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Sample records for adaptor protein complexes

  1. 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

  2. Identification of Cargo for Adaptor Protein (AP) Complexes 3 and 4 by Sucrose Gradient Profiling.

    PubMed

    Pertl-Obermeyer, Heidi; Wu, Xu Na; Schrodt, Jens; Müdsam, Christina; Obermeyer, Gerhard; Schulze, Waltraud X

    2016-09-01

    Intracellular vesicle trafficking is a fundamental process in eukaryotic cells. It enables cellular polarity and exchange of proteins between subcellular compartments such as the plasma membrane or the vacuole. Adaptor protein complexes participate in the vesicle formation by specific selection of the transported cargo. We investigated the role of the adaptor protein complex 3 (AP-3) and adaptor protein complex 4 (AP-4) in this selection process by screening for AP-3 and AP-4 dependent cargo proteins. Specific cargo proteins are expected to be mis-targeted in knock-out mutants of adaptor protein complex components. Thus, we screened for altered distribution profiles across a density gradient of membrane proteins in wild type versus ap-3β and ap-4β knock-out mutants. In ap-3β mutants, especially proteins with transport functions, such as aquaporins and plasma membrane ATPase, as well as vesicle trafficking proteins showed differential protein distribution profiles across the density gradient. In the ap-4β mutant aquaporins but also proteins from lipid metabolism were differentially distributed. These proteins also showed differential phosphorylation patterns in ap-3β and ap-4β compared with wild type. Other proteins, such as receptor kinases were depleted from the AP-3 mutant membrane system, possibly because of degradation after mis-targeting. In AP-4 mutants, membrane fractions were depleted for cytochrome P450 proteins, cell wall proteins and receptor kinases. Analysis of water transport capacity in wild type and mutant mesophyll cells confirmed aquaporins as cargo proteins of AP-3 and AP-4. The combination of organelle density gradients with proteome analysis turned out as a suitable experimental strategy for large-scale analyses of protein trafficking.

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

    SciTech Connect

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

    2005-05-13

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

  4. Roles of BLOC-1 and Adaptor Protein-3 Complexes in Cargo Sorting to Synaptic Vesicles

    PubMed Central

    Newell-Litwa, Karen; Salazar, Gloria; Smith, Yoland

    2009-01-01

    Neuronal lysosomes and their biogenesis mechanisms are primarily thought to clear metabolites and proteins whose abnormal accumulation leads to neurodegenerative disease pathology. However, it remains unknown whether lysosomal sorting mechanisms regulate the levels of membrane proteins within synaptic vesicles. Using high-resolution deconvolution microscopy, we identified early endosomal compartments where both selected synaptic vesicle and lysosomal membrane proteins coexist with the adaptor protein complex 3 (AP-3) in neuronal cells. From these early endosomes, both synaptic vesicle membrane proteins and characteristic AP-3 lysosomal cargoes can be similarly sorted to brain synaptic vesicles and PC12 synaptic-like microvesicles. Mouse knockouts for two Hermansky–Pudlak complexes involved in lysosomal biogenesis from early endosomes, the ubiquitous isoform of AP-3 (Ap3b1−/−) and muted, defective in the biogenesis of lysosome-related organelles complex 1 (BLOC-1), increased the content of characteristic synaptic vesicle proteins and known AP-3 lysosomal proteins in isolated synaptic vesicle fractions. These phenotypes contrast with those of the mouse knockout for the neuronal AP-3 isoform involved in synaptic vesicle biogenesis (Ap3b2−/−), in which the content of select proteins was reduced in synaptic vesicles. Our results demonstrate that lysosomal and lysosome-related organelle biogenesis mechanisms regulate steady-state synaptic vesicle protein composition from shared early endosomes. PMID:19144828

  5. 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.

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

    PubMed

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

    2016-06-30

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

  7. Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons

    PubMed Central

    Kyung, Jae Won; Cho, In Ha; Lee, Sukmook; Song, Woo Keun; Ryan, Timothy A.; Hoppa, Michael B.; Kim, Sung Hyun

    2017-01-01

    The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment. PMID:28139716

  8. The small G-protein Arf6GTP recruits the AP-2 adaptor complex to membranes.

    PubMed

    Paleotti, Olivia; Macia, Eric; Luton, Frederic; Klein, Stephanie; Partisani, Mariagrazia; Chardin, Pierre; Kirchhausen, Tom; Franco, Michel

    2005-06-03

    The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

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

    PubMed

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

    2011-06-10

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

  10. Identification of an adaptor protein that facilitates Nrf2-Keap1 complex formation and modulates antioxidant response.

    PubMed

    Zhang, Yuxue; Hou, Yongfan; Liu, Chunchun; Li, Yinlong; Guo, Weiwei; Wu, Jiu-Lin; Xu, Daqian; You, Xue; Pan, Yi; Chen, Yan

    2016-08-01

    Nrf2 plays a key role in the protection of the body against environmental stress via inducible expression of detoxification and antioxidant enzymes. Keap1 functions as a sensor for oxidative and electrophilic stresses and promotes Nrf2 degradation via its E3 ligase activity. Modulation of the Nrf2-Keap1 pathway has been extensively explored as a strategy to combat against drug toxicity and stress-induced diseases. Here we report a new player that modulates the Nrf2-Keap1 pathway. PAQR3, a membrane protein specifically localized in the Golgi apparatus, negatively regulates the expression of an array of Nrf2 target genes and alters cellular level of reactive oxygen species. PAQR3 tethers Nrf2 and Keap1, but not small MAF proteins to the Golgi apparatus. PAQR3 interacts with both Nrf2 and Keap1 and facilitates the interaction of Nrf2 with Keap1. PAQR3 promotes ubiquitination and degradation of Nrf2. Disruption of PAQR3 interaction with Nrf2 and Keap1 by a synthetic peptide reduces Nrf2 ubiquitination and elevates expression of Nrf2 target genes. At the animal level, deletion of PAQR3 increases Nrf2 protein level and the expression of Nrf2 target genes. In conclusion, our study pinpoints that PAQR3 functions as an adaptor protein to promote Nrf2-Keap1 complex formation, thereby modulating the Nrf2-Keap2 pathway and playing an important role in controlling antioxidant response of the cell.

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

    PubMed

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

    2015-12-25

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

  12. 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

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

    PubMed

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

    2015-08-28

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

  14. Targeting signals and subunit interactions in coated vesicle adaptor complexes

    PubMed Central

    1995-01-01

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma- adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane. PMID:7593184

  15. Studying multisite binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: application to adaptor protein complexes in cell signaling.

    PubMed

    Houtman, Jon C D; Brown, Patrick H; Bowden, Brent; Yamaguchi, Hiroshi; Appella, Ettore; Samelson, Lawrence E; Schuck, Peter

    2007-01-01

    Multisite interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and is frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multisite interactions, implemented as part of the public domain multimethod analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multisite interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multiprotein interactions, we have examined the interactions of three proteins that are critical for signal transduction after T-cell activation, LAT, Grb2, and Sos1. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multiprotein complexes important for T-cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.

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

    PubMed

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

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

  17. Adaptor protein ARH is recruited to the plasma membrane by low density lipoprotein (LDL) binding and modulates endocytosis of the LDL/LDL receptor complex in hepatocytes.

    PubMed

    Sirinian, Maria Isabella; Belleudi, Francesca; Campagna, Filomena; Ceridono, Mara; Garofalo, Tina; Quagliarini, Fabiana; Verna, Roberto; Calandra, Sebastiano; Bertolini, Stefano; Sorice, Maurizio; Torrisi, Maria Rosaria; Arca, Marcello

    2005-11-18

    ARH is a newly discovered adaptor protein required for the efficient activity of low density lipoprotein receptor (LDLR) in selected tissues. Individuals lacking ARH have severe hypercholesterolemia due to an impaired hepatic clearance of LDL. It has been demonstrated that ARH is required for the efficient internalization of the LDL-LDLR complex and to stabilize the association of the receptor with LDL in Epstein-Barr virus-immortalized B lymphocytes. However, little information is available on the role of ARH in liver cells. Here we provide evidence that ARH is codistributed with LDLR on the basolateral area in confluent HepG2-polarized cells. This distribution is not modified by the overexpression of LDLR. Conversely, the activation of the LDLR-mediated endocytosis, but not the binding of LDL to LDLR, promotes a significant colocalization of ARH with LDL-LDLR complex that peaked at 2 min at 37 degrees C. To further assess the role of ARH in LDL-LDLR complex internalization, we depleted ARH protein using the RNA interference technique. Twenty-four hours after transfection with ARH-specific RNA interference, ARH protein was depleted in HepG2 cells by more than 70%. Quantitative immunofluorescence analysis revealed that the depletion of ARH caused about 80% reduction in LDL internalization. Moreover, our findings indicate that ARH is associated with other proteins of the endocytic machinery. We suggest that ARH is an endocytic sorting adaptor that actively participates in the internalization of the LDL-LDLR complex, possibly enhancing the efficiency of its packaging into the endocytic vesicles.

  18. Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

    PubMed Central

    Berlioz-Torrent, Clarisse; Shacklett, Barbara L.; Erdtmann, Lars; Delamarre, Lelia; Bouchaert, Isabelle; Sonigo, Pierre; Dokhelar, Marie Christine; Benarous, Richard

    1999-01-01

    The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved. PMID:9882340

  19. The Adaptor Complex AP-4 Regulates Vacuolar Protein Sorting at the trans-Golgi Network by Interacting with VACUOLAR SORTING RECEPTOR1.

    PubMed

    Fuji, Kentaro; Shirakawa, Makoto; Shimono, Yuki; Kunieda, Tadashi; Fukao, Yoichiro; Koumoto, Yasuko; Takahashi, Hideyuki; Hara-Nishimura, Ikuko; Shimada, Tomoo

    2016-01-01

    Adaptor protein (AP) complexes play critical roles in protein sorting among different post-Golgi pathways by recognizing specific cargo protein motifs. Among the five AP complexes (AP-1-AP-5) in plants, AP-4 is one of the most poorly understood; the AP-4 components, AP-4 cargo motifs, and AP-4 functional mechanism are not known. Here, we identify the AP-4 components and show that the AP-4 complex regulates receptor-mediated vacuolar protein sorting by recognizing VACUOLAR SORTING RECEPTOR1 (VSR1), which was originally identified as a sorting receptor for seed storage proteins to target protein storage vacuoles in Arabidopsis (Arabidopsis thaliana). From the vacuolar sorting mutant library GREEN FLUORESCENT SEED (GFS), we isolated three gfs mutants that accumulate abnormally high levels of VSR1 in seeds and designated them as gfs4, gfs5, and gfs6. Their responsible genes encode three (AP4B, AP4M, and AP4S) of the four subunits of the AP-4 complex, respectively, and an Arabidopsis mutant (ap4e) lacking the fourth subunit, AP4E, also had the same phenotype. Mass spectrometry demonstrated that these four proteins form a complex in vivo. The four mutants showed defects in the vacuolar sorting of the major storage protein 12S globulins, indicating a role for the AP-4 complex in vacuolar protein transport. AP4M bound to the tyrosine-based motif of VSR1. AP4M localized at the trans-Golgi network (TGN) subdomain that is distinct from the AP-1-localized TGN subdomain. This study provides a novel function for the AP-4 complex in VSR1-mediated vacuolar protein sorting at the specialized domain of the TGN.

  20. Recruitment of the adaptor protein 2 complex by the human immunodeficiency virus type 2 envelope protein is necessary for high levels of virus release.

    PubMed

    Noble, Beth; Abada, Paolo; Nunez-Iglesias, Juan; Cannon, Paula M

    2006-03-01

    The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxxtheta) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxxtheta motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxxtheta motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxxtheta motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxxtheta motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can

  1. Recruitment of the Adaptor Protein 2 Complex by the Human Immunodeficiency Virus Type 2 Envelope Protein Is Necessary for High Levels of Virus Release†

    PubMed Central

    Noble, Beth; Abada, Paolo; Nunez-Iglesias, Juan; Cannon, Paula M.

    2006-01-01

    The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxxθ) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxxθ motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxxθ motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxxθ motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxxθ motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can functionally substitute

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

    SciTech Connect

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

    2010-10-08

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

  3. Protein Interaction Profiling of the p97 Adaptor UBXD1 Points to a Role for the Complex in Modulating ERGIC-53 Trafficking*

    PubMed Central

    Haines, Dale S.; Lee, J. Eugene; Beauparlant, Stephen L.; Kyle, Dane B.; den Besten, Willem; Sweredoski, Michael J.; Graham, Robert L. J.; Hess, Sonja; Deshaies, Raymond J.

    2012-01-01

    UBXD1 is a member of the poorly understood subfamily of p97 adaptors that do not harbor a ubiquitin association domain or bind ubiquitin-modified proteins. Of clinical importance, p97 mutants found in familial neurodegenerative conditions Inclusion Body Myopathy Paget's disease of the bone and/or Frontotemporal Dementia and Amyotrophic Lateral Sclerosis are defective at interacting with UBXD1, indicating that functions regulated by a p97-UBXD1 complex are altered in these diseases. We have performed liquid chromatography-mass spectrometric analysis of UBXD1-interacting proteins to identify pathways in which UBXD1 functions. UBXD1 displays prominent association with ERGIC-53, a hexameric type I integral membrane protein that functions in protein trafficking. The UBXD1-ERGIC-53 interaction requires the N-terminal 10 residues of UBXD1 and the C-terminal cytoplasmic 12 amino acid tail of ERGIC-53. Use of p97 and E1 enzyme inhibitors indicate that complex formation between UBXD1 and ERGIC-53 requires the ATPase activity of p97, but not ubiquitin modification. We also performed SILAC-based quantitative proteomic profiling to identify ERGIC-53 interacting proteins. This analysis identified known (e.g. COPI subunits) and novel (Rab3GAP1/2 complex involved in the fusion of vesicles at the cell membrane) interactions that are also mediated through the C terminus of the protein. Immunoprecipitation and Western blotting analysis confirmed the proteomic interaction data and it also revealed that an UBXD1-Rab3GAP association requires the ERGIC-53 binding domain of UBXD1. Localization studies indicate that UBXD1 modules the sub-cellular trafficking of ERGIC-53, including promoting movement to the cell membrane. We propose that p97-UBXD1 modulates the trafficking of ERGIC-53-containing vesicles by controlling the interaction of transport factors with the cytoplasmic tail of ERGIC-53. PMID:22337587

  4. Crystal structure of human programmed cell death 10 complexed with inositol-(1,3,4,5)-tetrakisphosphate: a novel adaptor protein involved in human cerebral cavernous malformation.

    PubMed

    Ding, Jingjin; Wang, Xiaoyan; Li, De-Feng; Hu, Yonglin; Zhang, Ying; Wang, Da-Cheng

    2010-09-03

    Programmed cell death 10 (PDCD10) is a novel adaptor protein involved in human cerebral cavernous malformation, a common vascular lesion mostly occurring in the central nervous system. By interacting with different signal proteins, PDCD10 could regulate various physiological processes in the cell. The crystal structure of human PDCD10 complexed with inositol-(1,3,4,5)-tetrakisphosphate has been determined at 2.3A resolution. The structure reveals an integrated dimer via a unique assembly that has never been observed before. Each PDCD10 monomer contains two independent domains: an N-terminal domain with a new fold involved in the tight dimer assembly and a C-terminal four-helix bundle domain that closely resembles the focal adhesion targeting domain of focal adhesion kinase. An eight-residue flexible linker connects the two domains, potentially conferring mobility onto the C-terminal domain, resulting in the conformational variability of PDCD10. A variable basic cleft on the top of the dimer interface binds to phosphatidylinositide and regulates the intracellular localization of PDCD10. Two potential sites, respectively located on the two domains, are critical for recruiting different binding partners, such as germinal center kinase III proteins and the focal adhesion protein paxillin.

