA and protein for FD and MASP-1/3 in synovial adipose tissue (SAT) and fibroblast-like synoviocytes (FLS) was determined using various techniques, including laser capture micro-dissection (LCM). SAT was the sole source of mRNA for pro-FD. Cultured differentiated 3T3 adipocytes, a surrogate for SAT, produced pro-FD but no mature FD. FLS were the main source of MASP-1/3 mRNA and protein. Using cartilage micro-particles (CMP) coated with anti-collagen mAb and serum from MASP-1/3−/− mice as a source of factor B, pro-FD in 3T3 supernatants was cleaved into mature FD by MASP-1/3 in FLS supernatants. The mature FD was eluted from the CMP, and was not present in the supernatants from the incubation with CMP, indicating that cleavage of pro-FD into mature FD by MASP-1 occurred on the CMP. These results demonstrate that pathogenic activation of the AP may occur in the joint through IC adherent to cartilage and the local production of necessary AP proteins by adipocytes and FLS. PMID:23650618

Variation in Empiric Coverage Versus Detection of Methicillin-Resistant Staphylococcus aureus and Pseudomonas aeruginosa in Hospitalizations for Community-Onset Pneumonia Across 128 US Veterans Affairs Medical Centers.

PubMed

Jones, Barbara E; Brown, Kevin Antoine; Jones, Makoto M; Huttner, Benedikt D; Greene, Tom; Sauer, Brian C; Madaras-Kelly, Karl; Rubin, Michael A; Bidwell Goetz, Matthew; Samore, Matthew H

2017-08-01

OBJECTIVE To examine variation in antibiotic coverage and detection of resistant pathogens in community-onset pneumonia. DESIGN Cross-sectional study. SETTING A total of 128 hospitals in the Veterans Affairs health system. PARTICIPANTS Hospitalizations with a principal diagnosis of pneumonia from 2009 through 2010. METHODS We examined proportions of hospitalizations with empiric antibiotic coverage for methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PAER) and with initial detection in blood or respiratory cultures. We compared lowest- versus highest-decile hospitals, and we estimated adjusted probabilities (AP) for patient- and hospital-level factors predicting coverage and detection using hierarchical regression modeling. RESULTS Among 38,473 hospitalizations, empiric coverage varied widely across hospitals (MRSA lowest vs highest, 8.2% vs 42.0%; PAER lowest vs highest, 13.9% vs 44.4%). Detection rates also varied (MRSA lowest vs highest, 0.5% vs 3.6%; PAER lowest vs highest, 0.6% vs 3.7%). Whereas coverage was greatest among patients with recent hospitalizations (AP for anti-MRSA, 54%; AP for anti-PAER, 59%) and long-term care (AP for anti-MRSA, 60%; AP for anti-PAER, 66%), detection was greatest in patients with a previous history of a positive culture (AP for MRSA, 7.9%; AP for PAER, 11.9%) and in hospitals with a high prevalence of the organism in pneumonia (AP for MRSA, 3.9%; AP for PAER, 3.2%). Low hospital complexity and rural setting were strong negative predictors of coverage but not of detection. CONCLUSIONS Hospitals demonstrated widespread variation in both coverage and detection of MRSA and PAER, but probability of coverage correlated poorly with probability of detection. Factors associated with empiric coverage (eg, healthcare exposure) were different from those associated with detection (eg, microbiology history). Providing microbiology data during empiric antibiotic decision making could better align coverage to risk for resistant pathogens and could promote more judicious use of broad-spectrum antibiotics. Infect Control Hosp Epidemiol 2017;38:937-944.

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.

Adaptor proteins GIR1 and GIR2. I. Interaction with the repressor GLABRA2 and regulation of root hair development.

PubMed

Wu, Renhong; Citovsky, Vitaly

2017-07-01

Plants use specialized root outgrowths, termed root hairs, to enhance acquisition of nutrients and water, help secure anchorage, and facilitate interactions with soil microbiome. One of the major regulators of this process is GLABRA2 (GL2), a transcriptional repressor of root hair differentiation. However, regulation of the GL2-function is relatively well characterized, it remains completely unknown whether GL2 itself functions in complex with other transcriptional regulators. We identified GIR1 and GIR2, a plant-specific two-member family of closely related proteins that interact with GL2. Loss-of-function mutants of GIR1 and GIR2 enhanced development of root hair whereas gain-of-function mutants repressed it. Thus, GIR1 and GIR2 might function as adaptor proteins that associate with GL2 and participate in control of root hair formation. Copyright © 2017 Elsevier Inc. All rights reserved.

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer.

PubMed

Zihni, Ceniz; Terry, Stephen James

2015-07-01

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

A 1:2 crystalline complex of ApA:proflavine: a model for binding to single-stranded regions in RNA.

PubMed Central

Neidle, S; Taylor, G; Sanderson, M

1978-01-01

The structure of a 1"2 complex of adenylyl-(3',5')-adenosine phosphate and proflavine hemisulfate has been determined using the methods of x-ray crystallography. Since the ApA does not form a mini double helix, it may serve as a model for the interaction of planar molecules with single stranded nucleic acids. The dinucleotide adopts an extended conformation with the adenines in adjacent molecules forming base pairs. A most unusual feature of the molecule is that it does not obey the "rigid nucleotide" concept although none of the torsion angles occur in energetically unfavourable regions. This is most probably due to the strong interactions between the proflavine and the oligonucleotide. PMID:724521

Gaze direction affects the magnitude of face identity aftereffects.

PubMed

Kloth, Nadine; Jeffery, Linda; Rhodes, Gillian

2015-02-20

The face perception system partly owes its efficiency to adaptive mechanisms that constantly recalibrate face coding to our current diet of faces. Moreover, faces that are better attended produce more adaptation. Here, we investigated whether the social cues conveyed by a face can influence the amount of adaptation that face induces. We compared the magnitude of face identity aftereffects induced by adaptors with direct and averted gazes. We reasoned that faces conveying direct gaze may be more engaging and better attended and thus produce larger aftereffects than those with averted gaze. Using an adaptation duration of 5 s, we found that aftereffects for adaptors with direct and averted gazes did not differ (Experiment 1). However, when processing demands were increased by reducing adaptation duration to 1 s, we found that gaze direction did affect the magnitude of the aftereffect, but in an unexpected direction: Aftereffects were larger for adaptors with averted rather than direct gaze (Experiment 2). Eye tracking revealed that differences in looking time to the faces between the two gaze directions could not account for these findings. Subsequent ratings of the stimuli (Experiment 3) showed that adaptors with averted gaze were actually perceived as more expressive and interesting than adaptors with direct gaze. Therefore it appears that the averted-gaze faces were more engaging and better attended, leading to larger aftereffects. Overall, our results suggest that naturally occurring facial signals can modulate the adaptive impact a face exerts on our perceptual system. Specifically, the faces that we perceive as most interesting also appear to calibrate the organization of our perceptual system most strongly. © 2015 ARVO.

VizieR Online Data Catalog: Spatial structure of young stellar clusters. I. (Kuhn+, 2014)

NASA Astrophysics Data System (ADS)

Kuhn, M. A.; Feigelson, E. D.; Getman, K. V.; Baddeley, A. J.; Broos, P. S.; Sills, A.; Bate, M. R.; Povich, M. S.; Luhman, K. L.; Busk, H. A.; Naylor, T.; King, R. R.

2015-07-01

The regions included in this study, listed in Table 1, are nearby sites of high-mass star formation that are included in the MYStIX sample (Feigelson et al. 2013ApJS..209...26F). These regions are covered in the near-IR by the UKIRT Infrared Deep Sky Survey (King et al. 2013, J/ApJS/209/28) or Two-Micron All Sky Survey (Skrutskie et al. 2006, VII/233), in the mid-IR by Spitzer IRAC observations (Kuhn et al. 2013, J/ApJS/209/29), and in the X-ray by the ACIS-I array on the Chandra X-Ray Observatory in the 0.5-8.0keV band (Kuhn et al. 2013, J/ApJS/209/27; Townsley et al. 2014, J/ApJS/213/1). Other sources to produce multiwavelength catalogs are described in section 2. The multiwavelength point-source catalogs are combined with a list of spectroscopic OB stars from the literature, and sources are classified probabilistically to obtain a list of MYStIX Probable Complex Members (MPCMs) (Broos et al. 2013, J/ApJS/209/32). (3 data files).

NETosing Neutrophils Activate Complement Both on Their Own NETs and Bacteria via Alternative and Non-alternative Pathways

PubMed Central

Yuen, Joshua; Pluthero, Fred G.; Douda, David N.; Riedl, Magdalena; Cherry, Ahmed; Ulanova, Marina; Kahr, Walter H. A.; Palaniyar, Nades; Licht, Christoph

2016-01-01

Neutrophils deposit antimicrobial proteins, such as myeloperoxidase and proteases on chromatin, which they release as neutrophil extracellular traps (NETs). Neutrophils also carry key components of the complement alternative pathway (AP) such as properdin or complement factor P (CFP), complement factor B (CFB), and C3. However, the contribution of these complement components and complement activation during NET formation in the presence and absence of bacteria is poorly understood. We studied complement activation on NETs and a Gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa (PA01, PAKwt, and PAKgfp). Here, we show that anaphylatoxin C5a, formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol myristate acetate (PMA), which activates NADPH oxidase, induce the release of CFP, CFB, and C3 from neutrophils. In response to PMA or P. aeruginosa, neutrophils secrete CFP, deposit it on NETs and bacteria, and induce the formation of terminal complement complexes (C5b–9). A blocking anti-CFP antibody inhibited AP-mediated but not non-AP-mediated complement activation on NETs and P. aeruginosa. Therefore, NET-mediated complement activation occurs via both AP- and non AP-based mechanisms, and AP-mediated complement activation during NETosis is dependent on CFP. These findings suggest that neutrophils could use their “AP tool kit” to readily activate complement on NETs and Gram-negative bacteria, such as P. aeruginosa, whereas additional components present in the serum help to fix non-AP-mediated complement both on NETs and bacteria. This unique mechanism may play important roles in host defense and help to explain specific roles of complement activation in NET-related diseases. PMID:27148258

Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes.