  5. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

    PubMed

    Carty, Michael; Goodbody, Rory; Schröder, Martina; Stack, Julianne; Moynagh, Paul N; Bowie, Andrew G

    2006-10-01

    Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

  6. Modulation of lipoprotein receptor functions by intracellular adaptor proteins.

    PubMed

    Stolt, Peggy C; Bock, Hans H

    2006-10-01

    Members of the low density lipoprotein (LDL) receptor gene family are critically involved in a wide range of physiological processes including lipid and vitamin homeostasis, cellular migration, neurodevelopment, and synaptic plasticity, to name a few. Lipoprotein receptors exert these diverse biological functions by acting as cellular uptake receptors or by inducing intracellular signaling cascades. It was discovered that a short sequence in the intracellular region of all lipoprotein receptors, Asn-Pro-X-Tyr (NPXY) is important for mediating either endocytosis or signal transduction events, and that this motif serves as a binding site for phosphotyrosine-binding (PTB) domain containing scaffold proteins. These molecular adaptors connect the transmembrane receptors with the endocytosis machinery and regulate cellular trafficking, or function as assembly sites for dynamic multi-protein signaling complexes. Whereas the LDL receptor represents the archetype of an endocytic lipoprotein receptor, the structurally closely related apolipoprotein E receptor 2 (apoER2) and very low density lipoprotein (VLDL) receptor activate a kinase-dependent intracellular signaling cascade after binding to the neuronal signaling molecule Reelin. This review focuses on two related PTB domain containing adaptor proteins that mediate these divergent lipoprotein receptor responses, ARH (autosomal recessive hypercholesterolemia protein) and Dab1 (disabled-1), and discusses the structural and molecular basis of this different behaviour.

  7. Calcyon, a mammalian specific NEEP21 family member, interacts with adaptor protein complex 3 (AP-3) and regulates targeting of AP-3 cargoes.

    PubMed

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2012-10-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain and stimulates clathrin assembly and clathrin-mediated endocytosis. A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (μ) subunits interact with a YXXØ-type tyrosine motif located at residues 133-136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of μ3, and also impacted μ1 and μ2 binding to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with μ3A and μ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes.

  8. CALCYON, A MAMMALIAN SPECIFIC NEEP21 FAMILY MEMBER, INTERACTS WITH ADAPTOR PROTEIN COMPLEX 3 (AP-3) AND REGULATES TARGETING OF AP-3 CARGOES

    PubMed Central

    Muthusamy, Nagendran; Faundez, Victor; Bergson, Clare

    2013-01-01

    Calcyon is a neural enriched, single transmembrane protein that interacts with clathrin light chain (CLC) and stimulates clathrin assembly and clathrin mediated endocytosis (CME). A similar property is shared by the heterotetrameric adaptor protein (AP) complexes AP-1, AP-2, and AP-3 which recruit cargoes for insertion into clathrin coated transport vesicles. Here we report that AP medium (µ) subunits interact with a YXXØ-type tyrosine motif located at residues 133–136 in the cytoplasmic domain of calcyon. Site specific mutagenesis of the critical tyrosine and bulky hydrophobic residues tyrosine 133 and methionine 136 preferentially abrogated binding of the ubiquitous and neuronal isoforms of µ3, and also impacted µ1 and µ2 binding but to a lesser degree. The relevance of these interactions was explored in vivo using mice harboring null-alleles of calcyon. As seen in the mutagenesis studies, calcyon deletion in mice preferentially altered the subcellular distribution of AP-3 suggesting that calcyon could regulate membrane-bound pools of AP-3 and AP-3 function. To test this hypothesis, we focused on the hilar region of hippocampus, where levels of calcyon, AP-3, and AP-3 cargoes are abundant. We analyzed brain cryosections from control and calcyon null mice for zinc transporter 3 (ZnT3), and phosphatidylinositol-4-kinase type II alpha (PI4KIIα), two well-defined AP-3 cargoes. Confocal microscopy indicated that ZnT3 and PI4KIIα are significantly reduced in the hippocampal mossy fibers of calcyon knock-out brain, a phenotype previously described in AP-3 deficiencies. Altogether, our data suggest that calcyon directly interacts with µ3A and µ3B, and regulates the subcellular distribution of AP-3 and the targeting of AP-3 cargoes. PMID:22650988

  9. Direct interactions of adaptor protein complexes 1 and 2 with the copper transporter ATP7A mediate its anterograde and retrograde trafficking

    PubMed Central

    Yi, Ling; Kaler, Stephen G.

    2015-01-01

    ATP7A is a P-type ATPase in which diverse mutations lead to X-linked recessive Menkes disease or occipital horn syndrome. Recently, two previously unknown ATP7A missense mutations, T994I and P1386S, were shown to cause an isolated distal motor neuropathy without clinical or biochemical features of other ATP7A disorders. These mutant alleles cause subtle defects in ATP7A intracellular trafficking, resulting in preferential plasma membrane localization compared with wild-type ATP7A. We reported previously that ATP7AP1386S causes unstable insertion of the eighth and final transmembrane segment, preventing proper position of the carboxyl-terminal tail in a proportion of mutant molecules. Here, we utilize this and other naturally occurring and engineered mutant ATP7A alleles to identify mechanisms of normal ATP7A trafficking. We show that adaptor protein (AP) complexes 1 and 2 physically interact with ATP7A and that binding is mediated in part by a carboxyl-terminal di-leucine motif. In contrast to other ATP7A missense mutations, ATP7AP1386S partially disturbs interactions with both APs, leading to abnormal axonal localization in transfected NSC-34 motor neurons and altered calcium-signaling following glutamate stimulation. Our results imply that AP-1 normally tethers ATP7A at the trans-Golgi network in the somatodendritic segments of motor neurons and that alterations affecting the ATP7A carboxyl-terminal tail induce release of the copper transporter to the axons or axonal membranes. The latter effects are intensified by diminished interaction with AP-2, impeding ATP7A retrograde trafficking. Taken together, these findings further illuminate the normal molecular mechanisms of ATP7A trafficking and suggest a pathophysiological basis for ATP7A-related distal motor neuropathy. PMID:25574028

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

    PubMed

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

    2013-01-01

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

  11. Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

    PubMed Central

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

    2013-01-01

    Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth. PMID:24116125

  12. Adaptor Protein 1A Facilitates Dengue Virus Replication

    PubMed Central

    Yasamut, Umpa; Tongmuang, Nopprarat; Yenchitsomanus, Pa-thai; Junking, Mutita; Noisakran, Sansanee; Puttikhunt, Chunya; Chu, Justin Jang Hann; Limjindaporn, Thawornchai

    2015-01-01

    Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication. PMID:26090672

  13. Molecular cloning and characterization of GhAPm, a gene encoding the μ subunit of the clathrin-associated adaptor protein complex that is associated with cotton (Gossypium hirsutum) fiber development.

    PubMed

    Zhou, Tao; Zhang, Rui; Yang, Dawei; Guo, Sandui

    2011-06-01

    The clathrin-associated adaptor protein (AP) complexes are the primary clathrin adaptors that contribute to the formation of clathrin-coated vesicles (CCVs). The GhAPm gene (GenBank accession number: GU359054), which encodes the medium subunit of the AP complexes, was cloned from cotton by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA was 1590 bp in size and encoded an open reading frame (ORF) of 416 amino acids with a molecular weight of 46 kDa. The GhAPm protein shared 81-85% identity at the amino acid level with the AP complex μ subunits isolated from Vitis vinifera, Glycine max, Populus trichocarpa, Ricinus communis and Arabidopsis thaliana, respectively. The corresponding genomic DNA, containing eight exons and seven introns, was isolated and analyzed. Also, a 5'-flanking region was analyzed, and a group of putative cis-acting elements were identified. DNA gel blot analysis showed that there is only one GhAPm gene in the cotton genome. Real-time RT-PCR analysis revealed that GhAPm is expressed in the root, stem, leaf, petal, ovule, and fiber. However, the interesting finding is that GhAPm expression level was shown to increase steadily as the cotton fiber develops. In 30 DPA fibers, expression increases sharply and arrives at a peak then the expression levels decrease rapidly. Based on these data, we propose that GhAPm has a critical role in cotton membrane trafficking and fiber development.

  14. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

    PubMed

    Theos, Alexander C; Tenza, Danièle; Martina, José A; Hurbain, Ilse; Peden, Andrew A; Sviderskaya, Elena V; Stewart, Abigail; Robinson, Margaret S; Bennett, Dorothy C; Cutler, Daniel F; Bonifacino, Juan S; Marks, Michael S; Raposo, Graça

    2005-11-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

  15. Adaptor protein Nck1 interacts with p120 Ras GTPase-activating protein and regulates its activity.

    PubMed

    Ger, Marija; Zitkus, Zigmantas; Valius, Mindaugas

    2011-10-01

    Adaptor protein Nck1 binds a number of intracellular proteins and influences various signaling pathways. Here we show that Nck1 directly binds and activates the GTPase-activating protein of Ras (RasGAP), which is responsible for the down-regulation of Ras. The first and the third SH3 domains of Nck1 and the NH(2)-terminal proline-rich sequence of RasGAP contribute most to the complex formation causing direct molecular interaction between the two proteins. Cell adhesion to the substrate is obligatory for the Nck1 and RasGAP association, as cell detachment makes RasGAP incapable of associating with Nck1. This leads to the complex dissipation, decrease of RasGAP activity and the increase of H-Ras-GTP level in the detached cells. Our findings reveal unexpected feature of adaptor protein Nck1 as the regulator of RasGAP activity.

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

    PubMed Central

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

    2016-01-01

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

  17. The adaptor protein ARH escorts megalin to and through endosomes.

    PubMed

    Nagai, Masaaki; Meerloo, Timo; Takeda, Tetsuro; Farquhar, Marilyn Gist

    2003-12-01

    Megalin is an endocytic receptor that binds multiple ligands and is essential for many physiological processes such as brain development and uptake of proteins by the kidney tubule, yolk sac, and thyroid. The cytoplasmic tail of megalin contains two FXNPXY motifs. Autosomal recessive hypercholesterolemia (ARH) is an adaptor protein that binds to the FXNPXY motif of the low-density lipoprotein receptor as well as clathrin and AP-2. We found that ARH also binds to the first FXNPXY motif of megalin in two-hybrid, pull-down and coimmunoprecipitation assays. ARH colocalizes with megalin in clathrin coated pits and in recycling endosomes in the Golgi region. When cells are treated with nocodazole, the recycling endosomes containing megalin and ARH disperse. On internalization of megalin, ARH and megalin are first seen in clathrin coated pits followed by sequential localization in early endosomes and tubular recycling endosomes in the pericentriolar region followed by their reappearance at the cell surface. Expression of ARH in Madin-Darby canine kidney cells expressing megalin mini-receptors enhances megalin-mediated uptake of 125I-lactoferrin, a megalin ligand. These results show that ARH facilitates endocytosis of megalin, escorts megalin along its endocytic route and raise the possibility that transport through the endosomal system is selective and requires interaction with specific adaptor proteins.

  18. The interaction between the adaptor protein APS and Enigma is involved in actin organisation.

    PubMed

    Barrès, Romain; Gonzalez, Teresa; Le Marchand-Brustel, Yannick; Tanti, Jean-François

    2005-08-15

    APS (adaptor protein with PH and SH2 domains) is an adaptor protein phosphorylated by several tyrosine kinase receptors including the insulin receptor. To identify novel binding partners of APS, we performed yeast two-hybrid screening. We identified Enigma, a PDZ and LIM domain-containing protein that was previously shown to be associated with the actin cytoskeleton. In HEK 293 cells, Enigma interacted specifically with APS, but not with the APS-related protein SH2-B. This interaction required the NPTY motif of APS and the LIM domains of Enigma. In NIH-3T3 cells that express the insulin receptor, Enigma and APS were partially co-localised with F-actin in small ruffling structures. Insulin increased the complex formation between APS and Enigma and their co-localisation in large F-actin containing ruffles. While in NIH-3T3 and HeLa cells the co-expression of both Enigma and APS did not modify the actin cytoskeleton organisation, expression of Enigma alone led to the formation of F-actin clusters. Similar alteration in actin cytoskeleton organisation was observed in cells expressing both Enigma and APS with a mutation in the NPTY motif. These results identify Enigma as a novel APS-binding protein and suggest that the APS/Enigma complex plays a critical role in actin cytoskeleton organisation.

  19. Structure of the periplasmic adaptor protein from a major facilitator superfamily (MFS) multidrug efflux pump

    PubMed Central

    Hinchliffe, Philip; Greene, Nicholas P.; Paterson, Neil G.; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

    2014-01-01

    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. PMID:24996185

  20. 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

  1. 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.

  2. Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

    PubMed Central

    Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

    2005-01-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

  3. Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release

    PubMed Central

    da Silva, Elaine Zayas Marcelino; Freitas-Filho, Edismauro Garcia; de Souza-Júnior, Devandir Antonio; daSilva, Luis Lamberti Pinto; Jamur, Maria Celia

    2017-01-01

    Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs. PMID:28273137

  4. Interaction with the adaptor protein Shc prevents aberrant Erk activation in the absence of extracellular stimulus

    PubMed Central

    Suen, Kin Man; Lin, Chi-Chuan; George, Roger; Melo, Fernando A.; Biggs, Eleanor R.; Ahmed, Zamal; Drake, Melanie N.; Arur, Swathi; Arold, Stefan T.; Ladbury, John E.

    2014-01-01

    Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a novel mechanism by which the adaptor protein Shc binds directly to the MAP-kinase Erk, preventing its activation in the absence of extracellular stimulus. The Shc–Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex is formed through unique binding sites on both the Shc PTB domain and N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc—induced through interaction with the phosphorylated receptor—releases Erk allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP-kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered as a tumor suppressor in human cells. PMID:23584453

  5. Characterization of the adaptor protein ARH expression in the brain and ARH molecular interactions.

    PubMed

    Mameza, Marie Germaine; Lockard, Jon M; Zamora, Eduardo; Hillefors, Mi; Lavina, Zeno Scotto; Kaplan, Barry B

    2007-11-01

    Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.

  6. Probing heterobivalent binding to the endocytic AP-2 adaptor complex by DNA-based spatial screening.

    PubMed

    Diezmann, F; von Kleist, L; Haucke, V; Seitz, O

    2015-08-07

    The double helical DNA scaffold offers a unique set of properties, which are particularly useful for studies of multivalency in biomolecular interactions: (i) multivalent ligand displays can be formed upon nucleic acid hybridization in a self-assembly process, which facilitates spatial screening (ii) valency and spatial arrangement of the ligand display can be precisely controlled and (iii) the flexibility of the ligand display can be adjusted by integrating nick sites and unpaired template regions. Herein we describe the use of DNA-based spatial screening for the characterization of the adaptor complex 2 (AP-2), a central interaction hub within the endocytic protein network in clathrin-mediated endocytosis. AP-2 is comprised of a core domain and two, so-called appendage domains, the α- and the β2-ear, which associate with cytoplasmatic proteins required for the formation or maturation of clathrin/AP-2 coated pits. Each appendage domain has two binding grooves which recognize distinct peptide motives with micromolar affinity. This provides opportunities for enhanced interactions with protein molecules that contain two (or more) different peptide motives. To determine whether a particular, spatial arrangement of binding motifs is required for high affinity binding we probed the distance-affinity relationships by means of DNA-programmed spatial screening with self-assembled peptide-DNA complexes. By using trimolecular and tetramolecular assemblies two different peptides were positioned in 2-22 nucleotide distance. The binding data obtained with both recombinant protein in well-defined buffer systems and native AP-2 in brain extract suggests that the two binding sites of the AP-2 α-appendage can cooperate to provide up to 40-fold enhancement of affinity compared to the monovalent interaction. The distance between the two recognized peptide motives was less important provided that the DNA duplex segments were connected by flexible, single strand segments. By

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  8. Artificial Neural Network for the Prediction of Tyrosine-Based Sorting Signal Recognition by Adaptor Complexes

    PubMed Central

    Mukherjee, Debarati; Hanna, Claudia B.; Aguilar, R. Claudio

    2012-01-01

    Sorting of transmembrane proteins to various intracellular compartments depends on specific signals present within their cytosolic domains. Among these sorting signals, the tyrosine-based motif (YXXØ) is one of the best characterized and is recognized by μ-subunits of the four clathrin-associated adaptor complexes (AP-1 to AP-4). Despite their overlap in specificity, each μ-subunit has a distinct sequence preference dependent on the nature of the X-residues. Moreover, combinations of these residues exert cooperative or inhibitory effects towards interaction with the various APs. This complexity makes it impossible to predict a priori, the specificity of a given tyrosine-signal for a particular μ-subunit. Here, we describe the results obtained with a computational approach based on the Artificial Neural Network (ANN) paradigm that addresses the issue of tyrosine-signal specificity, enabling the prediction of YXXØ-μ interactions with accuracies over 90%. Therefore, this approach constitutes a powerful tool to help predict mechanisms of intracellular protein sorting. PMID:22505811

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

    PubMed

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

    2015-08-27

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

  10. 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.

  11. The structure and polymerase-recognition mechanism of the crucial adaptor protein AND-1 in the human replisome.

    PubMed

    Guan, Chengcheng; Li, Jun; Sun, Dapeng; Liu, Yingfang; Liang, Huanhuan

    2017-04-05

    DNA replication in eukaryotic cells is performed by a multi-protein complex called the replisome, which consists of helicases, polymerases and adaptor molecules. Human acidic nucleoplasmic DNA-binding protein 1 (AND-1), also known as WD repeat and HMG-box DNA binding protein 1 (WDHD1), is an adaptor molecule crucial for DNA replication. While structural information for the AND-1 yeast ortholog is available, the mechanistic details for how human AND-1 protein anchors the lagging-strand DNA polymerase α (Pol α) to the DNA helicase complex (Cdc45-MCM2-7-GINS, CMG) await elucidation. Here, we report the structures of the N-terminal WD40 and SepB domains of human AND-1, as well as a biochemical analysis of the C-terminal HMG domain. We show that AND-1 exists as a homo-trimer mediated by the SepB domain. Mutant study results suggested that a positively charged groove within the SepB domain provides binding sites for Pol α. Different from its ortholog protein in budding yeast, human AND-1 is recruited to the CMG complex mediated by unknown participants other than GINS. In addition, we show that AND-1 binds to DNA in vitro, using its C-terminal HMG domain. In conclusion, our findings provide important insights into the mechanistic details of human AND-1 function, advancing our understanding of replisome formation during eukaryotic replication.

  12. 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.

  13. The Grb2 adaptor.

    PubMed

    Chardin, P; Cussac, D; Maignan, S; Ducruix, A

    1995-08-01

    Grb2 is an 'adaptor' protein made of one SH2 and two SH3 domains. The SH3 domains bind to prolinerich motifs in the C-terminal part of the ras exchange factor Sos. Binding of the Grb2 SH2 domain to phosphotyrosine motifs on receptors, or other adaptor proteins such as Shc, recruits this Grb2/Sos complex at the plasma membrane where Sos stimulates nucleotide exchange on ras, then ras activates raf and leads to MAP kinase activation. The structure of Grb2, the precise motifs recognised by its SH2 and SH3 domains, the way Grb2 performs its function, a possible regulation of its association with Sos, and its ability to complex with other proteins in vivo, are discussed.

  14. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex.

    PubMed

    Ortega, Davi R; Zhulin, Igor B

    2016-02-01

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  15. 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

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

    SciTech Connect

    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 linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a "phosphate sink" possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Altogether, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex.

  17. Evolutionary Genomics Suggests That CheV Is an Additional Adaptor for Accommodating Specific Chemoreceptors within the Chemotaxis Signaling Complex

    PubMed Central

    Ortega, Davi R.; Zhulin, Igor B.

    2016-01-01

    Escherichia coli and Salmonella enterica are models for many experiments in molecular biology including chemotaxis, and most of the results obtained with one organism have been generalized to another. While most components of the chemotaxis pathway are strongly conserved between the two species, Salmonella genomes contain some chemoreceptors and an additional protein, CheV, that are not found in E. coli. The role of CheV was examined in distantly related species Bacillus subtilis and Helicobacter pylori, but its role in bacterial chemotaxis is still not well understood. We tested a hypothesis that in enterobacteria CheV functions as an additional adaptor linking the CheA kinase to certain types of chemoreceptors that cannot be effectively accommodated by the universal adaptor CheW. Phylogenetic profiling, genomic context and comparative protein sequence analyses suggested that CheV interacts with specific domains of CheA and chemoreceptors from an orthologous group exemplified by the Salmonella McpC protein. Structural consideration of the conservation patterns suggests that CheV and CheW share the same binding spot on the chemoreceptor structure, but have some affinity bias towards chemoreceptors from different orthologous groups. Finally, published experimental results and data newly obtained via comparative genomics support the idea that CheV functions as a “phosphate sink” possibly to off-set the over-stimulation of the kinase by certain types of chemoreceptors. Overall, our results strongly suggest that CheV is an additional adaptor for accommodating specific chemoreceptors within the chemotaxis signaling complex. PMID:26844549

  18. The role of Tks adaptor proteins in invadopodia formation, growth and metastasis of melanoma

    PubMed Central

    Iizuka, Shinji; Abdullah, Christopher; Buschman, Matthew D.; Diaz, Begoña; Courtneidge, Sara A.

    2016-01-01

    Metastatic cancer cells are characterized by their ability to degrade and invade through extracellular matrix. We previously showed that the Tks adaptor proteins, Tks4 and Tks5, are required for invadopodia formation and/or function in Src-transformed fibroblasts and a number of human cancer cell types. In this study, we investigated the role of Tks adaptor proteins in melanoma cell invasion and metastasis. Knockdown of either Tks4 or Tks5 in both mouse and human melanoma cell lines resulted in a decreased ability to form invadopodia and degrade extracellular matrix. In addition, Tks-knockdown melanoma cells had decreased proliferation in a 3-dimensional type l collagen matrix, but not in 2-dimensional culture conditions. We also investigated the role of Tks proteins in melanoma progression in vivo using xenografts and experimental metastasis assays. Consistent with our in vitro results, reduction of Tks proteins markedly reduced subcutaneous melanoma growth as well as metastatic growth in the lung. We explored the clinical relevance of Tks protein expression in human melanoma specimens using a tissue microarray. Compared to non-malignant nevi, both Tks proteins were highly expressed in melanoma tissues. Moreover, metastatic melanoma cases showed higher expression of Tks5 than primary melanoma cases. Taken together, these findings suggest the importance of Tks adaptor proteins in melanoma growth and metastasis in vivo, likely via functional invadopodia formation. PMID:27802184

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Velazquez, Laura

    2012-12-01

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

  1. Science Signaling Podcast for 12 July 2016: Adaptor proteins limit signaling.

    PubMed

    Wiley, H Steven; VanHook, Annalisa M

    2016-07-12

    This Podcast features an interview with Steven Wiley, senior author of a Research Article that appears in the 12 July 2016 issue of Science Signaling, about how the abundance of adaptor proteins and feedback regulators affect the flow of information downstream of the epidermal growth factor receptor (EGFR). Information flows through a signaling pathway by sequential interactions between core components of the pathway, many of which have enzymatic activity. Adaptor proteins do not directly participate in relaying the signal and do not have enzymatic activity, but are important for signaling because they facilitate interactions between the core components. Using quantitative methods, Shi et al demonstrated that core components of the EGFR pathway were highly abundant in both normal cells and cancer cells. However, adaptor proteins were present in much lower abundance in both cell types, indicating that it is the abundance of these proteins that limit signaling downstream of EGFR. The authors also found that differences in EGFR signaling between different cell types likely resulted from the variable abundance of feedback regulators.Listen to Podcast.

  2. Crk and CrkL adaptor proteins: networks for physiological and pathological signaling

    PubMed Central

    Birge, Raymond B; Kalodimos, Charalampos; Inagaki, Fuyuhiko; Tanaka, Shinya

    2009-01-01

    The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses. PMID:19426560

  3. Identification of actin binding protein, ABP-280, as a binding partner of human Lnk adaptor protein.

    PubMed

    He, X; Li, Y; Schembri-King, J; Jakes, S; Hayashi, J

    2000-08-01

    Human Lnk (hLnk) is an adaptor protein with multiple functional domains that regulates T cell activation signaling. In order to identify cellular Lnk binding partners, a yeast two-hybrid screening of human spleen cDNA library was carried out using human hLnk as bait. A polypeptide sequence identical to the C-terminal segment of the actin binding protein (ABP-280) was identified as a hLnk binding protein. The expressed hLnk and the FLAG tagged C-terminal 673 amino acid residues of ABP-280 or the endogenous ABP-280 in COS-7 cells could be co-immunoprecipitated using antibodies either to hLnk, FLAG or ABP-280, respectively. Furthermore, immunofluorescence confocal microscope showed that hLnk and ABP-280 co-localized at the plasma membrane and at juxtanuclear region of COS-7 cells. In Jurkat cells, the endogenous hLnk also associates with the endogenous ABP-280 indicating that the association of these two proteins is physiological. The interacting domains of both proteins were mapped using yeast two-hybrid assays. Our results indicate that hLnk binds to the residues 2006-2454 (repeats 19-23C) of ABP-280. The domain in hLnk that associates with ABP-280 was mapped to an interdomain region of 56 amino acids between pleckstrin homology and Src homology 2 domains. These results suggest that hLnk may exert its regulatory role through its association with ABP-280.

  4. Tetraspan cargo adaptors usher GPI-anchored proteins into multivesicular bodies

    PubMed Central

    MacDonald, Chris; Stamnes, Mark A; Katzmann, David J; Piper, Robert C

    2015-01-01

    Ubiquitinated membrane proteins are sorted into intralumenal endosomal vesicles on their way for degradation in lysosomes. Here we summarize the discovery of the Cos proteins, which work to organize and segregate ubiquitinated cargo prior to its incorporation into intralumenal vesicles of the multivesicular body (MVB). Importantly, cargoes such as GPI-anchored proteins (GPI-APs) that cannot undergo ubiquitination, rely entirely on Cos proteins for sorting into intralumenal vesicles using the same pathway that depends on ESCRTs and ubiquitin ligases that typical polytopic membrane proteins do. Here we show Cos proteins provide functions as not only adaptor proteins for ubiquitin ligases, but also as cargo carriers that can physically usher a variety of other proteins into the MVB pathway. We then discuss the significance of this new sorting model and the broader implications for this cargo adaptor mechanism, whereby yeast Cos proteins, and their likely animal analogs, provide a ubiquitin sorting signal in trans to enable sorting of a membrane protein network into intralumenal vesicles. PMID:26505929

  5. The polarity protein Par3 regulates APP trafficking and processing through the endocytic adaptor protein Numb.

    PubMed

    Sun, Miao; Asghar, Suwaiba Z; Zhang, Huaye

    2016-09-01

    The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.

  6. A role for the adaptor proteins TRAM and TRIF in toll-like receptor 2 signaling.

    PubMed

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

    2015-02-06

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

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

    PubMed Central

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

    2016-01-01

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

  8. The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH.

    PubMed

    Maurer, Meghan E; Cooper, Jonathan A

    2006-10-15

    Clathrin-mediated endocytosis requires cargo-specific adaptor proteins that recognize specific receptors and recruit them into coated pits. ARH [also called low-density lipoprotein receptor (LDLR) adaptor protein] serves as an adaptor for LDLR endocytosis in liver. However, ARH is dispensable for LDL uptake by some other cell types. Here, we show that the adaptor Dab2 plays a major role in LDLR internalization in HeLa cells and fibroblasts. Dab2 mediates internalization of LDLRs but not transferrin receptors independently of ARH and the classic clathrin adaptor AP-2. If Dab2 is absent, ARH can mediate LDLR endocytosis, but its action requires AP-2. Furthermore, the rate of LDLR endocytosis is decreased when Dab2 is absent and Dab2, but not ARH, catalyzes the efficient clustering of LDLR into coated pits. Dab2 activity requires its binding to clathrin, LDLR and phospholipids. Dab2 is also involved in moving LDLRs off filopodia. We suggest that Dab2 is a cargo-specific endocytic adaptor protein, stably associating with phospholipids and clathrin to sort LDLR to nascent-coated pits, whereas ARH might accelerate later steps in LDLR endocytosis in cooperation with AP-2.

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

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

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

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

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

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

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

    PubMed Central

    Horn, Anselm H. C.; Sticht, Heinrich

    2015-01-01

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

  12. A highly versatile adaptor protein for the tethering of growth factors to gelatin-based biomaterials.

    PubMed

    Addi, Cyril; Murschel, Frédéric; Liberelle, Benoît; Riahi, Nesrine; De Crescenzo, Gregory

    2017-03-01

    In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed.

  13. Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1.

    PubMed

    Ren, Xuefeng; Farías, Ginny G; Canagarajah, Bertram J; Bonifacino, Juan S; Hurley, James H

    2013-02-14

    AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.

  14. The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins.

    PubMed

    Chum, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Malínský, Jan; Brdička, Tomáš; Cebecauer, Marek

    2016-01-01

    Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.

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

    PubMed

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

    2010-08-27

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

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

    SciTech Connect

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

    2009-06-19

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

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

    PubMed Central

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

    2003-01-01

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

  18. The Rai (Shc C) adaptor protein regulates the neuronal stress response and protects against cerebral ischemia

    PubMed Central

    Troglio, Flavia; Echart, Cinara; Gobbi, Alberto; Pawson, Tony; Pelicci, Pier Giuseppe; De Simoni, Maria Grazia; Pelicci, Giuliana

    2004-01-01

    Rai (Shc C or N-Shc) is a neuron-specific member of the family of Shc-like adaptor proteins. Rai functions in the cytoplasmic propagation of Ret-dependent survival signals and regulates, in vivo, the number of sympathetic neurons. We report here a function of Rai, i.e., the regulation of the neuronal adaptive response to environmental stresses. We demonstrate that (i) primary cultures of cortical neurons from Rai-/- mice are more sensitive to apoptosis induced by hypoxia or oxidative stress; (ii) in Rai-/- mice, ischemia/reperfusion injury induces severe neurological deficits, increased apoptosis and size of the infarct area, and significantly higher mortality; and (iii) Rai functions as a stress-response gene that increases phosphatidylinositol 3-kinase activation and Akt phosphorylation after hypoxic or oxidation insults. These data suggest that Rai has a functional neuroprotective role in brain injury, with possible implications in the treatment of stroke. PMID:15494442

  19. Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors.

    PubMed

    Pei, Jimin; Grishin, Nick V

    2012-03-01

    The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors.

  20. SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other.

    PubMed

    Maures, Travis J; Kurzer, Jason H; Carter-Su, Christin

    2007-01-01

    Src homology 2 (SH2) B adaptor protein 1 (SH2B1; originally named SH2-B) is a member of a family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase (JAK) and receptor tyrosine kinases. Although SH2B1 performs classical adaptor functions, such as recruitment of specific proteins to activated receptors, it also demonstrates a unique ability to enhance the kinase activity of the cytokine receptor-associated tyrosine kinase JAK2, as well as that of several receptor tyrosine kinases. SH2B1 is also among a small number of adaptor proteins shown to undergo nucleocytoplasmic shuttling, although its exact role within the nucleus is not yet clear. Deletion of the SH2B1 gene results in severe obesity and both leptin and insulin resistance, as well as infertility, which might be a consequence of resistance to insulin-like growth factor I. Thus, knockout mice support a role for SH2B1 as a positive regulator of JAK2 signaling pathways initiated by leptin, as well as of pathways initiated by insulin and, potentially, by insulin-like growth factor I.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2015-07-16

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

  3. 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

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

    PubMed Central

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

    2015-01-01

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

  5. ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2.

    PubMed

    He, Guocheng; Gupta, Sarita; Yi, Ming; Michaely, Peter; Hobbs, Helen H; Cohen, Jonathan C

    2002-11-15

    Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.