PubMed

Puri, Madhu; La Pietra, Luigi; Mraheil, Mobarak Abu; Lucas, Rudolf; Chakraborty, Trinad; Pillich, Helena

2017-09-05

Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes . Host cell recognition results in ubiquitination of L . monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein 1 light chain 3 (LC3). Although studies have indicated that L . monocytogenes induces autophagy, the significance of this process in the infectious cycle and the mechanisms involved remain poorly understood. Here, we examined the role of the autophagy adaptor optineurin (OPTN), the phosphorylation of which by the TANK binding kinase 1 (TBK1) enhances its affinity for LC3 and promotes autophagosomal degradation, during L . monocytogenes infection. In LC3- and OPTN-depleted host cells, intracellular replicating L . monocytogenes increased, an effect not seen with a mutant lacking the pore-forming toxin listeriolysin O (LLO). LLO induced the production of OPTN. In host cells expressing an inactive TBK1, bacterial replication was also inhibited. Our studies have uncovered an OPTN-dependent pathway in which L . monocytogenes uses LLO to restrict bacterial growth. Hence, manipulation of autophagy by L . monocytogenes , either through induction or evasion, represents a key event in its intracellular life style and could lead to either cytosolic growth or persistence in intracellular vacuolar structures.

77 FR 28238 - Airworthiness Directives; Airbus Airplanes

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-14

... adaptor and the wing skin. This AD requires performing an electrical bonding test between the gravity fill re-fuel adaptor and the top skin panels on the left-hand and right-hand wings, and if necessary... repairing the gravity fuel adaptor if any corrosion is found. We are issuing this AD to detect and correct...

Some properties of the action potentials conducted in the spines of the sea urchin Diadema antillarum.

PubMed

Berrios, A; Brink, D; del Castillo, J; Smith, D S

1985-01-01

Brief (2-5 msec) electrical pulses applied to the primary spines of the sea urchin Diadema antillarum elicit graded action potentials (ap's). These ap's can be attributed to the electrical activity of a set of 14-21 bundles of neurites, each comprising 1000 processes near the spine base and tapering towards the spine tip. The shape of the ap's varies from a simple diphasic deflection to a complex waveform with 6 or more components. Peak-to-peak amplitude is less than 1mV. The ap's are conducted at a uniform speed of ca. 27 cm/sec. The ap's are not affected by tetrodotoxin (1 microgram/ml) and continue to be produced in Na-free artificial sea water (ASW). The amplitude of the ap's is greatly reduced or totally abolished in Ca-free ASW. However, some electrical activity may continue in the absence of external Ca, due to release of Ca2+ ions from the calcium carbonate crystals of the spine shaft. Replacing the Ca content of ASW by barium ions causes an irreversible blockade of the ap's. Spines equilibrated with ASW containing Sr2+ ions instead of Ca2+ produce ap's of increased amplitude (up to X 2). The ap's are blocked by La3+, Co2+, Cd2+ (2-5 mM) and by the organic Ca channel blocker Bepridil (2 mM). We conclude that the spinal ap's are due to the summation of Ca spikes produced by the activation of Ca channels which are blocked by barium and have a high affinity for, or permeability to Sr vs Ca.

The complex magnetic field topology of the cool Ap star 49 Cam

NASA Astrophysics Data System (ADS)

Silvester, J.; Kochukhov, O.; Rusomarov, N.; Wade, G. A.

2017-10-01

49 Cam is a cool magnetic chemically peculiar star that has been noted for showing strong, complex Zeeman linear polarization signatures. This paper describes magnetic and chemical surface maps obtained for 49 Cam using the Invers10 magnetic Doppler imaging code and high-resolution spectropolarimetric data in all four Stokes parameters collected with the ESPaDOnS and Narval spectropolarimeters at the Canada-France-Hawaii Telescope and Pic du Midi Observatory. The reconstructed magnetic field maps of 49 Cam show a relatively complex structure. Describing the magnetic field topology in terms of spherical harmonics, we find significant contributions of modes up to ℓ = 3, including toroidal components. Observations cannot be reproduced using a simple low-order multipolar magnetic field structure. 49 Cam exhibits a level of field complexity that has not been seen in magnetic maps of other cool Ap stars. Hence, we concluded that relatively complex magnetic fields are observed in Ap stars at both low and high effective temperatures. In addition to mapping the magnetic field, we also derive surface abundance distributions of nine chemical elements, including Ca, Sc, Ti, Cr, Fe, Ce, Pr, Nd and Eu. Comparing these abundance maps with the reconstructed magnetic field geometry, we find no clear relationship of the abundance distributions with the magnetic field for some elements. However, for other elements some distinct patterns are found. We discuss these results in the context of other recent magnetic mapping studies and theoretical predictions of radiative diffusion.

An adaptive technique for multiscale approximate entropy (MAEbin) threshold (r) selection: application to heart rate variability (HRV) and systolic blood pressure variability (SBPV) under postural stress.

PubMed

Singh, Amritpal; Saini, Barjinder Singh; Singh, Dilbag

2016-06-01

Multiscale approximate entropy (MAE) is used to quantify the complexity of a time series as a function of time scale τ. Approximate entropy (ApEn) tolerance threshold selection 'r' is based on either: (1) arbitrary selection in the recommended range (0.1-0.25) times standard deviation of time series (2) or finding maximum ApEn (ApEnmax) i.e., the point where self-matches start to prevail over other matches and choosing the corresponding 'r' (rmax) as threshold (3) or computing rchon by empirically finding the relation between rmax, SD1/SD2 ratio and N using curve fitting, where, SD1 and SD2 are short-term and long-term variability of a time series respectively. None of these methods is gold standard for selection of 'r'. In our previous study [1], an adaptive procedure for selection of 'r' is proposed for approximate entropy (ApEn). In this paper, this is extended to multiple time scales using MAEbin and multiscale cross-MAEbin (XMAEbin). We applied this to simulations i.e. 50 realizations (n = 50) of random number series, fractional Brownian motion (fBm) and MIX (P) [1] series of data length of N = 300 and short term recordings of HRV and SBPV performed under postural stress from supine to standing. MAEbin and XMAEbin analysis was performed on laboratory recorded data of 50 healthy young subjects experiencing postural stress from supine to upright. The study showed that (i) ApEnbin of HRV is more than SBPV in supine position but is lower than SBPV in upright position (ii) ApEnbin of HRV decreases from supine i.e. 1.7324 ± 0.112 (mean ± SD) to upright 1.4916 ± 0.108 due to vagal inhibition (iii) ApEnbin of SBPV increases from supine i.e. 1.5535 ± 0.098 to upright i.e. 1.6241 ± 0.101 due sympathetic activation (iv) individual and cross complexities of RRi and systolic blood pressure (SBP) series depend on time scale under consideration (v) XMAEbin calculated using ApEnmax is correlated with cross-MAE calculated using ApEn (0.1-0.26) in steps of 0.02 at each time scale in supine and upright position and is concluded that ApEn0.26 has highest correlation at most scales (vi) choice of 'r' is critical in interpreting interactions between RRi and SBP and in ascertaining true complexity of the individual RRi and SBP series.

RIOK3 Is an Adaptor Protein Required for IRF3-Mediated Antiviral Type I Interferon Production

PubMed Central

Feng, Jun; De Jesus, Paul D.; Su, Victoria; Han, Stephanie; Gong, Danyang; Wu, Nicholas C.; Tian, Yuan; Li, Xudong; Wu, Ting-Ting; Chanda, Sumit K.

2014-01-01

ABSTRACT Detection of cytosolic nucleic acids by pattern recognition receptors leads to the induction of type I interferons (IFNs) and elicits the innate immune response. We report here the identification of RIOK3 as a novel adaptor protein that is essential for the cytosolic nucleic acid-induced type I IFN production and for the antiviral response to gammaherpesvirus through two independent kinome-wide RNA interference screens. RIOK3 knockdown blocks both cytosolic double-stranded B-form DNA and double-stranded RNA-induced IRF3 activation and IFN-β production. In contrast, the overexpression of RIOK3 activates IRF3 and induces IFN-β. RIOK3 functions downstream of TBK1 and upstream of IRF3 activation. Furthermore, RIOK3 physically interacts with both IRF3 and TBK1 and is necessary for the interaction between TBK1 and IRF3. In addition, global transcriptome analysis shows that the expression of many gene involved antiviral responses is dependent on RIOK3. Thus, knockdown of RIOK3 inhibits cellular antiviral responses against both DNA and RNA viruses (herpesvirus and influenza A virus). Our data suggest that RIOK3 plays a critical role in the antiviral type I IFN pathway by bridging TBK1 and IRF3. IMPORTANCE The innate immune response, such as the production of type I interferons, acts as the first line of defense, limiting infectious pathogens directly and shaping the adaptive immune response. In this study, we identified RIOK3 as a novel regulator of the antiviral type I interferon pathway. Specifically, we found that RIOK3 physically interacts with TBK1 and IRF3 and bridges the functions between TBK1 and IRF3 in the activation of type I interferon pathway. The identification of a cellular kinase that plays a role the type I interferon pathway adds another level of complexity in the regulation of innate immunity and will have implications for developing novel strategies to combat viral infection. PMID:24807708

Quantum dynamics of nuclear spins and spin relaxation in organic semiconductors

DOE PAGES

Mkhitaryan, V. V.; Dobrovitski, V. V.