  6. The Pch2 AAA+ ATPase promotes phosphorylation of the Hop1 meiotic checkpoint adaptor in response to synaptonemal complex defects

    PubMed Central

    Herruzo, Esther; Ontoso, David; González-Arranz, Sara; Cavero, Santiago; Lechuga, Ana; San-Segundo, Pedro A.

    2016-01-01

    Meiotic cells possess surveillance mechanisms that monitor critical events such as recombination and chromosome synapsis. Meiotic defects resulting from the absence of the synaptonemal complex component Zip1 activate a meiosis-specific checkpoint network resulting in delayed or arrested meiotic progression. Pch2 is an evolutionarily conserved AAA+ ATPase required for the checkpoint-induced meiotic block in the zip1 mutant, where Pch2 is only detectable at the ribosomal DNA array (nucleolus). We describe here that high levels of the Hop1 protein, a checkpoint adaptor that localizes to chromosome axes, suppress the checkpoint defect of a zip1 pch2 mutant restoring Mek1 activity and meiotic cell cycle delay. We demonstrate that the critical role of Pch2 in this synapsis checkpoint is to sustain Mec1-dependent phosphorylation of Hop1 at threonine 318. We also show that the ATPase activity of Pch2 is essential for its checkpoint function and that ATP binding to Pch2 is required for its localization. Previous work has shown that Pch2 negatively regulates Hop1 chromosome abundance during unchallenged meiosis. Based on our results, we propose that, under checkpoint-inducing conditions, Pch2 also possesses a positive action on Hop1 promoting its phosphorylation and its proper distribution on unsynapsed chromosome axes. PMID:27257060

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2005-02-01

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

  9. Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

    PubMed

    Halova, Ivana; Draber, Petr

    2016-01-01

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

  10. Tyrosine phosphorylation-independent regulation of lipopolysaccharide-mediated response by the transmembrane adaptor protein LAB.

    PubMed

    Zhu, Minghua; Fuller, Deirdre M; Ou-Yang, Chih-wen; Sullivan, Sarah A; Zhang, Weiguo

    2012-03-15

    Linker for activation of B cells (LAB)/non-T cell activation linker is a transmembrane adaptor protein that functions in immunoreceptor-mediated signaling. Published studies have shown that LAB has both positive and negative roles in regulating TCR and high-affinity Fc receptor-mediated signaling and cellular function. In this study, we showed that LAB was also expressed in dendritic cells and that LAB deficiency affected LPS-mediated signaling and cytokine production. LPS-mediated MAPK activation was enhanced in LAB(-/-) bone marrow-derived dendritic cells. These bone marrow-derived dendritic cells also produced more TNF-α, IL-6, and IL-10 than wild-type cells. Moreover, LAB(-/-) mice were hyperresponsive to LPS-induced septic shock. These data indicated that LAB has a negative role in LPS-mediated responses. By using LAB knockin mice, which harbor mutations at five membrane-distal tyrosines, we further showed that, in contrast to its role in immunoreceptor-mediated signaling, LAB function in LPS-mediated signaling pathway did not depend on its tyrosine phosphorylation. Our study suggested a novel mechanism by which LAB functions in the regulation of innate immunity.

  11. The p66(Shc) adaptor protein controls oxidative stress response in early bovine embryos.

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. Bcl-XL cooperatively associates with the Bap31 complex in the endoplasmic reticulum, dependent on procaspase-8 and Ced-4 adaptor.

    PubMed

    Ng, F W; Shore, G C

    1998-02-06

    Bap31 is a polytopic integral membrane protein of the endoplasmic reticulum and forms a complex with Bcl-2/Bcl-XL and procaspase-8 (Ng, F. W. H., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, W. D., Cromlish, J. A., and Shore, G. C. (1997) J. Cell Biol. 139, 327-338). In co-transfected human cells, procaspase-8 is capable of interacting with Ced-4, an important adaptor molecule in Caenorhabditis elegans that binds to and activates the C. elegans procaspase, proCed-3. Here, we show that the predicted death effector homology domain within the cytosolic region of Bap31 interacts with Ced-4 and contributes to recruitment of procaspase-8. Bcl-XL, which binds directly but weakly to the polytopic transmembrane region of Bap31, indirectly and cooperatively associates with the Bap31 cytosolic domain, dependent on the presence of procaspase-8 and Ced-4. Ced-4Deltac does not interact with Bcl-XL but rather displaces it from Bap31, suggesting that an endogenous Ced-4-like adaptor is a normal constituent of the Bap31 complex and is required for stable association of Bcl-XL with Bap31 in vivo. These findings indicate that Bap31 is capable of recruiting essential components of a core death regulatory machinery.

  14. The SH2B1 adaptor protein associates with a proximal region of the erythropoietin receptor.

    PubMed

    Javadi, Mojib; Hofstätter, Edda; Stickle, Natalie; Beattie, Bryan K; Jaster, Robert; Carter-Su, Christin; Barber, Dwayne L

    2012-07-27

    Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1β. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1β preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.

  15. The endocytic adaptor protein ARH associates with motor and centrosomal proteins and is involved in centrosome assembly and cytokinesis.

    PubMed

    Lehtonen, Sanna; Shah, Mehul; Nielsen, Rikke; Iino, Noriaki; Ryan, Jennifer J; Zhou, Huilin; Farquhar, Marilyn G

    2008-07-01

    Numerous proteins involved in endocytosis at the plasma membrane have been shown to be present at novel intracellular locations and to have previously unrecognized functions. ARH (autosomal recessive hypercholesterolemia) is an endocytic clathrin-associated adaptor protein that sorts members of the LDL receptor superfamily (LDLR, megalin, LRP). We report here that ARH also associates with centrosomes in several cell types. ARH interacts with centrosomal (gamma-tubulin and GPC2 and GPC3) and motor (dynein heavy and intermediate chains) proteins. ARH cofractionates with gamma-tubulin on isolated centrosomes, and gamma-tubulin and ARH interact on isolated membrane vesicles. During mitosis, ARH sequentially localizes to the nuclear membrane, kinetochores, spindle poles and the midbody. Arh(-/-) embryonic fibroblasts (MEFs) show smaller or absent centrosomes suggesting ARH plays a role in centrosome assembly. Rat-1 fibroblasts depleted of ARH by siRNA and Arh(-/-) MEFs exhibit a slower rate of growth and prolonged cytokinesis. Taken together the data suggest that the defects in centrosome assembly in ARH depleted cells may give rise to cell cycle and mitotic/cytokinesis defects. We propose that ARH participates in centrosomal and mitotic dynamics by interacting with centrosomal proteins. Whether the centrosomal and mitotic functions of ARH are related to its endocytic role remains to be established.

  16. 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

  17. 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.

  18. Ste50 adaptor protein governs sexual differentiation of Cryptococcus neoformans via the pheromone response MAPK signaling pathway

    PubMed Central

    Jung, Kwang-Woo; Kim, Seo-Young; Okagaki, Laura H.; Nielsen, Kirsten; Bahn, Yong-Sun

    2010-01-01

    The mitogen-activated protein kinase (MAPK) pathways control diverse cellular functions in pathogenic fungi, including sexual differentiation, stress-response, and maintenance of cell wall integrity. Here we characterized a C. neoformans gene, which is homologous to the yeast Ste50 that is known to play an important role in mating pheromone response and stress response as an adaptor protein to the Ste11 MAPK kinase kinase in Saccharomyces cerevisiae. The C. neoformans Ste50 was not involved in any of the stress responses or virulence factor production (capsule and melanin) that are controlled by the HOG and Ras/cAMP signaling pathways. However, Ste50 was required for mating in both serotype A and serotype D C. neoformans strains. The ste50Δ mutant was completely defective in cell-cell fusion and mating pheromone production. Double mutation of the STE50 gene blocked increased production of pheromone and the hyper-filamentation phenotype of cells deleted of the CRG1 gene, which encodes the RGS protein that negatively regulates pheromone responsive G-protein signaling via the MAPK pathway. Regardless of the presence of the basidiomycota-specific SH3 domains of Ste50 that are known to be required for full virulence of Ustilago maydis, Ste50 was dispensable for virulence of C. neoformans in a murine model of cryptococcosis. In conclusion, the Ste50 adaptor protein controls sexual differentiation of C. neoformans via the pheromone-responsive MAPK pathway but is not required for virulence. PMID:20971202

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-14

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5(GTP)-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

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

  3. The modular adaptor protein autosomal recessive hypercholesterolemia (ARH) promotes low density lipoprotein receptor clustering into clathrin-coated pits.

    PubMed

    Garuti, Rita; Jones, Christopher; Li, Wei-Ping; Michaely, Peter; Herz, Joachim; Gerard, Robert D; Cohen, Jonathan C; Hobbs, Helen H

    2005-12-09

    Autosomal recessive hypercholesterolemia is characterized by a cell type-specific defect in low density lipoprotein receptor (LDLR) endocytosis. LDLR-mediated uptake of LDL is impaired in the liver, but not in fibroblasts of subjects with this disorder. The disease is caused by mutations in ARH, which encodes a putative adaptor protein that interacts with the cytoplasmic tail of the LDLR, phospholipids, and two components of the clathrin endocytic machinery, clathrin and adaptor protein-2 (AP-2) in vitro. To determine the physiological relevance of these interactions, we examined the effect of mutations in the ARH on LDLR location and function in polarized hepatocytes (WIF-B). The integrity of the FDNPVY sequence in the LDLR cytoplasmic tail was required for ARH-associated LDLR clustering into clathrin-coated pits. The phosphotyrosine binding domain of ARH plus either the clathrin box or the AP-2 binding region were required for both clustering and internalization of the LDLR. Parallel studies performed in vivo with the same recombinant forms of ARH in livers of Arh(-/-) mice confirmed the relevance of the cell culture findings. These results demonstrate that ARH must bind the LDLR tail and either clathrin or AP-2 to promote receptor clustering and internalization of LDL.

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

    DOE PAGES

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

    2015-07-06

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

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

    PubMed Central

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

    2015-01-01

    Cyclic GMP-AMP containing a unique combination of mixed phosphodiester linkages (2′3′-cGAMP) is an endogenous second messenger molecule that activates the type-I IFN pathway upon binding to the homodimer of the adaptor protein STING on the surface of endoplasmic reticulum membrane. However, the preferential binding of the asymmetric ligand 2′3′-cGAMP to the symmetric dimer of STING represents a physicochemical enigma. Here we show that 2′3′-cGAMP, but not its linkage isomers, adopts an organized free-ligand conformation that resembles the STING-bound conformation and pays low entropy and enthalpy costs in converting into the active conformation. Our results demonstrate that analyses of free-ligand conformations can be as important as analyses of protein conformations in understanding protein–ligand interactions. PMID:26150511

  6. Deficiency of the adaptor protein SLy1 results in a natural killer cell ribosomopathy affecting tumor clearance.

    PubMed

    Arefanian, Saeed; Schäll, Daniel; Chang, Stephanie; Ghasemi, Reza; Higashikubo, Ryuji; Zheleznyak, Alex; Guo, Yizhan; Yu, Jinsheng; Asgharian, Hosseinali; Li, Wenjun; Gelman, Andrew E; Kreisel, Daniel; French, Anthony R; Zaher, Hani; Plougastel-Douglas, Beatrice; Maggi, Leonard; Yokoyama, Wayne; Beer-Hammer, Sandra; Krupnick, Alexander S

    2016-01-01

    Individuals with robust natural killer (NK) cell function incur lower rates of malignancies. To expand our understanding of genetic factors contributing to this phenomenon, we analyzed NK cells from cancer resistant and susceptible strains of mice. We identified a correlation between NK levels of the X-chromosome-located adaptor protein SLy1 and immunologic susceptibility to cancer. Unlike the case for T or B lymphocytes, where SLy1 shuttles between the cytoplasm and nucleus to facilitate signal transduction, in NK cells SLy1 functions as a ribosomal protein and is located solely in the cytoplasm. In its absence, ribosomal instability results in p53-mediated NK cell senescence and decreased clearance of malignancies. NK defects are reversible under inflammatory conditions and viral clearance is not impacted by SLy1 deficiency. Our work defines a previously unappreciated X-linked ribosomopathy that results in a specific and subtle NK cell dysfunction leading to immunologic susceptibility to cancer.

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

    SciTech Connect

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

    2015-07-06

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

  8. Regulation of in vitro and in vivo immune functions by the cytosolic adaptor protein SKAP-HOM.

    PubMed

    Togni, M; Swanson, K D; Reimann, S; Kliche, S; Pearce, A C; Simeoni, L; Reinhold, D; Wienands, J; Neel, B G; Schraven, B; Gerber, A

    2005-09-01

    SKAP-HOM is a cytosolic adaptor protein representing a specific substrate for the Src family protein tyrosine kinase Fyn. Previously, several groups have provided experimental evidence that SKAP-HOM (most likely in cooperation with the cytosolic adaptor protein ADAP) is involved in regulating leukocyte adhesion. To further assess the physiological role of SKAP-HOM, we investigated the immune system of SKAP-HOM-deficient mice. Our data show that T-cell responses towards a variety of stimuli are unaffected in the absence of SKAP-HOM. Similarly, B-cell receptor (BCR)-mediated total tyrosine phosphorylation and phosphorylation of Erk, p38, and JNK, as well as immunoreceptor-mediated Ca(2+) responses, are normal in SKAP-HOM(-/-) animals. However, despite apparently normal membrane-proximal signaling events, BCR-mediated proliferation is strongly attenuated in the absence of SKAP-HOM(-/-). In addition, adhesion of activated B cells to fibronectin (a ligand for beta1 integrins) as well as to ICAM-1 (a ligand for beta2 integrins) is strongly reduced. In vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental autoimmune encephalomyelitis following immunization of mice with the encephalitogenic peptide of MOG (myelin oligodendrocyte glycoprotein). This is accompanied by strongly reduced serum levels of MOG-specific antibodies and lower MOG-specific T-cell responses. In summary, our data suggest that SKAP-HOM is required for proper activation of the immune system, likely by regulating the cross-talk between immunoreceptors and integrins.

  9. 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

  10. 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.

  11. Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

    PubMed

    Mehlitz, Adrian; Banhart, Sebastian; Mäurer, André P; Kaushansky, Alexis; Gordus, Andrew G; Zielecki, Julia; Macbeath, Gavin; Meyer, Thomas F

    2010-07-12

    Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

  12. DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

    PubMed Central

    Hoang, Ha Thi; Schlager, Max A.; Carter, Andrew P.

    2017-01-01

    Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility. PMID:28196890

  13. DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes.

    PubMed

    Hoang, Ha Thi; Schlager, Max A; Carter, Andrew P; Bullock, Simon L

    2017-02-28

    Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein's core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

  14. Inflammasome adaptor protein Apoptosis-associated speck-like protein containing CARD (ASC) is critical for the immune response and survival in west Nile virus encephalitis.

    PubMed

    Kumar, Mukesh; Roe, Kelsey; Orillo, Beverly; Muruve, Daniel A; Nerurkar, Vivek R; Gale, Michael; Verma, Saguna

    2013-04-01

    West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis in humans. The WNV-induced innate immune response, including production of antiviral cytokines, is critical for controlling virus infection. The adaptor protein ASC mediates a critical step in innate immune signaling by bridging the interaction between the pathogen recognition receptors and caspase 1 in inflammasome complexes, but its role in WNV immunopathogenesis is not defined. Here, we demonstrate that ASC is essential for interleukin-1β (IL-1β) production and development of effective host immunity against WNV. ASC-deficient mice exhibited increased susceptibility to WNV infection, and reduced survival was associated with enhanced virus replication in the peripheral tissues and central nervous system (CNS). Infection of cultured bone marrow-derived dendritic cells showed that ASC was essential for the activation of caspase 1, a key component of inflammasome assembly. ASC(-/-) mice exhibited attenuated levels of proinflammatory cytokines in the serum. Intriguingly, infected ASC(-/-) mice also displayed reduced levels of alpha interferon (IFN-α) and IgM in the serum, indicating the overall protective role of ASC in restricting WNV infection. However, brains from ASC(-/-) mice displayed unrestrained inflammation, including elevated levels of proinflammatory cytokines and chemokines, such as IFN-γ, CCL2, and CCL5, which correlated with more pronounced activation of the astrocytes, enhanced infiltration of peripheral immune cells in the CNS, and increased neuronal cell death. Collectively, our data provide new insights into the role of ASC as an essential modulator of inflammasome-dependent and -independent immune responses to effectively control WNV infection.

  15. Rap1-GTP-interacting Adaptor Molecule (RIAM) Protein Controls Invasion and Growth of Melanoma Cells*

    PubMed Central

    Hernández-Varas, Pablo; Coló, Georgina P.; Bartolomé, Ruben A.; Paterson, Andrew; Medraño-Fernández, Iria; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Sánchez-Mateos, Paloma; Lafuente, Esther M.; Boussiotis, Vassiliki A.; Strömblad, Staffan; Teixidó, Joaquin

    2011-01-01

    The Mig-10/RIAM/lamellipodin (MRL) family member Rap1-GTP-interacting adaptor molecule (RIAM) interacts with active Rap1, a small GTPase that is frequently activated in tumors such as melanoma and prostate cancer. We show here that RIAM is expressed in metastatic human melanoma cells and that both RIAM and Rap1 are required for BLM melanoma cell invasion. RIAM silencing in melanoma cells led to inhibition of tumor growth and to delayed metastasis in a severe combined immunodeficiency xenograft model. Defective invasion of RIAM-silenced melanoma cells arose from impairment in persistent cell migration directionality, which was associated with deficient activation of a Vav2-RhoA-ROCK-myosin light chain pathway. Expression of constitutively active Vav2 and RhoA in cells depleted for RIAM partially rescued their invasion, indicating that Vav2 and RhoA mediate RIAM function. These results suggest that inhibition of cell invasion in RIAM-silenced melanoma cells is likely based on altered cell contractility and cell polarization. Furthermore, we show that RIAM depletion reduces β1 integrin-dependent melanoma cell adhesion, which correlates with decreased activation of both Erk1/2 MAPK and phosphatidylinositol 3-kinase, two central molecules controlling cell growth and cell survival. In addition to causing inhibition of cell proliferation, RIAM silencing led to higher susceptibility to cell apoptosis. Together, these data suggest that defective activation of these kinases in RIAM-silenced cells could account for inhibition of melanoma cell growth and that RIAM might contribute to the dissemination of melanoma cells. PMID:21454517

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

    PubMed Central

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

    2016-01-01

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

  17. 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

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

    PubMed Central

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

    2015-01-01

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

  19. Signaling adaptor protein SH2B1 enhances neurite outgrowth and accelerates the maturation of human induced neurons.

    PubMed

    Hsu, Yi-Chao; Chen, Su-Liang; Wang, Ya-Jean; Chen, Yun-Hsiang; Wang, Dan-Yen; Chen, Linyi; Chen, Chia-Hsiang; Chen, Hwei-Hsien; Chiu, Ing-Ming

    2014-06-01

    Recent advances in somatic cell reprogramming have highlighted the plasticity of the somatic epigenome, particularly through demonstrations of direct lineage reprogramming of adult mouse and human fibroblasts to induced pluripotent stem cells (iPSCs) and induced neurons (iNs) under defined conditions. However, human cells appear to be less plastic and have a higher epigenetic hurdle for reprogramming to both iPSCs and iNs. Here, we show that SH2B adaptor protein 1β (SH2B1) can enhance neurite outgrowth of iNs reprogrammed from human fibroblasts as early as day 14, when combined with miR124 and transcription factors BRN2 and MYT1L (IBM) under defined conditions. These SH2B1-enhanced iNs (S-IBM) showed canonical neuronal morphology, and expressed multiple neuronal markers, such as TuJ1, NeuN, and synapsin, and functional proteins for neurotransmitter release, such as GABA, vGluT2, and tyrosine hydroxylase. Importantly, SH2B1 accelerated mature process of functional neurons and exhibited action potentials as early as day 14; without SH2B1, the IBM iNs do not exhibit action potentials until day 21. Our data demonstrate that SH2B1 can enhance neurite outgrowth and accelerate the maturation of human iNs under defined conditions. This approach will facilitate the application of iNs in regenerative medicine and in vitro disease modeling.

  20. 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.

  1. Adaptor protein disabled-2 modulates low density lipoprotein receptor synthesis in fibroblasts from patients with autosomal recessive hypercholesterolaemia.

    PubMed

    Eden, Emily R; Sun, Xi-Ming; Patel, Dilipkumar D; Soutar, Anne K

    2007-11-15

    Autosomal recessive hypercholesterolaemia (ARH), characterized clinically by severe inherited hypercholesterolaemia, is caused by recessive null mutations in LDLRAP1 (formerly ARH). Immortalized lymphocytes and monocyte-macrophages, and presumably hepatocytes, from ARH patients fail to take up and degrade plasma low density lipoproteins (LDL) because they lack LDLRAP1, a cargo-specific adaptor required for clathrin-mediated endocytosis of the LDL receptor. Surprisingly, LDL-receptor function is normal in ARH patients' skin fibroblasts in culture. Disabled-2 (Dab2) has been implicated previously in clathrin-mediated internalization of LDL-receptor family members, and we show here that Dab2 is highly expressed in skin fibroblasts, but not in lymphocytes. SiRNA-depletion of Dab2 profoundly reduced LDL-receptor activity in ARH fibroblasts as a result of profound reduction in LDL-receptor protein, but not mRNA; heterologous expression of murine Dab2 reversed this effect. In contrast, LDL-receptor protein content was unchanged in Dab-2-depleted control cells. Incorporation of 35S-labelled amino acids into LDL receptor protein revealed a corresponding apparent reduction in accumulation of newly synthesized LDL-receptor protein on depletion of Dab2 in ARH, but not in control, cells. This reduction in LDL-receptor protein in Dab2-depleted ARH cells could not be reversed by treatment of the cells with proteasomal or lysosomal inhibitors. Thus, we propose a novel role for Dab2 in ARH fibroblasts, where it is apparently required to allow normal translation of LDL receptor mRNA.

  2. New insight into the functions of the interleukin-17 receptor adaptor protein Act1 in psoriatic arthritis

    PubMed Central

    2012-01-01

    Recent genome-wide association studies have implicated the tumor necrosis factor receptor-associated factor 3-interacting protein 2 (TRAF3IP2) gene and its product, nuclear factor-kappa-B activator 1 (Act1), in the development of psoriatic arthritis (PsA). The high level of sequence homology of the TRAF3IP2 (Act1) gene across the animal kingdom and the presence of the Act1 protein in multiple cell types strongly suggest that the protein is of importance in normal cellular function. Act1 is an adaptor protein for the interleukin-17 (IL-17) receptor, and recent observations have highlighted the significance of IL-17 signaling and localized inflammation in autoimmune diseases. This review summarizes data from recent genome-wide association studies as well as immunological and molecular investigations of Act1. Together, these studies provide new insight into the role of IL-17 signaling in PsA. It is well established that IL-17 activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) signaling pathways normally leads to nuclear factor-kappa-B-mediated inflammation. However, the dominant PsA-associated TRAF3IP2 (Act1) gene single-nucleotide polymorphism (rs33980500) results in decreased binding of Act1 to TRAF6. This key mutation in Act1 could lead to a greater association of the IL-17 receptor with TRAF2/TRAF5 and this in turn suggests an alternative function for IL-17 in PsA. The recent observations described and discussed in this review raise the clinically significant possibility of redefining the immunological role of IL-17 in PsA and provide a basis for defining future studies to elucidate the molecular and cellular functions of Act1. PMID:23116200

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

    PubMed Central

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

    2015-01-01

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

  4. The modular adaptor protein ARH is required for low density lipoprotein (LDL) binding and internalization but not for LDL receptor clustering in coated pits.

    PubMed

    Michaely, Peter; Li, Wei-Ping; Anderson, Richard G W; Cohen, Jonathan C; Hobbs, Helen H

    2004-08-06

    ARH is an adaptor protein required for efficient endocytosis of low density lipoprotein (LDL) receptors (LDLRs) in selected tissues. Individuals lacking ARH (ARH-/-) have severe hypercholesterolemia due to impaired hepatic clearance of LDL. Immortalized lymphocytes, but not fibroblasts, from ARH-deficient subjects fail to internalize LDL. To further define the role of ARH in LDLR function, we compared the subcellular distribution of the LDLR in lymphocytes from normal and ARH-/- subjects. In normal lymphocytes LDLRs were predominantly located in intracellular compartments, whereas in ARH-/- cells the receptors were almost exclusively on the plasma membrane. Biochemical assays and quantification of LDLR by electron microscopy indicated that ARH-/- lymphocytes had >20-fold more LDLR on the cell surface and a approximately 27-fold excess of LDLR outside of coated pits. The accumulation of LDLR on the cell surface was not due to failure of receptors to localize in coated pits since the number of LDLRs in coated pits was similar in ARH-/- and normal cells. Despite the dramatic increase in cell surface receptors, LDL binding was only 2-fold higher in the ARH-/- lymphocytes. These findings indicate that ARH is required not only for internalization of the LDL.LDLR complex but also for efficient binding of LDL to the receptor and suggest that ARH stabilizes the associations of the receptor with LDL and with the invaginating portion of the budding pit, thereby increasing the efficiency of LDL internalization.

  5. 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

  6. Comparative analysis of adaptor-mediated clathrin assembly reveals general principles for adaptor clustering.

    PubMed

    Pucadyil, Thomas J; Holkar, Sachin S

    2016-10-15

    Clathrin-mediated endocytosis (CME) manages the sorting and uptake of the bulk of membrane proteins (or cargo) from the plasma membrane. CME is initiated by the formation of clathrin-coated pits (CCPs), in which adaptors nucleate clathrin assembly. Clathrin adaptors display diversity in both the type and number of evolutionarily conserved clathrin-binding boxes. How this diversity relates to the process of adaptor clustering as clathrin assembles around a growing pit remains unclear. Using real-time, fluorescence microscopy-based assays, we compare the formation kinetics and distribution of clathrin assemblies on membranes that display five unique clathrin adaptors. Correlations between equilibrium and kinetic parameters of clathrin assembly to the eventual adaptor distribution indicate that adaptor clustering is determined not by the amount of clathrin recruited or the degree of clathrin clustered but instead by the rate of clathrin assembly. Together our results emphasize the need to analyze kinetics of protein interactions to better understand mechanisms that regulate CME.

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

    PubMed

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

    2013-07-01

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

  8. 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.

  9. Endocytosis of megalin by visceral endoderm cells requires the Dab2 adaptor protein.

    PubMed

    Maurer, Meghan E; Cooper, Jonathan A

    2005-11-15

    Rapid endocytosis of lipoprotein receptors involves NPxY signals contained in their cytoplasmic tails. Several proteins, including ARH and Dab2, can bind these sequences, but their importance for endocytosis may vary in different cell types. The lipoprotein receptor megalin is expressed in the visceral endoderm (VE), a polarized epithelium that supplies maternal nutrients to the early mammalian embryo. Dab2 is also expressed in the VE, and is required for embryo growth and gastrulation. Here, we show that ARH is absent from the VE, and Dab2 is required for uptake of megalin, its co-receptor cubilin, and a cubilin ligand, transferrin, from the brush border of the VE into intracellular vesicles. By making isoform-specific knock-in mice, we show that the p96 splice form of Dab2, which binds endocytic proteins, can fully rescue endocytosis. The more abundant p67 isoform, which lacks some endocytic protein binding sites, only partly rescues endocytosis. Endocytosis of cubilin is also impaired in VE and in mid-gestation visceral yolk sac when p96 is absent. These studies suggest that Dab2 p96 mediates endocytosis of megalin in the VE. In addition, rescue of embryonic viability correlates with endocytosis, suggesting that endocytosis mediated by Dab2 is important for normal development.

  10. Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry.

    PubMed

    Snyder, Greg A; Deredge, Daniel; Waldhuber, Anna; Fresquez, Theresa; Wilkins, David Z; Smith, Patrick T; Durr, Susi; Cirl, Christine; Jiang, Jiansheng; Jennings, William; Luchetti, Timothy; Snyder, Nathaniel; Sundberg, Eric J; Wintrode, Patrick; Miethke, Thomas; Xiao, T Sam

    2014-01-10

    The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys(89) and Cys(134). A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.

  11. New function of the adaptor protein SH2B1 in brain-derived neurotrophic factor-induced neurite outgrowth.

    PubMed

    Shih, Chien-Hung; Chen, Chien-Jen; Chen, Linyi

    2013-01-01

    Neurite outgrowth is an essential process for the establishment of the nervous system. Brain-derived neurotrophic factor (BDNF) binds to its receptor TrkB and regulates axonal and dendritic morphology of neurons through signal transduction and gene expression. SH2B1 is a signaling adaptor protein that regulates cellular signaling in various physiological processes. The purpose of this study is to investigate the role of SH2B1 in the development of the central nervous system. In this study, we show that knocking down SH2B1 reduces neurite formation of cortical neurons whereas overexpression of SH2B1β promotes the development of hippocampal neurons. We further demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth and signaling using the established PC12 cells stably expressing TrkB, SH2B1β or SH2B1β mutants. Our data indicate that overexpressing SH2B1β enhances BDNF-induced MEK-ERK1/2, and PI3K-AKT signaling pathways. Inhibition of MEK-ERK1/2 and PI3K-AKT pathways by specific inhibitors suggest that these two pathways are required for SH2B1β-promoted BDNF-induced neurite outgrowth. Moreover, SH2B1β enhances BDNF-stimulated phosphorylation of signal transducer and activator of transcription 3 at serine 727. Finally, our data indicate that the SH2 domain and tyrosine phosphorylation of SH2B1β contribute to BDNF-induced signaling pathways and neurite outgrowth. Taken together, these findings demonstrate that SH2B1β promotes BDNF-induced neurite outgrowth through enhancing pathways involved MEK-ERK1/2 and PI3K-AKT.

  12. The adaptor protein SH2B3 (Lnk) negatively regulates neurite outgrowth of PC12 cells and cortical neurons.

    PubMed

    Wang, Tien-Cheng; Chiu, Hsun; Chang, Yu-Jung; Hsu, Tai-Yu; Chiu, Ing-Ming; Chen, Linyi

    2011-01-01

    SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

  13. 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.

  14. Dengue Virus Subverts Host Innate Immunity by Targeting Adaptor Protein MAVS

    PubMed Central

    He, Zhenjian; Zhu, Xun; Wen, Weitao; Yuan, Jie; Hu, Yiwen; Chen, Jiahui; An, Shu; Dong, Xinhuai; Lin, Cuiji; Yu, Jianchen; Wu, Jueheng; Yang, Yi; Cai, Junchao; Li, Jun

    2016-01-01

    ABSTRACT Dengue virus (DENV) is the most common mosquito-borne virus infecting humans and is currently a serious global health challenge. To establish infection in its host cells, DENV must subvert the production and/or antiviral effects of interferon (IFN). The aim of this study was to understand the mechanisms by which DENV suppresses IFN production. We determined that DENV NS4A interacts with mitochondrial antiviral signaling protein (MAVS), which was previously found to activate NF-κB and IFN regulatory factor 3 (IRF3), thus inducing type I IFN in the mitochondrion-associated endoplasmic reticulum membranes (MAMs). We further demonstrated that NS4A is associated with the N-terminal CARD-like (CL) domain and the C-terminal transmembrane (TM) domain of MAVS. This association prevented the binding of MAVS to RIG-I, resulting in the repression of RIG-I-induced IRF3 activation and, consequently, the abrogation of IFN production. Collectively, our findings illustrate a new molecular mechanism by which DENV evades the host immune system and suggest new targets for anti-DENV strategies. IMPORTANCE Type I interferon (IFN) constitutes the first line of host defense against invading viruses. To successfully establish infection, dengue virus (DENV) must counteract either the production or the function of IFN. The mechanism by which DENV suppresses IFN production is poorly understood and characterized. In this study, we demonstrate that the DENV NS4A protein plays an important role in suppressing interferon production through binding MAVS and disrupting the RIG-I–MAVS interaction in mitochondrion-associated endoplasmic reticulum membranes (MAMs). Our study reveals that MAVS is a novel host target of NS4A and provides a molecular mechanism for DENV evasion of the host innate immune response. These findings have important implications for understanding the pathogenesis of DENV and may provide new insights into using NS4A as a therapeutic and/or prevention target. PMID

  15. Cell-based Fluorescence Complementation Reveals a Role for HIV-1 Nef Protein Dimerization in AP-2 Adaptor Recruitment and CD4 Co-receptor Down-regulation.