2017-06-12

3D printing of materials with active functional groups can provide custom-designed structures that promote chemical conversions. Herein, catalytically active architectures were produced by photopolymerizing bifunctional molecules using a commercial stereolithographic 3D printer. Functionalities in the monomers included a polymerizable vinyl group to assemble the 3D structures and a secondary group to provide them with active sites. The 3D-printed architectures containing accessible carboxylic acid, amine, and copper carboxylate functionalities were catalytically active for the Mannich, aldol, and Huisgen cycloaddition reactions, respectively. The functional groups in the 3D-printed structures were also amenable to post-printing chemical modification. As proof of principle, chemically activemore » cuvette adaptors were 3D printed and used to measure in situ the kinetics of a heterogeneously catalyzed Mannich reaction in a conventional solution spectrophotometer. In addition, 3D-printed millifluidic devices with catalytically active copper carboxylate complexes were used to promote azide-alkyne cycloaddition under flow conditions. The importance of controlling the 3D architecture of the millifluidic devices was evidenced by enhancing reaction conversion upon increasing the complexity of the 3D prints.« less

The use of intelligent database systems in acute pancreatitis--a systematic review.

PubMed

van den Heever, Marc; Mittal, Anubhav; Haydock, Matthew; Windsor, John

2014-01-01

Acute pancreatitis (AP) is a complex disease with multiple aetiological factors, wide ranging severity, and multiple challenges to effective triage and management. Databases, data mining and machine learning algorithms (MLAs), including artificial neural networks (ANNs), may assist by storing and interpreting data from multiple sources, potentially improving clinical decision-making. 1) Identify database technologies used to store AP data, 2) collate and categorise variables stored in AP databases, 3) identify the MLA technologies, including ANNs, used to analyse AP data, and 4) identify clinical and non-clinical benefits and obstacles in establishing a national or international AP database. Comprehensive systematic search of online reference databases. The predetermined inclusion criteria were all papers discussing 1) databases, 2) data mining or 3) MLAs, pertaining to AP, independently assessed by two reviewers with conflicts resolved by a third author. Forty-three papers were included. Three data mining technologies and five ANN methodologies were reported in the literature. There were 187 collected variables identified. ANNs increase accuracy of severity prediction, one study showed ANNs had a sensitivity of 0.89 and specificity of 0.96 six hours after admission--compare APACHE II (cutoff score ≥8) with 0.80 and 0.85 respectively. Problems with databases were incomplete data, lack of clinical data, diagnostic reliability and missing clinical data. This is the first systematic review examining the use of databases, MLAs and ANNs in the management of AP. The clinical benefits these technologies have over current systems and other advantages to adopting them are identified. Copyright © 2013 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Evaluation of influence of Ap4A analogues on Fhit-positive HEK293T cells; cytotoxicity and ability to induce apoptosis.

PubMed

Krakowiak, Agnieszka; Pęcherzewska, Róża; Kaczmarek, Renata; Tomaszewska, Agnieszka; Nawrot, Barbara; Stec, Wojciech J

2011-08-15

Fragile histidine triad (Fhit) protein encoded by tumour suppressor FHIT gene is a proapoptotic protein with diadenosine polyphosphate (Ap(n)A, n=2-6) hydrolase activity. It has been hypothesised that formation of Fhit-substrate complex results in an apoptosis initiation signal while subsequent hydrolysis of Ap(n)A terminates this action. A series of Ap(n)A analogues have been identified in vitro as strong Fhit ligands [Varnum, J. M.; Baraniak, J.; Kaczmarek, R.; Stec, W. J.; Brenner, C. BMC Chem. Biol.2001, 1, 3]. We assumed that in Fhit-positive cells these compounds might preferentially bind to Fhit and inhibit its hydrolytic activity what would prolong the lifetime of apoptosis initiation signalling complex. Therefore, several Fhit inhibitors were tested for their cytotoxicity and ability to induce apoptosis in Fhit-positive HEK293T cells. These experiments have shown that Ap(4)A analogue, containing a glycerol residue instead of the central pyrophosphate and two terminal phosphorothioates [A(PS)-CH(2)CH(OH)CH(2)-(PS)A (1)], is the most cytotoxic among test compounds (IC(50)=17.5±4.2 μM) and triggers caspase-dependent cell apoptosis. The Fhit-negative HEK293T cells (in which Fhit was silenced by RNAi) were not sensitive to compound 1. These results indicate that the Ap(4)A analogue 1 induces Fhit-dependent apoptosis and therefore, it can be considered as a drug candidate for anticancer therapy in Fhit-positive cancer cells and in Fhit-negative cancer cells, in which re-expression of Fhit was accomplished by gene therapy. Copyright © 2011 Elsevier Ltd. All rights reserved.

A design of coaxial-to-radial line adaptors in radial line slot antennas

NASA Astrophysics Data System (ADS)

Natori, Makoto; Ando, Makoto; Goto, Naohisa

1990-11-01

A numerical design of a coaxial-to-radial line adaptor is presented for the use as a feed in a radial line slot antenna. To realize stable performances in mass production, the reflection from a probe type adaptor in which only the outer conductor of a coaxial line is in contact with the waveguide, is analyzed and suppressed. The tolerance for the change and the errors in the height of the waveguide as well as the bandwidth is highlighted; the advantages of the conical probe over the conventional shorting post and the coax-gap adaptor are emphasized.

Image statistics and the perception of surface gloss and lightness.

PubMed

Kim, Juno; Anderson, Barton L

2010-07-01

Despite previous data demonstrating the critical importance of 3D surface geometry in the perception of gloss and lightness, I. Motoyoshi, S. Nishida, L. Sharan, and E. H. Adelson (2007) recently proposed that a simple image statistic--histogram or sub-band skew--is computed by the visual system to infer the gloss and albedo of surfaces. One key source of evidence used to support this claim was an experiment in which adaptation to skewed image statistics resulted in opponent aftereffects in observers' judgments of gloss and lightness. We report a series of adaptation experiments that were designed to assess the cause of these aftereffects. We replicated their original aftereffects in gloss but found no consistent aftereffect in lightness. We report that adaptation to zero-skew adaptors produced similar aftereffects as positively skewed adaptors, and that negatively skewed adaptors induced no reliable aftereffects. We further find that the adaptation effect observed with positively skewed adaptors is not robust to changes in mean luminance that diminish the intensity of the luminance extrema. Finally, we show that adaptation to positive skew reduces (rather than increases) the apparent lightness of light pigmentation on non-uniform albedo surfaces. These results challenge the view that the adaptation results reported by Motoyoshi et al. (2007) provide evidence that skew is explicitly computed by the visual system.

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae.

PubMed Central

Avila, D M; Robinson, A K; Kaushal, V; Barnes, L D

1991-01-01

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product. PMID:1660456

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae.

PubMed

Avila, D M; Robinson, A K; Kaushal, V; Barnes, L D

1991-12-01

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.

75 FR 57920 - Kerr-Philpott System

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-23

... schedules VA-1-B, VA-2-B, VA-3- B, VA-4-B, CP&L-1-B, CP&L-2-B, CP&L-3-B, CP&L-4-B, AP-1-B, AP-2-B, AP- 3-B..., CP&L-1-A, CP&L-2-A, CP&L-3-A, CP&L-4-A, AP-1-A, AP-2-A, AP-3-A, AP-4-A, NC-1-A, and Replacement-2... Schedules VA-1-A, VA-2-A, VA-3-A, VA-4-A, CP&L-1- A, CP&L-2-A, CP&L-3-A, CP&L-4-A, AP-1-A, AP-2-A, AP-3-A...

An Interaction between KSHV ORF57 and UIF Provides mRNA-Adaptor Redundancy in Herpesvirus Intronless mRNA Export

PubMed Central

Jackson, Brian R.; Boyne, James R.; Noerenberg, Marko; Taylor, Adam; Hautbergue, Guillaume M.; Walsh, Matthew J.; Wheat, Rachel; Blackbourn, David J.; Wilson, Stuart A.; Whitehouse, Adrian

2011-01-01

The hTREX complex mediates cellular bulk mRNA nuclear export by recruiting the nuclear export factor, TAP, via a direct interaction with the export adaptor, Aly. Intriguingly however, depletion of Aly only leads to a modest reduction in cellular mRNA nuclear export, suggesting the existence of additional mRNA nuclear export adaptor proteins. In order to efficiently export Kaposi's sarcoma-associated herpesvirus (KSHV) intronless mRNAs from the nucleus, the KSHV ORF57 protein recruits hTREX onto viral intronless mRNAs allowing access to the TAP-mediated export pathway. Similarly however, depletion of Aly only leads to a modest reduction in the nuclear export of KSHV intronless mRNAs. Herein, we identify a novel interaction between ORF57 and the cellular protein, UIF. We provide the first evidence that the ORF57-UIF interaction enables the recruitment of hTREX and TAP to KSHV intronless mRNAs in Aly-depleted cells. Strikingly, depletion of both Aly and UIF inhibits the formation of an ORF57-mediated nuclear export competent ribonucleoprotein particle and consequently prevents ORF57-mediated mRNA nuclear export and KSHV protein production. Importantly, these findings highlight that redundancy exists in the eukaryotic system for certain hTREX components involved in the mRNA nuclear export of intronless KSHV mRNAs. PMID:21814512

Association of red complex, A. actinomycetemcomitans and non-oral bacteria with periodontal diseases.

PubMed

da Silva-Boghossian, Carina Maciel; do Souto, Renata Martins; Luiz, Ronir R; Colombo, Ana Paula Vieira

2011-09-01

Pathogens related to systemic infections have been detected in the periodontal microbiota. The relationship amongst these pathogens, periodontal bacteria and periodontal clinical status is poorly understood. This study evaluated the association amongst red complex, A. actinomycetemcomitans (A.a) and non-oral pathogenic bacteria in subjects with good periodontal health (PH), gingivitis (G), chronic (CP) and aggressive (AP) periodontitis. Subgingival biofilm samples were obtained from 51 PH, 42 G, 219 CP and 90 AP subjects. The presence and levels of A.a, red complex (Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola), Acinetobacter baumannii, Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Staphylococcus aureus were determined by DNA probes and DNA-DNA hybridization technique. CP and AP subjects presented significantly higher prevalence and levels of A.a, red complex and A. baumannii than G and PH individuals (p<0.01), whereas S. aureus was detected in lower frequency and counts in AP as compared to the other groups (p<0.001). The predictor variables age, prevalence of red complex, and the presence of A. baumannii and P. aeruginosa were strongly associated with the frequency of sites with PD and CAL ≥5 mm. Increasing age (OR 1.08), high frequency of red complex (OR 6.10), and the presence of A.a with P. aeruginosa (OR 1.90) were associated with periodontal disease (p<0.001). Subjects harbouring a high prevalence of A.a, A. baumannii, and red complex with P. aeruginosa were more likely to have AP than CP (p<0.001). Putative periodontal pathogens and non-oral bacteria alone or in association were strongly associated with periodontitis. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Sharpin interactome reveals a role for Sharpin in lamellipodium formation via the Arp2/3 complex.

PubMed

Khan, Meraj H; Salomaa, Siiri I; Jacquemet, Guillaume; Butt, Umar; Miihkinen, Mitro; Deguchi, Takahiro; Kremneva, Elena; Lappalainen, Pekka; Humphries, Martin J; Pouwels, Jeroen

2017-09-15

Sharpin, a multifunctional adaptor protein, regulates several signalling pathways. For example, Sharpin enhances signal-induced NF-κB signalling as part of the linear ubiquitin assembly complex (LUBAC) and inhibits integrins, the T cell receptor, caspase 1 and PTEN. However, despite recent insights into Sharpin and LUBAC function, a systematic approach to identify the signalling pathways regulated by Sharpin has not been reported. Here, we present the first 'Sharpin interactome', which identifies a large number of novel potential Sharpin interactors in addition to several known ones. These data suggest that Sharpin and LUBAC might regulate a larger number of biological processes than previously identified, such as endosomal trafficking, RNA processing, metabolism and cytoskeleton regulation. Importantly, using the Sharpin interactome, we have identified a novel role for Sharpin in lamellipodium formation. We demonstrate that Sharpin interacts with Arp2/3, a protein complex that catalyses actin filament branching. We have identified the Arp2/3-binding site in Sharpin and demonstrate using a specific Arp2/3-binding deficient mutant that the Sharpin-Arp2/3 interaction promotes lamellipodium formation in a LUBAC-independent fashion.This article has an associated First Person interview with the first author of the paper. © 2017. Published by The Company of Biologists Ltd.

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

PubMed Central

Goraczniak, Rafal; Wall, Brian A; Behlke, Mark A; Lennox, Kim A; Ho, Eric S; Zaphiros, Nikolas H; Jakubowski, Christopher; Patel, Neil R; Zhao, Steven; Magaway, Carlo; Subbie, Stacey A; Jenny Yu, Lumeng; LaCava, Stephanie; Reuhl, Kenneth R; Chen, Suzie; Gunderson, Samuel I

2013-01-01

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases. PMID:23673539

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

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

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

2013-01-01

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. 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. 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. 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. © 2013 S. Karger AG, Basel.

The Bcr-Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway.

PubMed

Preisinger, Christian; Kolch, Walter

2010-05-01

Bcr-Abl is the transforming principle underlying chronic myelogenous leukaemia (CML). Here, we use a functional interaction proteomics approach to map pathways by which Bcr-Abl regulates defined cellular processes. The results show that Bcr-Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/Slp-76/Nck1 adaptor protein pathway. The binding of GADS to Bcr-Abl requires Bcr-Abl tyrosine kinase activity and is sensitive to the Bcr-Abl inhibitor imatinib, while the GADS/Slp-76 and Slp-76/Nck interactions are tyrosine phosphorylation independent. All three adaptor proteins co-localize with cortical actin in membrane blebs. Downregulation of each adaptor protein disrupts the actin cytoskeleton and membrane blebbing in a similar fashion and similar to imatinib. These findings highlight the importance of protein interaction dependent adaptor protein pathways in oncogenic kinase signaling. 2010 Elsevier Inc. All rights reserved.

An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.

PubMed

Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F

2008-04-01

Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species.

Biogenesis of zinc storage granules in Drosophila melanogaster.

PubMed

Tejeda-Guzmán, Carlos; Rosas-Arellano, Abraham; Kroll, Thomas; Webb, Samuel M; Barajas-Aceves, Martha; Osorio, Beatriz; Missirlis, Fanis

2018-03-19

Membrane transporters and sequestration mechanisms concentrate metal ions differentially into discrete subcellular microenvironments for use in protein cofactors, signalling, storage or excretion. Here we identify zinc storage granules as the insect's major zinc reservoir in principal Malpighian tubule epithelial cells of Drosophila melanogaster The concerted action of Adaptor Protein-3, Rab32, HOPS and BLOC complexes as well as of the white-scarlet (ABCG2-like) and ZnT35C (ZnT2/ZnT3/ZnT8-like) transporters is required for zinc storage granule biogenesis. Due to lysosome-related organelle defects caused by mutations in the homologous human genes, patients with Hermansky-Pudlak syndrome may lack zinc granules in beta pancreatic cells, intestinal paneth cells and presynaptic vesicles of hippocampal mossy fibers. © 2018. Published by The Company of Biologists Ltd.

High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays.

PubMed

Masschaele, Delphine; Wauman, Joris; Vandemoortele, Giel; De Sutter, Delphine; De Ceuninck, Leentje; Eyckerman, Sven; Tavernier, Jan

2018-04-06

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification-mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.

Structures of yeast Apa2 reveal catalytic insights into a canonical AP₄A phosphorylase of the histidine triad superfamily.

PubMed

Hou, Wen-Tao; Li, Wen-Zhe; Chen, Yuxing; Jiang, Yong-Liang; Zhou, Cong-Zhao

2013-08-09

The homeostasis of intracellular diadenosine 5',5″'-P(1),P(4)-tetraphosphate (Ap4A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap4A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap3A (5',5″'-P(1),P(3)-tetraphosphate), Ap4A, and Ap5A (5',5″'-P(1),P(5)-tetraphosphate), and Ap4A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap4A-complexed forms at 2.30, 2.80, and 2.70Å resolution, respectively. Apa2 is an α/β protein with a core domain of a twisted eight-stranded antiparallel β-sheet flanked by several α-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap4A, the AMP and ATP moieties of which are perpendicular to each other, leaving the α-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap4A phosphorylases, which could be grouped as a unique branch in the GalT family. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

PubMed

Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

2015-02-01

The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

A View on the Function of Self-Adaptors and Their Communication Consequences.

ERIC Educational Resources Information Center

Genova, B. K. L.

The purpose of this paper is to examine the function served by self-adaptor type behaviors (defined here as "hand touch" on the face, body, the other hand, and the fingers) in order to discover why people perform self-adaptors and what happens when they do. Following an extensive review of the literature, it is proposed that…

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)

High-torque open-end wrench

NASA Technical Reports Server (NTRS)

Giandomenico, A.; Dame, J. M.; Behimer, H. (Inventor)

1978-01-01

A wrench is described that is usable where limited access normally requires an open-end wrench, but which has substantially the high-torque capacity and small radial clearance characteristics of a closed-end wrench. The wrench includes a sleeve forming a nut-engageable socket with a gap in its side, and an adaptor forming a socket with a gap in its side, the adaptor closely surrounding the sleeve and extending across the gap in the sleeve. The sleeve and adaptor have surfaces that become fully engaged when a wrench handle is applied to the adaptor to turn it so as to tighten a nut engaged by the sleeve.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

PubMed

Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

2015-12-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. © 2015 Wiley Periodicals, Inc.

Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium.