    PubMed

    Shu, Sherry T; Emert-Sedlak, Lori A; Smithgall, Thomas E

    2017-02-17

    The HIV-1 Nef accessory factor enhances viral infectivity, immune evasion, and AIDS progression. Nef triggers rapid down-regulation of CD4 via the endocytic adaptor protein 2 (AP-2) complex, a process linked to enhanced viral infectivity and immune escape. Here, we describe a bimolecular fluorescence complementation (BiFC) assay to visualize the interaction of Nef with AP-2 and CD4 in living cells. Interacting protein pairs were fused to complementary non-fluorescent fragments of YFP and co-expressed in 293T cells. Nef interactions with both CD4 and AP-2 resulted in complementation of YFP and a bright fluorescent signal by confocal microcopy that localized to the cell periphery. Co-expression of the AP-2 α subunit enhanced the Nef·AP-2 σ2 subunit BiFC signal and vice versa, suggesting that the AP-2 α-σ2 hemicomplex interacts cooperatively with Nef. Mutagenesis of Nef amino acids Arg-134, Glu-174, and Asp-175, which stabilize Nef for AP-2 α-σ2 binding in a recent co-crystal structure, substantially reduced AP-2 interaction without affecting CD4 binding. A dimerization-defective mutant of Nef failed to interact with either CD4 or AP-2 in the BiFC assay, indicating that Nef quaternary structure is required for CD4 and AP-2 recruitment as well as CD4 down-regulation. A small molecule previously shown to bind the Nef dimerization interface also reduced Nef interactions with AP-2 and CD4 and restored CD4 expression to the surface of HIV-infected cells. Our findings provide a mechanistic explanation for previous observations that dimerization-defective Nef mutants fail to down-regulate CD4 and validate the Nef dimerization interface as a target site for antiretroviral drug development.

  16. The adaptor protein soc-1/Gab1 modifies growth factor receptor output in Caenorhabditis elegans.

    PubMed

    Hopper, Neil A

    2006-05-01

    Previous genetic analysis has shown that dos/soc-1/Gab1 functions positively in receptor tyrosine kinase (RTK)-stimulated Ras/Map kinase signaling through the recruitment of csw/ptp-2/Shp2. Using sensitized assays in Caenorhabditis elegans for let-23/Egfr and daf-2/InsR (insulin receptor-like) signaling, it is shown that soc-1/Gab1 inhibits phospholipase C-gamma (PLCgamma) and phosphatidylinositol 3'-kinase (PI3K)-mediated signaling. Furthermore, as well as stimulating Ras/Map kinase signaling, soc-1/Gab1 stimulates a poorly defined signaling pathway that represses class 2 daf-2 phenotypes. In addition, it is shown that SOC-1 binds the C-terminal SH3 domain of SEM-5. This binding is likely to be functional as the sem-5(n2195)G201R mutation, which disrupts SOC-1 binding, behaves in a qualitatively similar manner to a soc-1 null allele in all assays for let-23/Egfr and daf-2/InsR signaling that were examined. Further genetic analysis suggests that ptp-2/Shp2 mediates the negative function of soc-1/Gab1 in PI3K-mediated signaling, as well as the positive function in Ras/Map kinase signaling. Other effectors of soc-1/Gab1 are likely to inhibit PLCgamma-mediated signaling and stimulate the poorly defined signaling pathway that represses class 2 daf-2 phenotypes. Thus, the recruitment of soc-1/Gab1, and its effectors, into the RTK-signaling complex modifies the cellular response by enhancing Ras/Map kinase signaling while inhibiting PI3K and PLCgamma-mediated signaling.

  17. 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.

  18. 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.

  19. Phosphorylation of APP-CTF-AICD domains and interaction with adaptor proteins: signal transduction and/or transcriptional role--relevance for Alzheimer pathology.

    PubMed

    Schettini, Gennaro; Govoni, Stefano; Racchi, Marco; Rodriguez, Guido

    2010-12-01

    In recent decades, the study of the amyloid precursor protein (APP) and of its proteolytic products carboxy terminal fragment (CTF), APP intracellular C-terminal domain (AICD) and amyloid beta has been mostly focussed on the role of APP as a producer of the toxic amyloid beta peptide. Here, we reconsider the role of APP suggesting, in a provocative way, the protein as a central player in a putative signalling pathway. We highlight the presence in the cytosolic tail of APP of the YENPTY motif which is typical of tyrosine kinase receptors, the phosphorylation of the tyrosine, serine and threonine residues, the kinases involved and the interaction with intracellular adaptor proteins. In particular, we examine the interaction with Shc and Grb2 regulators, which through the activation of Ras proteins elicit downstream signalling events such as the MAPK pathway. The review also addresses the interaction of APP, CTFs and AICD with other adaptor proteins and in particular with Fe65 for nuclear transcriptional activity and the importance of phosphorylation for sorting the secretases involved in the amyloidogenic or non-amyloidogenic pathways. We provide a novel perspective on Alzheimer's disease pathogenesis, focussing on the perturbation of the physiological activities of APP-CTFs and AICD as an alternative perspective from that which normally focuses on the accumulation of neurotoxic proteolytic fragments.

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

    PubMed Central

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

    2016-01-01

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

  1. Cargo adaptors: structures illuminate mechanisms regulating vesicle biogenesis.

    PubMed

    Paczkowski, Jon E; Richardson, Brian C; Fromme, J Christopher

    2015-07-01

    Cargo adaptors sort transmembrane protein cargos into nascent vesicles by binding directly to their cytosolic domains. Recent studies have revealed previously unappreciated roles for cargo adaptors and regulatory mechanisms governing their function. The adaptor protein (AP)-1 and AP-2 clathrin adaptors switch between open and closed conformations that ensure they function at the right place at the right time. The exomer cargo adaptor has a direct role in remodeling the membrane for vesicle fission. Several different cargo adaptors functioning in distinct trafficking pathways at the Golgi are similarly regulated through bivalent binding to the ADP-ribosylation factor 1 (Arf1) GTPase, potentially enabling regulation by a threshold concentration of Arf1. Taken together, these studies highlight that cargo adaptors do more than just adapt cargos.

  2. The Mu Subunit of Plasmodium falciparum Clathrin-Associated Adaptor Protein 2 Modulates In Vitro Parasite Response to Artemisinin and Quinine

    PubMed Central

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

    2015-01-01

    The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance. PMID:25691625

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

    PubMed

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

    2015-05-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. Structural determinants for binding of sorting nexin 17 (SNX17) to the cytoplasmic adaptor protein Krev interaction trapped 1 (KRIT1).

    PubMed

    Stiegler, Amy L; Zhang, Rong; Liu, Weizhi; Boggon, Titus J

    2014-09-05

    Sorting nexin 17 (SNX17) is a member of the family of cytoplasmic sorting nexin adaptor proteins that regulate endosomal trafficking of cell surface proteins. SNX17 localizes to early endosomes where it directly binds NPX(Y/F) motifs in the cytoplasmic tails of its target receptors to mediate their rates of endocytic internalization, recycling, and/or degradation. SNX17 has also been implicated in mediating cell signaling and can interact with cytoplasmic proteins. KRIT1 (Krev interaction trapped 1), a cytoplasmic adaptor protein associated with cerebral cavernous malformations, has previously been shown to interact with SNX17. Here, we demonstrate that SNX17 indeed binds directly to KRIT1 and map the binding to the second Asn-Pro-Xaa-Tyr/Phe (NPX(Y/F)) motif in KRIT1. We further characterize the interaction as being mediated by the FERM domain of SNX17. We present the co-crystal structure of SNX17-FERM with the KRIT1-NPXF2 peptide to 3.0 Å resolution and demonstrate that the interaction is highly similar in structure and binding affinity to that between SNX17 and P-selectin. We verify the molecular details of the interaction by site-directed mutagenesis and pulldown assay and thereby confirm that the major binding site for SNX17 is confined to the NPXF2 motif in KRIT1. Taken together, our results verify a direct interaction between SNX17 and KRIT1 and classify KRIT1 as a SNX17 binding partner.

  7. Mitochondrial antiviral signaling adaptor mediated apoptosis in H3N2 swine influenza virus infection is inhibited by viral protein NS1 in vitro.

    PubMed

    Zhang, Jinqiu; Miao, Jinfeng; Hou, Jibo; Lu, Chengping

    2015-05-15

    We investigated the in vitro role of mitochondrial antiviral signaling adaptor (MAVS) in apoptosis induced by H3N2 swine influenza virus infection and the influence of viral NS1 (nonstructural protein 1) protein on this process. H3N2 swine influenza virus (SIV, A/Swine/Shandong/3/2005) was co-cultured with human lung epithelial A549 cells. The relationship of MAVS expression to SIV replication and apoptosis, and the influence of viral proteins on MAVS functions were studied. The data indicate that in response to SIV infection, MAVS was significantly upregulated at both the transcriptional and protein levels in the early stages of infection. Its expression and localization to mitochondria are necessary for apoptosis of epithelial cells induced by H3N2 swine influenza virus. Viral protein NS1 can antagonize MAVS-mediated apoptosis. These findings indicate that MAVS have a role in regulating innate mitochondrial responses to viral infection.

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

    PubMed Central

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

    2015-01-01

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

  9. The Cell Signaling Adaptor Protein EPS-8 Is Essential for C. elegans Epidermal Elongation and Interacts with the Ankyrin Repeat Protein VAB-19

    PubMed Central

    Ding, Mei; King, Ryan S.; Berry, Emily C.; Wang, Ying; Hardin, Jeff; Chisholm, Andrew D.

    2008-01-01

    Background The epidermal cells of the C. elegans embryo undergo coordinated cell shape changes that result in the morphogenetic process of elongation. The cytoskeletal ankyrin repeat protein VAB-19 is required for cell shape changes and localizes to cell-matrix attachment structures. The molecular functions of VAB-19 in this process are obscure, as no previous interactors for VAB-19 have been described. Methodology/Principal Findings In screens for VAB-19 binding proteins we identified the signaling adaptor EPS-8. Within C. elegans epidermal cells, EPS-8 and VAB-19 colocalize at cell-matrix attachment structures. The central domain of EPS-8 is necessary and sufficient for its interaction with VAB-19. eps-8 null mutants, like vab-19 mutants, are defective in epidermal elongation and in epidermal-muscle attachment. The eps-8 locus encodes two isoforms, EPS-8A and EPS-8B, that appear to act redundantly in epidermal elongation. The function of EPS-8 in epidermal development involves its N-terminal PTB and central domains, and is independent of its C-terminal SH3 and actin-binding domains. VAB-19 appears to act earlier in the biogenesis of attachment structures and may recruit EPS-8 to these structures. Conclusions/Significance EPS-8 and VAB-19 define a novel pathway acting at cell-matrix attachments to regulate epithelial cell shape. This is the first report of a role for EPS-8 proteins in cell-matrix attachments. The existence of EPS-8B-like isoforms in Drosophila suggests this function of EPS-8 proteins could be conserved among other organisms. PMID:18833327

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

    SciTech Connect

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

    2011-11-11

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

  11. Proteins : paradigms of complexity /

    SciTech Connect

    Frauenfelder, Hans,

    2001-01-01

    Proteins are the working machines of living systems. Directed by the DNA, of the order of a few hundred building blocks, selected from twenty different amino acids, are covalently linked into a linear polypeptide chain. In the proper environment, the chain folds into the working protein, often a globule of linear dimensions of a few nanometers. The biologist considers proteins units from which living systems are built. Many physical scientists look at them as systems in which the laws of complexity can be studied better than anywhere else. Some of the results of such studies will be sketched.

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

    SciTech Connect

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

    2010-08-12

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

  13. Cross-talk between Tetraspanin CD9 and Transmembrane Adaptor Protein Non-T Cell Activation Linker (NTAL) in Mast Cell Activation and Chemotaxis*

    PubMed Central

    Hálová, Ivana; Dráberová, Lubica; Bambousková, Monika; Machyna, Martin; Stegurová, Lucie; Smrž, Daniel; Dráber, Petr

    2013-01-01

    Chemotaxis, a process leading to movement of cells toward increasing concentrations of chemoattractants, is essential, among others, for recruitment of mast cells within target tissues where they play an important role in innate and adaptive immunity. Chemotaxis is driven by chemoattractants, produced by various cell types, as well as by intrinsic cellular regulators, which are poorly understood. In this study we prepared a new mAb specific for the tetraspanin CD9. Binding of the antibody to bone marrow-derived mast cells triggered activation events that included cell degranulation, Ca2+ response, dephosphorylation of ezrin/radixin/moesin (ERM) family proteins, and potent tyrosine phosphorylation of the non-T cell activation linker (NTAL) but only weak phosphorylation of the linker for activation of T cells (LAT). Phosphorylation of the NTAL was observed with whole antibody but not with its F(ab)2 or Fab fragments. This indicated involvement of the Fcγ receptors. As documented by electron microscopy of isolated plasma membrane sheets, CD9 colocalized with the high-affinity IgE receptor (FcϵRI) and NTAL but not with LAT. Further tests showed that both anti-CD9 antibody and its F(ab)2 fragment inhibited mast cell chemotaxis toward antigen. Experiments with bone marrow-derived mast cells deficient in NTAL and/or LAT revealed different roles of these two adaptors in antigen-driven chemotaxis. The combined data indicate that chemotaxis toward antigen is controlled in mast cells by a cross-talk among FcϵRI, tetraspanin CD9, transmembrane adaptor proteins NTAL and LAT, and cytoskeleton-regulatory proteins of the ERM family. PMID:23443658

  14. Cargo adaptors: structures illuminate mechanisms regulating vesicle biogenesis

    PubMed Central

    Paczkowski, Jon E.; Richardson, Brian C.; Fromme, J. Christopher

    2015-01-01

    Cargo adaptors sort transmembrane protein cargos into nascent vesicles by binding directly to their cytosolic domains. Recent studies have revealed previously unappreciated roles for cargo adaptors and regulatory mechanisms governing their function. The AP-1 and AP-2 clathrin adaptors switch between open and closed conformations that ensure they function at the right place at the right time. The exomer cargo adaptor plays a direct role in remodeling the membrane for vesicle fission. Several different cargo adaptors functioning in distinct trafficking pathways at the Golgi are similarly regulated through bivalent binding to the Arf1 GTPase, potentially enabling regulation by a threshold concentration of Arf1. Taken together, these studies highlight that cargo adaptors do more than just adapt cargos. PMID:25795254

  15. Endocytic adaptors – social networking at the plasma membrane

    PubMed Central

    Reider, Amanda; Wendland, Beverly

    2011-01-01

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

  16. A putative TIR domain protein from Helicobacter pylori is dimeric in solution and interacts with human TLR adaptor Myeloid Differentiation Primary Response 88.