PubMed

Rapetti-Mauss, Raphael; O'Mahony, Fiona; Sepulveda, Francisco V; Urbach, Valerie; Harvey, Brian J

2013-06-01

The cAMP-regulated potassium channel KCNQ1:KCNE3 plays an essential role in transepithelial Cl(-) secretion. Recycling of K(+) across the basolateral membrane provides the driving force necessary to maintain apical Cl(-) secretion. The steroid hormone oestrogen (17β-oestradiol; E2), produces a female-specific antisecretory response in rat distal colon through the inhibition of the KCNQ1:KCNE3 channel. It has previously been shown that rapid inhibition of the channel conductance results from E2-induced uncoupling of the KCNE3 regulatory subunit from the KCNQ1 channel pore complex. The purpose of this study was to determine the mechanism required for sustained inhibition of the channel function. We found that E2 plays a role in regulation of KCNQ1 cell membrane abundance by endocytosis. Ussing chamber experiments have shown that E2 inhibits both Cl(-) secretion and KCNQ1 current in a colonic cell line, HT29cl.19A, when cultured as a confluent epithelium. Following E2 treatment, KCNQ1 was retrieved from the plasma membrane by a clathrin-mediated endocytosis, which involved the association between KCNQ1 and the clathrin adaptor, AP-2. Following endocytosis, KCNQ1 was accumulated in early endosomes. Following E2-induced endocytosis, rather than being degraded, KCNQ1 was recycled by a biphasic mechanism involving Rab4 and Rab11. Protein kinase Cδ and AMP-dependent kinase were rapidly phosphorylated in response to E2 on their activating phosphorylation sites, Ser643 and Thr172, respectively (as previously shown). Both kinases are necessary for the E2-induced endocytosis, because E2 failed to induce KCNQ1 internalization following pretreatment with specific inhibitors of both protein kinase Cδ and AMP-dependent kinase. The ubiquitin ligase Nedd4.2 binds KCNQ1 in response to E2 to induce channel internalization. This study has provided the first demonstration of hormonal regulation of KCNQ1 trafficking. In conclusion, we propose that internalization of KCNQ1 is a key event in the sustained antisecretory response to oestrogen.

The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

PubMed Central

Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A.; Nash, Piers; Tafuri, Anna; Gertler, Frank B.; Pawson, Tony

2003-01-01

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 β-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. PMID:12808099

Characterizing the complexity of spontaneous motor unit patterns of amyotrophic lateral sclerosis using approximate entropy

NASA Astrophysics Data System (ADS)

Zhou, Ping; Barkhaus, Paul E.; Zhang, Xu; Zev Rymer, William

2011-10-01

This paper presents a novel application of the approximate entropy (ApEn) measurement for characterizing spontaneous motor unit activity of amyotrophic lateral sclerosis (ALS) patients. High-density surface electromyography (EMG) was used to record spontaneous motor unit activity bilaterally from the thenar muscles of nine ALS subjects. Three distinct patterns of spontaneous motor unit activity (sporadic spikes, tonic spikes and high-frequency repetitive spikes) were observed. For each pattern, complexity was characterized by calculating the ApEn values of the representative signal segments. A sliding window over each segment was also introduced to quantify the dynamic changes in complexity for the different spontaneous motor unit patterns. We found that the ApEn values for the sporadic spikes were the highest, while those of the high-frequency repetitive spikes were the lowest. There is a significant difference in mean ApEn values between two arbitrary groups of the three spontaneous motor unit patterns (P < 0.001). The dynamic ApEn curve from the sliding window analysis is capable of tracking variations in EMG activity, thus providing a vivid, distinctive description for different patterns of spontaneous motor unit action potentials in terms of their complexity. These findings expand the existing knowledge of spontaneous motor unit activity in ALS beyond what was previously obtained using conventional linear methods such as firing rate or inter-spike interval statistics.

Atmospheric Pressure-Thermal Desorption (AP-TD)/Electrospray Ionization-Mass Spectrometry for the Rapid Analysis of Bacillus Spores

USDA-ARS?s Scientific Manuscript database

A technique is described where an atmospheric pressure-thermal desorption (AP-TD) device and electrospray ionization (ESI)-mass spectrometry are coupled and used for the rapid analysis of Bacillus spores in complex matrices. The resulting AP-TD/ESI-MS technique combines the generation of volatile co...

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex.

PubMed

Ortega-Roldan, Jose Luis; Jensen, Malene Ringkjøbing; Brutscher, Bernhard; Azuaga, Ana I; Blackledge, Martin; van Nuland, Nico A J

2009-05-01

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 muM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (K(d) = 132 +/- 13 muM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein-protein complexes.

Accurate characterization of weak macromolecular interactions by titration of NMR residual dipolar couplings: application to the CD2AP SH3-C:ubiquitin complex

PubMed Central

Ortega-Roldan, Jose Luis; Jensen, Malene Ringkjøbing; Brutscher, Bernhard; Azuaga, Ana I.; Blackledge, Martin; van Nuland, Nico A. J.

2009-01-01

The description of the interactome represents one of key challenges remaining for structural biology. Physiologically important weak interactions, with dissociation constants above 100 μM, are remarkably common, but remain beyond the reach of most of structural biology. NMR spectroscopy, and in particular, residual dipolar couplings (RDCs) provide crucial conformational constraints on intermolecular orientation in molecular complexes, but the combination of free and bound contributions to the measured RDC seriously complicates their exploitation for weakly interacting partners. We develop a robust approach for the determination of weak complexes based on: (i) differential isotopic labeling of the partner proteins facilitating RDC measurement in both partners; (ii) measurement of RDC changes upon titration into different equilibrium mixtures of partially aligned free and complex forms of the proteins; (iii) novel analytical approaches to determine the effective alignment in all equilibrium mixtures; and (iv) extraction of precise RDCs for bound forms of both partner proteins. The approach is demonstrated for the determination of the three-dimensional structure of the weakly interacting CD2AP SH3-C:Ubiquitin complex (Kd = 132 ± 13 μM) and is shown, using cross-validation, to be highly precise. We expect this methodology to extend the remarkable and unique ability of NMR to study weak protein–protein complexes. PMID:19359362

Approximate Entropy in the Electroencephalogram During Wake and Sleep

PubMed Central

Burioka, Naoto; Miyata, Masanori; Cornélissen, Germaine; Halberg, Franz; Takeshima, Takao; Kaplan, Daniel T.; Suyama, Hisashi; Endo, Masanori; Maegaki, Yoshihiro; Nomura, Takashi; Tomita, Yutaka; Nakashima, Kenji; Shimizu, Eiji

2006-01-01

Entropy measurement can discriminate among complex systems, including deterministic, stochastic and composite systems. We evaluated the changes of approximate entropy (ApEn) in signals of the electroencephalogram (EEG) during sleep. EEG signals were recorded from eight healthy volunteers during nightly sleep. We estimated the values of ApEn in EEG signals in each sleep stage. The ApEn values for EEG signals (mean ± SD) were 0.896 ± 0.264 during eyes-closed waking state, 0.738 ± 0.089 during Stage I, 0.615 ± 0.107 during Stage II, 0.487 ± 0.101 during Stage III, 0.397 ± 0.078 during Stage IV and 0.789 ± 0.182 during REM sleep. The ApEn values were found to differ with statistical significance among the six different stages of consciousness (ANOVA, p<0.001). ApEn of EEG was statistically significantly lower during Stage IV and higher during wake and REM sleep. We conclude that ApEn measurement can be useful to estimate sleep stages and the complexity in brain activity. PMID:15683194

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

PubMed

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-03-22

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology

PubMed Central

Sudhir, Putty-Reddy; Chen, Chung-Hsuan

2016-01-01

A protein complex consists of two or more proteins that are linked together through protein–protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples. PMID:27011181

A20 restricts ubiquitination of pro-interleukin-1β protein complexes and suppresses NLRP3 inflammasome activity

PubMed Central

Duong, Bao H.; Onizawa, Michio; Oses-Prieto, Juan A.; Advincula, Rommel; Burlingame, Alma; Malynn, Barbara A.; Ma, Averil

2015-01-01

SUMMARY Inappropriate inflammasome activation contributes to multiple human diseases, but the mechanisms by which inflammasomes are suppressed are poorly understood. The NFκB inhibitor A20 is a ubiquitin-modifying enzyme that may prevent human inflammatory diseases and lymphomas. Here, we report that A20-deficient macrophages, unlike normal cells, exhibit spontaneous NLRP3 inflammasome activity to LPS alone. The kinase RIPK3, but not the adaptor MyD88, is required for this response. In normal cells, A20 constitutively associates with caspase-1 and pro-IL-1β, and NLRP3 activation further promotes A20 recruitment to the inflammasome. Pro-IL-1β also co-immunoprecipitates with RIPK1, RIPK3, caspase-1 and caspase-8 in a complex that is modified with K63-linked and unanchored polyubiquitin. In A20-deficient macrophages, this pro-IL-1β-associated ubiquitination is markedly increased in a RIPK3-dependent manner. Mass spectrometric and mutational analyses reveal that K133 of pro-IL-1β is a physiological ubiquitination site that supports processing. Our study reveals a novel mechanism by which A20 prevents inflammatory diseases. PMID:25607459

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hua, Fang, E-mail: fhua2@emory.edu; Wang, Jun; Sayeed, Iqbal

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-{kappa}B). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF's role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined themore » activation of NF-{kappa}B and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-{kappa}B activity and phosphorylation of the inhibitor of kappa B (I{kappa}B{alpha}) increased in ischemic brains, but IRF3, inhibitor of {kappa}B kinase complex-{epsilon} (IKK{epsilon}), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-{kappa}B activity or p-I{kappa}B{alpha} induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-{kappa}B signaling and brain injury after acute cerebral I/R.« less

Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

PubMed Central

Bulatov, Emil; Ciulli, Alessio

2015-01-01

In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Gadwal, Shyam; Sosinowski, Tomasz; Gu, Hua; Weiss, Arthur

2006-11-28

Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation.

Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner

PubMed Central

Dragone, Leonard L.; Myers, Margaret D.; White, Carmen; Gadwal, Shyam; Sosinowski, Tomasz; Gu, Hua; Weiss, Arthur

2006-01-01

Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation. PMID:17110436

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation.

PubMed

Abraham, Libin; Bankhead, Peter; Pan, Xiaoyu; Engel, Ulrike; Fackler, Oliver T

2012-08-15

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

PubMed Central

Ophir, Michael J.; Liu, Beiyun C.

2013-01-01

The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

ATG13

PubMed Central

Alers, Sebastian; Wesselborg, Sebastian; Stork, Björn

2014-01-01

During the past 20 years, autophagy signaling has entered the main stage of the cell biological theater. Autophagy represents an intracellular degradation process that is involved in both the bulk recycling of cytoplasmic components and the selective removal of organelles, protein aggregates, or intracellular pathogens. The understanding of autophagy has been greatly facilitated by the characterization of the molecular machinery governing this process. In yeast, initiation of autophagy is controlled by the Atg1 kinase complex, which is composed of the Ser/Thr kinase Atg1, the adaptor protein Atg13, and the ternary complex of Atg17-Atg31-Atg29. In vertebrates, the orthologous ULK1 kinase complex contains the Ser/Thr kinase ULK1 and the accessory proteins ATG13, RB1CC1, and ATG101. Among these components, Atg1/ULK1 have gained major attention in the past, i.e., for the identification of upstream regulatory kinases, the characterization of downstream substrates controlling the autophagic flux, or as a druggable target for the modulation of autophagy. However, accumulating data indicate that the function of Atg13/ATG13 has been likely underestimated so far. In addition to ensuring proper Atg1/ULK1 recruitment and activity, this adaptor molecule has been implicated in ULK1-independent autophagy processes. Furthermore, recent data have identified additional binding partners of Atg13/ATG13 besides the components of the Atg1/ULK1 complex, e.g., Atg8 family proteins or acidic phospholipids. Therefore, in this review we will center the spotlight on Atg13/ATG13 and summarize the role that Atg13/ATG13 assumes in the autophagy stage play. PMID:24879146

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

PubMed

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses.

PubMed

Lambert, Sylviane; Swindell, William R; Tsoi, Lam C; Stoll, Stefan W; Elder, James T

2017-07-01

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The Legionella IcmSW Complex Directly Interacts with DotL to Mediate Translocation of Adaptor-Dependent Substrates

PubMed Central

Sutherland, Molly C.; Nguyen, Thuy Linh; Tseng, Victor; Vogel, Joseph P.

2012-01-01

Legionella pneumophila is a Gram-negative bacterium that replicates within human alveolar macrophages by evasion of the host endocytic pathway through the formation of a replicative vacuole. Generation of this vacuole is dependent upon the secretion of over 275 effector proteins into the host cell via the Dot/Icm type IVB secretion system (T4SS). The type IV coupling protein (T4CP) subcomplex, consisting of DotL, DotM, DotN, IcmS and IcmW, was recently defined. DotL is proposed to be the T4CP of the L. pneumophila T4SS based on its homology to known T4CPs, which function as inner-membrane receptors for substrates. As a result, DotL is hypothesized to play an integral role(s) in the L. pneumophila T4SS for the engagement and translocation of substrates. To elucidate this role, a genetic approach was taken to screen for dotL mutants that were unable to survive inside host cells. One mutant, dotLY725Stop, did not interact with the type IV adaptor proteins IcmS/IcmW (IcmSW) leading to the identification of an IcmSW-binding domain on DotL. Interestingly, the dotLY725Stop mutant was competent for export of one class of secreted effectors, the IcmSW-independent substrates, but exhibited a specific defect in secretion of IcmSW-dependent substrates. This differential secretion illustrates that DotL requires a direct interaction with the type IV adaptor proteins for the secretion of a major class of substrates. Thus, by identifying a new target for IcmSW, we have discovered that the type IV adaptors perform an additional role in the export of substrates by the L. pneumophila Dot/Icm T4SS. PMID:23028312

Long-range ordering in the Bi 1-xAe xFeO 3-x/2 perovskites: Bi 1/3Sr 2/3FeO 2.67 and Bi 1/2Ca 1/2FeO 2.75

NASA Astrophysics Data System (ADS)

Lepoittevin, C.; Malo, S.; Barrier, N.; Nguyen, N.; Van Tendeloo, G.; Hervieu, M.

2008-10-01

Two-ordered perovskites, Bi 1/3Sr 2/3FeO 2.67 and Bi 1/2Ca 1/2FeO 2.75, have been stabilized and characterized by transmission electron microscopy, Mössbauer spectroscopy and X-ray powder diffraction techniques. They both exhibit orthorhombic superstructures, one with a≈ b≈2 ap and c≈3 ap (S.G.: Pb2 n or Pbmn) for the Sr-based compound and one with a≈ b≈2 ap and c≈8 ap (S.G.: B222, Bmm2, B2 mm or Bmmm) for the Ca-based one. The high-resolution transmission electron microscopy (HRTEM) images evidence the existence of one deficient [FeO x] ∞ layer, suggesting that Bi 1/3Sr 2/3FeO 2.67 and Bi 1/2Ca 1/2FeO 2.75 behave differently compared to their Ln-based homolog. The HAADF-STEM images allow to propose a model of cation ordering on the A sites of the perovskite. The Mössbauer analyses confirm the trivalent state of iron and its complex environment with three types of coordination. Both compounds exhibit a high value of resistivity and the inverse molar susceptibility versus temperature curves evidence a magnetic transition at about 730 K for the Bi 1/3Sr 2/3FeO 2.67 and a smooth reversible transition between 590 and 650 K for Bi 1/2Ca 1/2FeO 2.75.

Ageing introduces a complex pattern of changes in several rat brain transcription factors depending on gender and anatomical localization.

PubMed

Sanguino, Elena; Roglans, Núria; Rodríguez-Calvo, Ricardo; Alegret, Marta; Sánchez, Rosa M; Vázquez-Carrera, Manuel; Laguna, Juan C

2006-04-01

As ageing changes the activity of several transcription factors in the rat cortex, we were interested in determining whether similar changes also appear in the hippocampus of old rats. We determined by electrophoretic gel shift assays the binding activity of nuclear factor kappa B (NFkappaB), activator protein-1 (AP-1), peroxisome proliferator-activated receptor (PPAR), and liver X receptor (LXR) in cortex and hippocampus samples from young (3-month-old), and old (18-month-old) male and female Sprague-Dawley rats. NFkappaB activity increased in old male and female rats, though only in cortex samples, while AP-1 activity decreased only in the cortex and hippocampus of old female animals. LXR activity decreased in all conditions, except in old male cortexes; whereas PPAR activity only decreased in the hippocampus of old female rats. Decreases in AP-1 and PPAR activities restricted to old female rats did not result from an age-related decline in plasma 17beta-estradiol concentration, as their activities did not change in samples obtained from ovariectomized young female rats. Our results indicate that ageing induces a complex pattern of changes in the brain-binding activity of NFkappaB, AP-1, PPAR and LXR, depending on the anatomical origin of the samples (cortex or hippocampus), and the sex of the animals studied.

Conformational characteristics of dimeric subunits of RNA from energy minimization studies. Mixed sugar-puckered ApG, ApU, CpG, and CpU.

PubMed

Thiyagarajan, P; Ponnuswamy, P K

1981-09-01

Following the procedure described in the preceding article, the low energy conformations located for the four dimeric subunits of RNA, ApG, ApU, CpG, and CpU are presented. The A-RNA type and Watson-Crick type helical conformations and a number of different kinds of loop promoting ones were identified as low energy in all the units. The 3E-3E and 3E-2E pucker sequences are found to be more or less equally preferred; the 2E-2E sequence is occasionally preferred, while the 2E-3E is highly prohibited in all the units. A conformation similar to the one observed in the drug-dinucleoside monophosphate complex crystals becomes a low energy case only for the CpG unit. The low energy conformations obtained for the four model units were used to assess the stability of the conformational states of the dinucleotide segments in the four crystal models of the tRNAPhe molecule. Information on the occurrence of the less preferred sugar-pucker sequences in the various loop regions in the tRNAPhe molecule has been obtained. A detailed comparison of the conformational characteristics of DNA and RNA subunits at the dimeric level is presented on the basis of the results.

Conformational characteristics of dimeric subunits of RNA from energy minimization studies. Mixed sugar-puckered ApG, ApU, CpG, and CpU.

PubMed Central

Thiyagarajan, P; Ponnuswamy, P K

1981-01-01

Following the procedure described in the preceding article, the low energy conformations located for the four dimeric subunits of RNA, ApG, ApU, CpG, and CpU are presented. The A-RNA type and Watson-Crick type helical conformations and a number of different kinds of loop promoting ones were identified as low energy in all the units. The 3E-3E and 3E-2E pucker sequences are found to be more or less equally preferred; the 2E-2E sequence is occasionally preferred, while the 2E-3E is highly prohibited in all the units. A conformation similar to the one observed in the drug-dinucleoside monophosphate complex crystals becomes a low energy case only for the CpG unit. The low energy conformations obtained for the four model units were used to assess the stability of the conformational states of the dinucleotide segments in the four crystal models of the tRNAPhe molecule. Information on the occurrence of the less preferred sugar-pucker sequences in the various loop regions in the tRNAPhe molecule has been obtained. A detailed comparison of the conformational characteristics of DNA and RNA subunits at the dimeric level is presented on the basis of the results. PMID:6168312

MBD3L2 interacts with MBD3 and components of the NuRD complex and can oppose MBD2-MeCP1-mediated methylation silencing.