    PubMed

    Türköz, Burcu Kaplan

    2017-03-06

    Helicobacter pylori is an important human pathogen capable of causing persistent infection with minimal immune response. The first line of defense during H. pylori infection is through gastric epithelial cells that present Toll like receptors (TLR), a family of bacterial proteins which share homology with the Toll/IL1 receptor (TIR) domain. Bacterial TIR proteins (BTP) mimic human TIR domain proteins and act on MyD88 signaling pathways to suppress TLR signaling. H. pylori might also produce a similar protein. A putative H. pylori BTP was found based on sequence homology and the corresponding gene hp1437 was inserted into an expression vector in fusion with an N-terminal cleavable 6his-tag. The recombinant protein, 6his-HP1437 was purified using nickel affinity chromatography with a yield of 8 mg/ L culture. Oligomerization of HP1437 was investigated by size-exclusion chromatography. Our results show that HP1437 forms dimers in solution similar to other BTPs. Furthermore, GST pull down assays identify an interaction between HP1437 and human TIR domain adaptor MyD88. This study suggests that HP1437 has the characteristic features of BTPs and may play a direct role in reduced immune response against H. pylori by binding to MyD88 and pave the way for an in-depth characterization of this putative novel H. pylori virulence factor.

  17. Comparative analysis of adaptor-mediated clathrin assembly reveals general principles for adaptor clustering

    PubMed Central

    Pucadyil, Thomas J.; Holkar, Sachin S.

    2016-01-01

    Clathrin-mediated endocytosis (CME) manages the sorting and uptake of the bulk of membrane proteins (or cargo) from the plasma membrane. CME is initiated by the formation of clathrin-coated pits (CCPs), in which adaptors nucleate clathrin assembly. Clathrin adaptors display diversity in both the type and number of evolutionarily conserved clathrin-binding boxes. How this diversity relates to the process of adaptor clustering as clathrin assembles around a growing pit remains unclear. Using real-time, fluorescence microscopy–based assays, we compare the formation kinetics and distribution of clathrin assemblies on membranes that display five unique clathrin adaptors. Correlations between equilibrium and kinetic parameters of clathrin assembly to the eventual adaptor distribution indicate that adaptor clustering is determined not by the amount of clathrin recruited or the degree of clathrin clustered but instead by the rate of clathrin assembly. Together our results emphasize the need to analyze kinetics of protein interactions to better understand mechanisms that regulate CME. PMID:27559129

  18. Regulation of insulin and type 1 insulin-like growth factor signaling and action by the Grb10/14 and SH2B1/B2 adaptor proteins.

    PubMed

    Desbuquois, Bernard; Carré, Nadège; Burnol, Anne-Françoise

    2013-02-01

    The effects of insulin and type 1 insulin-like growth factor (IGF-1) on metabolism, growth and survival are mediated by their association with specific receptor tyrosine kinases, which results in both receptor and substrate phosphorylation. Phosphotyrosine residues on receptors and substrates provide docking sites for signaling proteins containing SH2 (Src homology 2) domains, including molecular adaptors. This review focuses on the regulation of insulin/IGF-1 signaling and action by two adaptor families with a similar domain organization: the growth factor receptor-bound proteins Grb7/10/14 and the SH2B proteins. Both Grb10/14 and SH2B1/B2 associate with the activation loop of insulin/IGF-1 receptors through their SH2 domains, but association of Grb10/14 also involves their unique BPS domain. Consistent with Grb14 binding as a pseudosubstrate to the kinase active site, insulin/IGF-induced activation of receptors and downstream signaling pathways in cultured cells is inhibited by Grb10/14 adaptors, but is potentiated by SH2B1/B2 adaptors. Accordingly, Grb10 and Grb14 knockout mice show improved insulin/IGF sensitivity in vivo, and, for Grb10, overgrowth and increased skeketal muscle and pancreatic β-cell mass. Conversely, SH2B1-depleted mice display insulin and IGF-1 resistance, with peripheral depletion leading to reduced adiposity and neuronal depletion leading to obesity through associated leptin resistance. Grb10/14 and SH2B1 adaptors also modulate insulin/IGF-1 action by interacting with signaling components downstream of receptors and exert several tissue-specific effects. The identification of Grb10/14 and SH2B1 as physiological regulators of insulin signaling and action, together with observations that variants at their gene loci are associated with obesity and/or insulin resistance, highlight them as potential therapeutic targets for these conditions.

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

    PubMed

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

    2014-10-01

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

  20. Induction of Androgen Formation in the Male by a TAT-VDAC1 Fusion Peptide Blocking 14-3-3ɛ Protein Adaptor and Mitochondrial VDAC1 Interactions

    PubMed Central

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

    2014-01-01

    Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production. PMID:24947306

  1. Molecular cloning of the mouse grb2 gene: differential interaction of the Grb2 adaptor protein with epidermal growth factor and nerve growth factor receptors.

    PubMed Central

    Suen, K L; Bustelo, X R; Pawson, T; Barbacid, M

    1993-01-01

    We report the isolation and molecular characterization of the mouse grb2 gene. The product of this gene, the Grb2 protein, is highly related to the Caenorhabditis elegans sem-5 gene product and the human GRB2 protein and displays the same SH3-SH2-SH3 structural motifs. In situ hybridization studies revealed that the mouse grb2 gene is widely expressed throughout embryonic development (E9.5 to P0). However, grb2 transcripts are not uniformly distributed, and in certain tissues (e.g., thymus) they appear to be regulated during development. Recent genetic and biochemical evidence has implicated the Grb2 protein in the signaling pathways that link cell surface tyrosine kinase receptors with Ras. We have investigated the association of the Grb2 protein with epidermal growth factor (EGF) and nerve growth factor (NGF) receptors in PC12 pheochromocytoma cells. EGF treatment of PC12 cells results in the rapid association of Grb2 with the activated EGF receptors, an interaction mediated by the Grb2 SH2 domain. However, Grb2 does not bind to NGF-activated Trk receptors. Mitogenic signaling of NGF in NIH 3T3 cells ectopically expressing Trk receptors also takes place without detectable association between Grb2 and Trk. These results suggest that whereas EGF and NGF can activate the Ras signaling pathway in PC12 cells, only the EGF receptor is likely to do so through a direct interaction with Grb2. Finally, binding studies with glutathione S-transferase fusion proteins indicate that Grb2 binds two distinct subsets of proteins which are individually recognized by its SH2 and SH3 domains. These observations add further support to the concept that Grb2 is a modular adaptor protein. Images PMID:7689150

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

    PubMed

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

    2008-01-01

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

  3. WAC regulates mTOR activity by acting as an adaptor for the TTT and Pontin/Reptin complexes

    PubMed Central

    David-Morrison, Gabriela; Xu, Zhen; Rui, Yan-Ning; Charng, Wu-Lin; Jaiswal, Manish; Yamamoto, Shinya; Xiong, Bo; Zhang, Ke; Sandoval, Hector; Duraine, Lita; Zuo, Zhongyuan; Zhang, Sheng; Bellen, Hugo J.

    2015-01-01

    SUMMARY The ability to sense energy status is crucial in the regulation of metabolism via the mechanistic Target of Rapamycin Complex 1 (mTORC1). The assembly of the TTT-Pontin/Reptin complex is responsive to changes in energy status. In energy sufficient conditions, the TTT-Pontin/Reptin complex promotes mTORC1 dimerization and mTORC1-Rag interaction, which are critical for mTORC1 activation. We show that WAC is a regulator of energy-mediated mTORC1 activity. In a Drosophila screen designed to isolate mutations that cause neuronal dysfunction, we identified wacky, the homolog of WAC. Loss of Wacky leads to neurodegeneration, defective mTOR activity and increased autophagy. Wacky and WAC have conserved physical interactions with mTOR and its regulators, including Pontin and Reptin which bind to the TTT complex to regulate energy-dependent activation of mTORC1. WAC promotes the interaction between TTT and Pontin/Reptin in an energy-dependent manner, thereby promoting mTORC1 activity by facilitating mTORC1 dimerization and mTORC1-Rag interaction. PMID:26812014

  4. The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

    SciTech Connect

    Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou . E-mail: mcutler@usuhs.mil

    2005-05-15

    Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion.

  5. Non-redundant and complementary functions of adaptor proteins TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-κB signaling

    PubMed Central

    Vallabhapurapu, Sivakumar; Matsuzawa, Atsushi; Zhang, WeiZhou; Tseng, Ping-Hui; Keats, Jonathan J.; Wang, Haopeng; Vignali, Dario A. A.; Bergsagel, P. Leif; Karin, Michael

    2009-01-01

    The adaptor and signaling proteins TRAF2, TRAF3 and cIAP1 and cIAP2 were suggested to inhibit alternative nuclear factor kappa B (NF-κB) signaling in resting cells by targeting NF-κB inducing kinase (NIK) to ubiquitin-dependent degradation, thus preventing processing of the NF-κB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-κB signaling has remained elusive. We now show that CD40 or BAFF receptor activation resulted in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2- dependent way due to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-κB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects. PMID:18997792

  6. 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

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

    PubMed

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

    2015-10-16

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

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

    PubMed

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

    2016-11-01

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

  9. Dystrophin complex functions as a scaffold for signalling proteins.

    PubMed

    Constantin, Bruno

    2014-02-01

    Dystrophin is a 427kDa sub-membrane cytoskeletal protein, associated with the inner surface membrane and incorporated in a large macromolecular complex of proteins, the dystrophin-associated protein complex (DAPC). In addition to dystrophin the DAPC is composed of dystroglycans, sarcoglycans, sarcospan, dystrobrevins and syntrophin. This complex is thought to play a structural role in ensuring membrane stability and force transduction during muscle contraction. The multiple binding sites and domains present in the DAPC confer the scaffold of various signalling and channel proteins, which may implicate the DAPC in regulation of signalling processes. The DAPC is thought for instance to anchor a variety of signalling molecules near their sites of action. The dystroglycan complex may participate in the transduction of extracellular-mediated signals to the muscle cytoskeleton, and β-dystroglycan was shown to be involved in MAPK and Rac1 small GTPase signalling. More generally, dystroglycan is view as a cell surface receptor for extracellular matrix proteins. The adaptor proteins syntrophin contribute to recruit and regulate various signalling proteins such as ion channels, into a macromolecular complex. Although dystrophin and dystroglycan can be directly involved in signalling pathways, syntrophins play a central role in organizing signalplex anchored to the dystrophin scaffold. The dystrophin associated complex, can bind up to four syntrophin through binding domains of dystrophin and dystrobrevin, allowing the scaffold of multiple signalling proteins in close proximity. Multiple interactions mediated by PH and PDZ domains of syntrophin also contribute to build a complete signalplex which may include ion channels, such as voltage-gated sodium channels or TRPC cation channels, together with, trimeric G protein, G protein-coupled receptor, plasma membrane calcium pump, and NOS, to enable efficient and regulated signal transduction and ion transport. This article is part

  10. Adaptor protein cerebral cavernous malformation 3 (CCM3) mediates phosphorylation of the cytoskeletal proteins ezrin/radixin/moesin by mammalian Ste20-4 to protect cells from oxidative stress.

    PubMed

    Fidalgo, Miguel; Guerrero, Ana; Fraile, María; Iglesias, Cristina; Pombo, Celia M; Zalvide, Juan

    2012-03-30

    While studying the functions of CCM3/PDCD10, a gene encoding an adaptor protein whose mutation results in vascular malformations, we have found that it is involved in a novel response to oxidative stress that results in phosphorylation and activation of the ezrin/radixin/moesin (ERM) family of proteins. This phosphorylation protects cells from accidental cell death induced by oxidative stress. We also present evidence that ERM phosphorylation is performed by the GCKIII kinase Mst4, which is activated and relocated to the cell periphery after oxidative stress. The cellular levels of Mst4 and its activation after oxidative stress depend on the presence of CCM3, as absence of the latter impairs the phosphorylation of ERM proteins and enhances death of cells exposed to reactive oxygen species. These findings shed new light on the response of cells to oxidative stress and identify an important pathophysiological situation in which ERM proteins and their phosphorylation play a significant role.

  11. Transforming growth factor-beta suppresses nonmetastatic colon cancer through Smad4 and adaptor protein ELF at an early stage of tumorigenesis.

    PubMed

    Tang, Yi; Katuri, Varalakshmi; Srinivasan, Radhika; Fogt, Franz; Redman, Robert; Anand, Girish; Said, Anan; Fishbein, Thomas; Zasloff, Michael; Reddy, E Premkumar; Mishra, Bibhuti; Mishra, Lopa

    2005-05-15

    Although transforming growth factor-beta (TGF-beta) is both a suppressor and promoter of tumorigenesis, its contribution to early tumor suppression and staging remains largely unknown. In search of the mechanism of early tumor suppression, we identified the adaptor protein ELF, a beta-spectrin from stem/progenitor cells committed to foregut lineage. ELF activates and modulates Smad4 activation of TGF-beta to confer cell polarity, to maintain cell architecture, and to inhibit epithelial-to-mesenchymal transition. Analysis of development of colon cancer in (adult) elf+/-/Smad4+/-, elf+/-, Smad4+/-, and gut epithelial cells from elf-/- mutant mouse embryos pinpoints the defect to hyperplasia/adenoma transition. Further analysis of the role of ELF in human colorectal cancer confirms reduced expression of ELF in Dukes' B1 stage tissues (P < 0.05) and of Smad4 in advanced colon cancers (P < 0.05). This study indicates that by modulating Smad 4, ELF has a key role in TGF-beta signaling in the suppression of early colon cancer.

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

    PubMed

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

    2014-04-01

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

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

    PubMed

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

    2010-04-22

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

  14. Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.

    PubMed

    Jung, SangYong; Maritzen, Tanja; Wichmann, Carolin; Jing, Zhizi; Neef, Andreas; Revelo, Natalia H; Al-Moyed, Hanan; Meese, Sandra; Wojcik, Sonja M; Panou, Iliana; Bulut, Haydar; Schu, Peter; Ficner, Ralf; Reisinger, Ellen; Rizzoli, Silvio O; Neef, Jakob; Strenzke, Nicola; Haucke, Volker; Moser, Tobias

    2015-11-03

    Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.

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

    SciTech Connect

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

    2012-09-17

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

  16. Length, protein protein interactions, and complexity

    NASA Astrophysics Data System (ADS)

    Tan, Taison; Frenkel, Daan; Gupta, Vishal; Deem, Michael W.

    2005-05-01

    The evolutionary reason for the increase in gene length from archaea to prokaryotes to eukaryotes observed in large-scale genome sequencing efforts has been unclear. We propose here that the increasing complexity of protein-protein interactions has driven the selection of longer proteins, as they are more able to distinguish among a larger number of distinct interactions due to their greater average surface area. Annotated protein sequences available from the SWISS-PROT database were analyzed for 13 eukaryotes, eight bacteria, and two archaea species. The number of subcellular locations to which each protein is associated is used as a measure of the number of interactions to which a protein participates. Two databases of yeast protein-protein interactions were used as another measure of the number of interactions to which each S. cerevisiae protein participates. Protein length is shown to correlate with both number of subcellular locations to which a protein is associated and number of interactions as measured by yeast two-hybrid experiments. Protein length is also shown to correlate with the probability that the protein is encoded by an essential gene. Interestingly, average protein length and number of subcellular locations are not significantly different between all human proteins and protein targets of known, marketed drugs. Increased protein length appears to be a significant mechanism by which the increasing complexity of protein-protein interaction networks is accommodated within the natural evolution of species. Consideration of protein length may be a valuable tool in drug design, one that predicts different strategies for inhibiting interactions in aberrant and normal pathways.