PubMed

Jin, Seung-Gi; Jiang, Chun-Ling; Rauch, Tibor; Li, Hongwei; Pfeifer, Gerd P

2005-04-01

MBD2 and MBD3 are two proteins that contain methyl-CpG binding domains and have a transcriptional repression function. Both proteins are components of a large CpG-methylated DNA binding complex named MeCP1, which consists of the nucleosome remodeling and histone deacetylase complex Mi2-NuRD and MBD2. MBD3L2 (methyl-CpG-binding protein 3-like 2) is a protein with substantial homology to MBD2 and MBD3, but it lacks the methyl-CpG-binding domain. Unlike MBD3L1, which is specifically expressed in haploid male germ cells, MBD3L2 expression is more widespread. MBD3L2 interacts with MBD3 in vitro and in vivo, co-localizes with MBD3 but not MBD2, and does not localize to methyl-CpG-rich regions in the nucleus. In glutathione S-transferase pull-down assays, MBD3L2 is found associated with several known components of the Mi2-NuRD complex, including HDAC1, HDAC2, MTA1, MBD3, p66, RbAp46, and RbAp48. Gel shift experiments with nuclear extracts and a CpG-methylated DNA probe indicate that recombinant MBD3L2 can displace a form of the MeCP1 complex from methylated DNA. MBD3L2 acts as a transcriptional repressor when tethered to a GAL4-DNA binding domain. Repression by GAL4-MBD3L2 is relieved by MBD2 and vice versa, and repression by MBD2 from a methylated promoter is relieved by MBD3L2. The data are consistent with a role of MBD3L2 as a transcriptional modulator that can interchange with MBD2 as an MBD3-interacting component of the NuRD complex. Thus, MBD3L2 has the potential to recruit the MeCP1 complex away from methylated DNA and reactivate transcription.

The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida

PubMed Central

Poole, Angela Z.; Kitchen, Sheila A.; Weis, Virginia M.

2016-01-01

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions. PMID:27148208

The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida.

PubMed

Poole, Angela Z; Kitchen, Sheila A; Weis, Virginia M

2016-01-01

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions.

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Statistical Evaluation of Airport Pavement Condition Survey Data for Washington, Oregon, and Idaho

DTIC Science & Technology

1988-01-01

21...COUNTY SQUIRE AIRPARK, OREGON 12 70 22...FLORENCE MUNICIPAL AP, OREGON 3 95 23...HERMISTON MUNICIPAL AP, OREGON 11 87 24...HOOD RIVER AP, OREGON...12 87 33...OROFINO MUNICIPAL AP, IDAHO 17 81 34...PRIEST RIVER MUNICIPAL AP, IDAHO 11 86 *NUgU**MmUU~tNUINNNNNNNffWUU0W NUU EU*UI** **-28 -28- TABLE 3...3 88 8...BAKER MUNICIPAL AP, OREGON 3 90 9...BEND MUNICIPAL AP, OREGON 9 89 10..CRESWELL MUNICIPAL AP, OREGON 1 98 11..HOOD RIVER AP, OREGON 1 96 12

NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

PubMed

Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

2012-05-01

The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

Pull rod assembly

DOEpatents

Cioletti, O.C.

1988-04-21

A pull rod assembly comprising a pull rod having three peripheral grooves, a piston device including an adaptor ring and a seal ring, said piston device being mounted on the pull rod by a split ring retainer situated in one groove and extending into an interior groove in the adaptor and a resilient split ring retained in another groove and positioned to engage the piston device and to retain the seal on its adaptor.

The approximate entropy concept extended to three dimensions for calibrated, single parameter structural complexity interrogation of volumetric images.

PubMed

Moore, Christopher; Marchant, Thomas

2017-07-12

Reconstructive volumetric imaging permeates medical practice because of its apparently clear depiction of anatomy. However, the tell tale signs of abnormality and its delineation for treatment demand experts work at the threshold of visibility for hints of structure. Hitherto, a suitable assistive metric that chimes with clinical experience has been absent. This paper develops the complexity measure approximate entropy (ApEn) from its 1D physiological origin into a three-dimensional (3D) algorithm to fill this gap. The first 3D algorithm for this is presented in detail. Validation results for known test arrays are followed by a comparison of fan-beam and cone-beam x-ray computed tomography image volumes used in image guided radiotherapy for cancer. Results show the structural detail down to individual voxel level, the strength of which is calibrated by the ApEn process itself. The potential for application in machine assisted manual interaction and automated image processing and interrogation, including radiomics associated with predictive outcome modeling, is discussed.

The approximate entropy concept extended to three dimensions for calibrated, single parameter structural complexity interrogation of volumetric images

NASA Astrophysics Data System (ADS)

Moore, Christopher; Marchant, Thomas

2017-08-01

Reconstructive volumetric imaging permeates medical practice because of its apparently clear depiction of anatomy. However, the tell tale signs of abnormality and its delineation for treatment demand experts work at the threshold of visibility for hints of structure. Hitherto, a suitable assistive metric that chimes with clinical experience has been absent. This paper develops the complexity measure approximate entropy (ApEn) from its 1D physiological origin into a three-dimensional (3D) algorithm to fill this gap. The first 3D algorithm for this is presented in detail. Validation results for known test arrays are followed by a comparison of fan-beam and cone-beam x-ray computed tomography image volumes used in image guided radiotherapy for cancer. Results show the structural detail down to individual voxel level, the strength of which is calibrated by the ApEn process itself. The potential for application in machine assisted manual interaction and automated image processing and interrogation, including radiomics associated with predictive outcome modeling, is discussed.

Experimental and Numerical Study of Ammonium Perchlorate Counterflow Diffusion Flames

NASA Technical Reports Server (NTRS)

Smooke, M. D.; Yetter, R. A.; Parr, T. P.; Hanson-Parr, D. M.; Tanoff, M. A.

1999-01-01

Many solid rocket propellants are based on a composite mixture of ammonium perchlorate (AP) oxidizer and polymeric binder fuels. In these propellants, complex three-dimensional diffusion flame structures between the AP and binder decomposition products, dependent upon the length scales of the heterogeneous mixture, drive the combustion via heat transfer back to the surface. Changing the AP crystal size changes the burn rate of such propellants. Large AP crystals are governed by the cooler AP self-deflagration flame and burn slowly, while small AP crystals are governed more by the hot diffusion flame with the binder and burn faster. This allows control of composite propellant ballistic properties via particle size variation. Previous measurements on these diffusion flames in the planar two-dimensional sandwich configuration yielded insight into controlling flame structure, but there are several drawbacks that make comparison with modeling difficult. First, the flames are two-dimensional and this makes modeling much more complex computationally than with one-dimensional problems, such as RDX self- and laser-supported deflagration. In addition, little is known about the nature, concentration, and evolution rates of the gaseous chemical species produced by the various binders as they decompose. This makes comparison with models quite difficult. Alternatively, counterflow flames provide an excellent geometric configuration within which AP/binder diffusion flames can be studied both experimentally and computationally.

RNA-directed activation of cytoplasmic dynein-1 in reconstituted transport RNPs.

PubMed

McClintock, Mark A; Dix, Carly I; Johnson, Christopher M; McLaughlin, Stephen H; Maizels, Rory J; Hoang, Ha Thi; Bullock, Simon L

2018-06-26

Polarised mRNA transport is a prevalent mechanism for spatial control of protein synthesis. However, the composition of transported ribonucleoprotein particles (RNPs) and the regulation of their movement are poorly understood. We have reconstituted microtubule minus end-directed transport of mRNAs using purified components. A Bicaudal-D (BicD) adaptor protein and the RNA-binding protein Egalitarian (Egl) are sufficient for long-distance mRNA transport by the dynein motor and its accessory complex dynactin, thus defining a minimal transport-competent RNP. Unexpectedly, the RNA is required for robust activation of dynein motility. We show that a cis -acting RNA localisation signal promotes the interaction of Egl with BicD, which licenses the latter protein to recruit dynein and dynactin. Our data support a model for BicD activation based on RNA-induced occupancy of two Egl-binding sites on the BicD dimer. Scaffolding of adaptor protein assemblies by cargoes is an attractive mechanism for regulating intracellular transport. © 2018, McClintock et al.

Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export.

PubMed

Fan, Jing; Kuai, Bin; Wu, Guifen; Wu, Xudong; Chi, Binkai; Wang, Lantian; Wang, Ke; Shi, Zhubing; Zhang, Heng; Chen, She; He, Zhisong; Wang, Siyuan; Zhou, Zhaocai; Li, Guohui; Cheng, Hong

2017-10-02

The exosome is a key RNA machine that functions in the degradation of unwanted RNAs. Here, we found that significant fractions of precursors and mature forms of mRNAs and long noncoding RNAs are degraded by the nuclear exosome in normal human cells. Exosome-mediated degradation of these RNAs requires its cofactor hMTR4. Significantly, hMTR4 plays a key role in specifically recruiting the exosome to its targets. Furthermore, we provide several lines of evidence indicating that hMTR4 executes this role by directly competing with the mRNA export adaptor ALYREF for associating with ARS2, a component of the cap-binding complex (CBC), and this competition is critical for determining whether an RNA is degraded or exported to the cytoplasm. Together, our results indicate that the competition between hMTR4 and ALYREF determines exosome recruitment and functions in creating balanced nuclear RNA pools for degradation and export. © 2017 The Authors.

Genetic errors of the human CARD-BCL10-MALT1 (CBM) complex: molecular, immunological, and clinical heterogeneity

PubMed Central

de Diego, Rebeca Pérez; Sánchez-Ramón, Silvia; López-Collazo, Eduardo; Martínez-Barricarte, Rubén; Cubillos-Zapata, Carolina; Cerdán, Antonio Ferreira; Casanova, Jean-Laurent; Puel, Anne

2016-01-01

Three members of the caspase recruitment domain (CARD) family of adaptors (CARD9, CARD10, and CARD11) are known to form heterotrimers with B-cell lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma-translocation gene 1 (MALT1). These three CARD-BCL10-MALT1 (CBM) complexes activate NF-κB in both the innate and adaptive arms of immunity. Human inherited defects of the three components of the CBM complex, including the two adaptors CARD9 and CARD11 and the two core components BCL10 and MALT1, have recently been reported. Bi-allelic loss-of-function (LOF) mutant alleles underlie several different immunological and clinical phenotypes, which can be assigned to two distinct categories. Isolated invasive fungal infections, of unclear cellular basis, are associated with CARD9 deficiency, whereas a broad range of clinical manifestations, including those characteristic of T- and B-lymphocyte defects, are associated with CARD11, MALT1 and BCL10 deficiencies. Interestingly, humans with these mutations have some features in common with the corresponding knockout mice, but other features are different between humans and mice. Moreover, germline and somatic gain-of-function (GOF) mutations of MALT1, BCL10 and CARD11 have also been found in other patients with lymphoproliferative disorders. This broad range of germline and somatic CBM lesions, including LOF and GOF mutations, highlights the contribution of each of the components of the CBM to human immunity. PMID:26277595

14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends.

PubMed

Bertolaccini, Maria Laura; Amengual, Olga; Andreoli, Laura; Atsumi, Tatsuya; Chighizola, Cecilia B; Forastiero, Ricardo; de Groot, Philip; Lakos, Gabriella; Lambert, Marc; Meroni, Pierluigi; Ortel, Thomas L; Petri, Michelle; Rahman, Anisur; Roubey, Robert; Sciascia, Savino; Snyder, Melissa; Tebo, Anne E; Tincani, Angela; Willis, Rohan

2014-09-01

Current classification criteria for definite Antiphospholipid Syndrome (APS) require the use of three laboratory assays to detect antiphospholipid antibodies (aCL, anti-β2GPI and LA) in the presence of at least one of the two major clinical manifestations (i.e. thrombosis or pregnancy morbidity) of the syndrome. However, several other autoantibodies shown to be directed to other proteins or their complex with phospholipids have been proposed to be relevant to APS but their clinical utility and their diagnostic value remains elusive. This report summarizes the findings, conclusions and recommendations of the "APS Task Force 3-Laboratory Diagnostics and Trends" meeting that took place during the 14th International Congress on Antiphospholipid Antibodies (APLA 2013, September 18-21, Rio de Janeiro, RJ, Brazil). Copyright © 2014 Elsevier B.V. All rights reserved.

VizieR Online Data Catalog: Virial analysis of the dense cores in Orion A (Kirk+, 2017)

NASA Astrophysics Data System (ADS)

Kirk, H.; Friesen, R. K.; Pineda, J. E.; Rosolowsky, E.; Offner, S. S. R.; Matzner, C. D.; Myers, P. C.; di, Francesco J.; Caselli, P.; Alves, F. O.; Chacon-Tanarro, A.; Chen, H.-H.; Chen, M. C.-Y.; Keown, J.; Punanova, A.; Seo, Y. M.; Shirley, Y.; Ginsburg, A.; Hall, C.; Singh, A.; Arce, H. G.; Goodman, A. A.; Martin, P.; Redaelli, E.

2018-05-01

NH3 observations were obtained through the Green Bank Ammonia (GAS), a large project to map the ammonia (1,1), (2,2), and (3,3) rotation-inversion transitions across the high-extinction regions of nearby Gould Belt molecular clouds using the Green Bank Telescope's K-band Focal Plane Array. The survey strategy, data reduction procedure, and basic data properties are described in detail in Friesen+ (2017ApJ...843...63F). The spatial resolution is 32" (0.064pc), while the spectral resolution is 0.07km/s. For our analysis, we use the dense core catalog presented in Lane+ (2016ApJ...833...44L), which covers the entire Orion A complex with the JCMT SCUBA-2 850um. See section 2.2. (1 data file).

Function of multiple Lis-Homology domain/WD-40 repeat-containing proteins in feed-forward transcriptional repression by silencing mediator for retinoic and thyroid receptor/nuclear receptor corepressor complexes.

PubMed

Choi, Hyo-Kyoung; Choi, Kyung-Chul; Kang, Hee-Bum; Kim, Han-Cheon; Lee, Yoo-Hyun; Haam, Seungjoo; Park, Hyoung-Gi; Yoon, Ho-Geun

2008-05-01

Lis-homology (LisH) motifs are involved in protein dimerization, and the discovery of the conserved N-terminal LisH domain in transducin beta-like protein 1 and its receptor (TBL1 and TBLR1) led us to examine the role of this domain in transcriptional repression. Here we show that multiple beta-transducin (WD-40) repeat-containing proteins interact to form oligomers in solution and that oligomerization depends on the presence of the LisH domain in each protein. Repression of transcription, as assayed using Gal4 fusion proteins, also depended on the presence of the LisH domain, suggesting that oligomerization is a prerequisite for efficient transcriptional repression. Furthermore, we show that the LisH domain is responsible for the binding to the hypoacetylated histone H4 tail and for stable chromatin targeting by the nuclear receptor corepressor complex. Mutations in conserved residues in the LisH motif of TBL1 and TBLR1 block histone binding, oligomerization, and transcriptional repression, supporting the functional importance of the LisH motif in transcriptional repression. Our results indicate that another WD-40 protein, TBL3, also preferentially binds to the N-terminal domain of TBL1 and TBLR1, and forms oligomers with other WD-40 proteins. Finally, we observed that the WD-40 proteins RbAp46 and RbAp48 of the sin3A corepressor complex failed to dimerize. We also found the specific interaction UbcH/E2 with TBL1, but not RbAp46/48. Altogether, our results thus indicate that the presence of multiple LisH/WD-40 repeat containing proteins is exclusive to nuclear receptor corepressor/ silencing mediator for retinoic and thyroid receptor complexes compared with other class 1 histone deacetylase-containing corepessor complexes.

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

PubMed Central

Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2.

PubMed

Lo, Te-Wen; Bennett, Daniel C; Goodman, S Jay; Stern, Michael J

2010-06-01

The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

Cell-fate determination by ubiquitin-dependent regulation of translation.

PubMed

Werner, Achim; Iwasaki, Shintaro; McGourty, Colleen A; Medina-Ruiz, Sofia; Teerikorpi, Nia; Fedrigo, Indro; Ingolia, Nicholas T; Rape, Michael

2015-09-24

Metazoan development depends on the accurate execution of differentiation programs that allow pluripotent stem cells to adopt specific fates. Differentiation requires changes to chromatin architecture and transcriptional networks, yet whether other regulatory events support cell-fate determination is less well understood. Here we identify the ubiquitin ligase CUL3 in complex with its vertebrate-specific substrate adaptor KBTBD8 (CUL3(KBTBD8)) as an essential regulator of human and Xenopus tropicalis neural crest specification. CUL3(KBTBD8) monoubiquitylates NOLC1 and its paralogue TCOF1, the mutation of which underlies the neurocristopathy Treacher Collins syndrome. Ubiquitylation drives formation of a TCOF1-NOLC1 platform that connects RNA polymerase I with ribosome modification enzymes and remodels the translational program of differentiating cells in favour of neural crest specification. We conclude that ubiquitin-dependent regulation of translation is an important feature of cell-fate determination.

Arabidopsis SEPALLATA proteins differ in cooperative DNA-binding during the formation of floral quartet-like complexes

PubMed Central

Jetha, Khushboo; Theißen, Günter; Melzer, Rainer

2014-01-01

The SEPALLATA (SEP) genes of Arabidopsis thaliana encode MADS-domain transcription factors that specify the identity of all floral organs. The four Arabidopsis SEP genes function in a largely yet not completely redundant manner. Here, we analysed interactions of the SEP proteins with DNA. All of the proteins were capable of forming tetrameric quartet-like complexes on DNA fragments carrying two sequence elements termed CArG-boxes. Distances between the CArG-boxes for strong cooperative DNA-binding were in the range of 4–6 helical turns. However, SEP1 also bound strongly to CArG-box pairs separated by smaller or larger distances, whereas SEP2 preferred large and SEP4 preferred small inter-site distances for binding. Cooperative binding of SEP3 was comparatively weak for most of the inter-site distances tested. All SEP proteins constituted floral quartet-like complexes together with the floral homeotic proteins APETALA3 (AP3) and PISTILLATA (PI) on the target genes AP3 and SEP3. Our results suggest an important part of an explanation for why the different SEP proteins have largely, but not completely redundant functions in determining floral organ identity: they may bind to largely overlapping, but not identical sets of target genes that differ in the arrangement and spacing of the CArG-boxes in their cis-regulatory regions. PMID:25183521