  17. The Sho1 Adaptor Protein Links Oxidative Stress to Morphogenesis and Cell Wall Biosynthesis in the Fungal Pathogen Candida albicans† ‡

    PubMed Central

    Román, Elvira; Nombela, César; Pla, Jesús

    2005-01-01

    The Sho1 adaptor protein is an important element of one of the two upstream branches of the high-osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase pathway in Saccharomyces cerevisiae, a signal transduction cascade involved in adaptation to stress. In the present work, we describe its role in the pathogenic yeast Candida albicans by the construction of mutants altered in this gene. We report here that sho1 mutants are sensitive to oxidative stress but that Sho1 has a minor role in the transmission of the phosphorylation signal to the Hog1 MAP kinase in response to oxidative stress, which mainly occurs through a putative Sln1-Ssk1 branch of the HOG pathway. Genetic analysis revealed that double ssk1 sho1 mutants were still able to grow on high-osmolarity media and activate Hog1 in response to this stress, indicating the existence of alternative inputs of the pathway. We also demonstrate that the Cek1 MAP kinase is constitutively active in hog1 and ssk1 mutants, a phenotypic trait that correlates with their resistance to the cell wall inhibitor Congo red, and that Sho1 is essential for the activation of the Cek1 MAP kinase under different conditions that require active cell growth and/or cell wall remodeling, such as the resumption of growth upon exit from the stationary phase. sho1 mutants are also sensitive to certain cell wall interfering compounds (Congo red, calcofluor white), presenting an altered cell wall structure (as shown by the ability to aggregate), and are defective in morphogenesis on different media, such as SLAD and Spider, that stimulate hyphal growth. These results reveal a role for the Sho1 protein in linking oxidative stress, cell wall biogenesis, and morphogenesis in this important human fungal pathogen. PMID:16287872

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

    PubMed

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

    2015-10-01

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

  19. Proteins, fluctuations and complexity

    SciTech Connect

    Frauenfelder, Hans; Chen, Guo; Fenimore, Paul W

    2008-01-01

    Glasses, supercooled liquids, and proteins share common properties, in particular the existence of two different types of fluctuations, {alpha} and {beta}. While the effect of the {alpha} fluctuations on proteins has been known for a few years, the effect of {beta} fluctuations has not been understood. By comparing neutron scattering data on the protein myoglobin with the {beta} fluctuations in the hydration shell measured by dielectric spectroscopy we show that the internal protein motions are slaved to these fluctuations. We also show that there is no 'dynamic transition' in proteins near 200 K. The rapid increase in the mean square displacement with temperature in many neutron scattering experiments is quantitatively predicted by the {beta} fluctuations in the hydration shell.

  20. Adaptor protein CRK induces epithelial–mesenchymal transition and metastasis of bladder cancer cells through HGF/c-Met feedback loop

    PubMed Central

    Matsumoto, Ryuji; Tsuda, Masumi; Wang, Lei; Maishi, Nako; Abe, Takashige; Kimura, Taichi; Tanino, Mishie; Nishihara, Hiroshi; Hida, Kyoko; Ohba, Yusuke; Shinohara, Nobuo; Nonomura, Katsuya; Tanaka, Shinya

    2015-01-01

    We have previously reported that an adaptor protein CRK, including CRK-I and CRK-II, plays essential roles in the malignant potential of various aggressive human cancers, suggesting the validity of targeting CRK in molecular targeted therapy of a wide range of cancers. Nevertheless, the role of CRK in human bladder cancer with marked invasion, characterized by distant metastasis and poor prognosis, remains obscure. In the present study, immunohistochemistry indicated a striking enhancement of CRK-I/-II, but not CRK-like, in human bladder cancer tissues compared to normal urothelium. We established CRK-knockdown bladder cancer cells using 5637 and UM-UC-3, which showed a significant decline in cell migration, invasion, and proliferation. It is noteworthy that an elimination of CRK conferred suppressed phosphorylation of c-Met and the downstream scaffold protein Gab1 in a hepatocyte growth factor-dependent and -independent manner. In epithelial–mesenchymal transition-related molecules, E-cadherin was upregulated by CRK elimination, whereas N-cadherin, vimentin, and Zeb1 were downregulated. A similar effect was observed following treatment with c-Met inhibitor SU11274. Depletion of CRK significantly decreased cell proliferation of 5637 and UM-UC-3, consistent with reduced activity of ERK. An orthotopic xenograft model with bioluminescent imaging revealed that CRK knockdown significantly attenuated not only tumor volume but also the number of circulating tumor cells, resulted in a complete abrogation of metastasis. Taken together, this evidence uncovered essential roles of CRK in invasive bladder cancer through the hepatocyte growth factor/c-Met/CRK feedback loop for epithelial–mesenchymal transition induction. Thus, CRK might be a potent molecular target in bladder cancer, particularly for preventing metastasis, leading to the resolution of clinically longstanding critical issues. PMID:25816892

  1. SH2 domain–containing adaptor protein B expressed in dendritic cells is involved in T-cell homeostasis by regulating dendritic cell–mediated Th2 immunity

    PubMed Central

    2017-01-01

    Purpose The Src homology 2 domain–containing adaptor protein B (SHB) is widely expressed in immune cells and acts as an important regulator for hematopoietic cell function. SHB silencing induces Th2 immunity in mice. SHB is also involved in T-cell homeostasis in vivo. However, SHB has not yet been studied and addressed in association with dendritic cells (DCs). Materials and Methods The effects of SHB expression on the immunogenicity of DCs were assessed by Shb gene silencing in mouse bone marrow–derived DCs (BMDCs). After silencing, surface phenotype, cytokine expression profile, and T-cell stimulation capacity of BMDCs were examined. We investigated the signaling pathways involved in SHB expression during BMDC development. We also examined the immunogenicity of SHB-knockdown (SHBKD) BMDCs in a mouse atopic dermatitis model. Results SHB was steadily expressed in mouse splenic DCs and in in vitro–generated BMDCs in both immature and mature stages. SHB expression was contingent on activation of the mitogen- activated protein kinase/Foxa2 signaling pathway during DC development. SHBKD increased the expression of MHC class II and costimulatory molecules without affecting the cytokine expression of BMDCs. When co-cultured with T cells, SHBKD in BMDCs significantly induced CD4+ T-cell proliferation and the expression of Th2 cytokines, while the regulatory T cell (Treg) population was downregulated. In mouse atopic dermatitis model, mice inoculated with SHBKD DCs developed more severe symptoms of atopic dermatitis compared with mice injected with control DCs. Conclusion SHB expression in DCs plays an important role in T-cell homeostasis in vivo by regulating DC-mediated Th2 polarization. PMID:28168174

  2. An adaptor hierarchy regulates proteolysis during a bacterial cell cycle

    PubMed Central

    Joshi, Kamal Kishore; Bergé, Matthieu; Radhakrishnan, Sunish Kumar; Viollier, Patrick Henri; Chien, Peter

    2015-01-01

    Summary Regulated protein degradation is essential. The timed destruction of crucial proteins by the ClpXP protease drives cell-cycle progression in the bacterium Caulobacter crescentus. Although ClpXP is active alone, additional factors are inexplicably required for cell-cycle dependent proteolysis. Here, we show that these factors constitute an adaptor hierarchy where different substrates are destroyed based on the degree of adaptor assembly. The hierarchy builds upon priming of ClpXP by the adaptor CpdR, which promotes degradation of one class of substrates and also recruits the adaptor RcdA to degrade a second class of substrates. Adding the PopA adaptor promotes destruction of a third class of substrates, while inhibiting degradation of the second class. We dissect RcdA to generate bespoke adaptors, identifying critical substrate elements needed for RcdA recognition and uncovering additional cell-cycle dependent ClpXP substrates. Our work reveals how hierarchical adaptors and primed proteases orchestrate regulated proteolysis during bacterial cell-cycle progression. PMID:26451486

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2014-12-01

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

  6. Role of an adaptor protein Lin-7B in brain development: possible involvement in autism spectrum disorders.

    PubMed

    Mizuno, Makoto; Matsumoto, Ayumi; Hamada, Nanako; Ito, Hidenori; Miyauchi, Akihiko; Jimbo, Eriko F; Momoi, Mariko Y; Tabata, Hidenori; Yamagata, Takanori; Nagata, Koh-Ichi

    2015-01-01

    Using comparative genomic hybridization analysis for an autism spectrum disorder (ASD) patient, a 73-Kb duplication at 19q13.33 (nt. 49 562 755-49 635 956) including LIN7B and 5 other genes was detected. We then identified a novel frameshift mutation in LIN7B in another ASD patient. Since LIN7B encodes a scaffold protein essential for neuronal function, we analyzed the role of Lin-7B in the development of cerebral cortex. Acute knockdown of Lin-7B with in utero electroporation caused a delay in neuronal migration during corticogenesis. When Lin-7B was knocked down in cortical neurons in one hemisphere, their axons failed to extend efficiently into the contralateral hemisphere after leaving the corpus callosum. Meanwhile, enhanced expression of Lin-7B had no effects on both cortical neuron migration and axon growth. Notably, silencing of Lin-7B did not affect the proliferation of neuronal progenitors and stem cells. Taken together, Lin-7B was found to play a pivotal role in corticogenesis through the regulation of excitatory neuron migration and interhemispheric axon growth, while further analyses are required to directly link functional defects of Lin-7B to ASD pathophysiology. Lin-7 plays a pivotal role as a scaffold protein in synaptic development and plasticity. Based on genetic analyses we identified mutations in LIN-7B gene in some ASD (autism-spectrum disorder) patients. Functional defects in Lin-7B caused abnormal neuronal migration and interhemispheric axon growth during mouse brain development. Thus, functional deficiency in Lin-7B could be implicated in clinical phenotypes in some ASD patients through bringing about abnormal cortical architecture.

  7. A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia.

    PubMed

    Soufi, Muhidien; Rust, Stephan; Walter, Michael; Schaefer, Juergen R

    2013-05-25

    Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.

  8. Stac adaptor proteins regulate trafficking and function of muscle and neuronal L-type Ca2+ channels.

    PubMed

    Polster, Alexander; Perni, Stefano; Bichraoui, Hicham; Beam, Kurt G

    2015-01-13

    Excitation-contraction (EC) coupling in skeletal muscle depends upon trafficking of CaV1.1, the principal subunit of the dihydropyridine receptor (DHPR) (L-type Ca(2+) channel), to plasma membrane regions at which the DHPRs interact with type 1 ryanodine receptors (RyR1) in the sarcoplasmic reticulum. A distinctive feature of this trafficking is that CaV1.1 expresses poorly or not at all in mammalian cells that are not of muscle origin (e.g., tsA201 cells), in which all of the other nine CaV isoforms have been successfully expressed. Here, we tested whether plasma membrane trafficking of CaV1.1 in tsA201 cells is promoted by the adapter protein Stac3, because recent work has shown that genetic deletion of Stac3 in skeletal muscle causes the loss of EC coupling. Using fluorescently tagged constructs, we found that Stac3 and CaV1.1 traffic together to the tsA201 plasma membrane, whereas CaV1.1 is retained intracellularly when Stac3 is absent. Moreover, L-type Ca(2+) channel function in tsA201 cells coexpressing Stac3 and CaV1.1 is quantitatively similar to that in myotubes, despite the absence of RyR1. Although Stac3 is not required for surface expression of CaV1.2, the principle subunit of the cardiac/brain L-type Ca(2+) channel, Stac3 does bind to CaV1.2 and, as a result, greatly slows the rate of current inactivation, with Stac2 acting similarly. Overall, these results indicate that Stac3 is an essential chaperone of CaV1.1 in skeletal muscle and that in the brain, Stac2 and Stac3 may significantly modulate CaV1.2 function.

  9. The Clathrin Adaptor Proteins ARH, Dab2, and Numb Play Distinct Roles in Niemann-Pick C1-Like 1 Versus Low Density Lipoprotein Receptor-mediated Cholesterol Uptake*

    PubMed Central

    Wei, Jian; Fu, Zhen-Yan; Li, Pei-Shan; Miao, Hong-Hua; Li, Bo-Liang; Ma, Yi-Tong; Song, Bao-Liang

    2014-01-01

    The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake. PMID:25331956

  10. The clathrin adaptor proteins ARH, Dab2, and numb play distinct roles in Niemann-Pick C1-Like 1 versus low density lipoprotein receptor-mediated cholesterol uptake.

    PubMed

    Wei, Jian; Fu, Zhen-Yan; Li, Pei-Shan; Miao, Hong-Hua; Li, Bo-Liang; Ma, Yi-Tong; Song, Bao-Liang

    2014-11-28

    The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake.

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

    PubMed Central

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

    2015-01-01

    The adaptor protein-2 sigma subunit (AP2σ2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2σ2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca2+o) homeostasis. To elucidate the role of AP2σ2 in Ca2+o regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2σ2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2σ2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype–phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2σ2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2σ2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2σ2 mutations to result in a more severe FHH phenotype with genotype–phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue. PMID:26082470

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

    PubMed Central

    Birtwistle, Marc R.

    2015-01-01

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

  13. Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B, Type I (SR-BI)*

    PubMed Central

    Tsukamoto, Kosuke; Wales, Thomas E.; Daniels, Kathleen; Pal, Rinku; Sheng, Ren; Cho, Wonhwa; Stafford, Walter; Engen, John R.; Krieger, Monty; Kocher, Olivier

    2013-01-01

    The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or target peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical target peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical target peptide binding and noncanonical (peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. PMID:23720744

  14. 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

  15. 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.

  16. A Repressor Protein Complex Regulates Leaf Growth in Arabidopsis

    PubMed Central

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

    2015-01-01

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

  17. 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

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

    PubMed

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

    2015-09-01

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

  19. Purifying protein complexes for mass spectrometry: applications to protein translation.

    PubMed

    Link, Andrew J; Fleischer, Tracey C; Weaver, Connie M; Gerbasi, Vincent R; Jennings, Jennifer L

    2005-03-01

    Proteins control and mediate most of the biological activities in the cell. In most cases, proteins either interact with regulatory proteins or function in large molecular assemblies to carryout biological processes. Understanding the functions of individual proteins requires the identification of these interacting proteins. With its speed and sensitivity, mass spectrometry has become the dominant method for identifying components of protein complexes. This article reviews and discusses various approaches to purify protein complexes and analyze the proteins using mass spectrometry. As examples, methods to isolate and analyze protein complexes responsible for the translation of messenger RNAs into polypeptides are described.

  20. The tyrosine-sorting motif of the vacuolar sorting receptor VSR4 from Arabidopsis thaliana, which is involved in the interaction between VSR4 and AP1M2, μ1-adaptin type 2 of clathrin adaptor complex 1 subunits, participates in the post-Golgi sorting of VSR4.

    PubMed

    Nishimura, Kohji; Matsunami, Erika; Yoshida, Shohei; Kohata, Shuhei; Yamauchi, Junji; Jisaka, Mitsuo; Nagaya, Tsutomu; Yokota, Kazushige; Nakagawa, Tsuyoshi

    2016-01-01

    μ1-Adaptin of adaptor protein (AP) 1 complex, AP1M, is generally accepted to load cargo proteins into clathrin-coated vesicles (CCVs) at the trans-Golgi network through its binding to cargo-recognition sequences (CRSs). Plant vacuolar-sorting receptors (VSRs) function in sorting vacuolar proteins, which are reportedly mediated by CCV. We herein investigated the involvement of CRSs of Arabidopsis thaliana VSR4 in the sorting of VSR4. The results obtained showed the increased localization of VSR4 at the plasma membrane or vacuoles by mutations in CRSs including the tyrosine-sorting motif YMPL or acidic dileucine-like motif EIRAIM, respectively. Interaction analysis using the bimolecular fluorescence complementation (BiFC) system, V10-BiFC, which we developed, indicated an interaction between VSR4 and AP1M2, AP1M type 2, which was attenuated by a YMPL mutation, but not influenced by an EIRAIM mutation. These results demonstrated the significance of the recognition of YMPL in VSR4 by AP1M2 for the post-Golgi sorting of VSR4.

  1. Cell-surface Attachment of Bacterial Multienzyme Complexes Involves Highly Dynamic Protein-Protein Anchors*

    PubMed Central

    Cameron, Kate; Najmudin, Shabir; Alves, Victor D.; Bayer, Edward A.; Smith, Steven P.; Bule, Pedro; Waller, Helen; Ferreira, Luís M. A.; Gilbert, Harry J.; Fontes, Carlos M. G. A.

    2015-01-01

    Protein-protein interactions play a pivotal role in the assembly of the cellulosome, one of nature's most intricate nanomachines dedicated to the depolymerization of complex carbohydrates. The integration of cellulosomal components usually occurs through the binding of type I dockerin modules located at the C terminus of the enzymes to cohesin modules located in the primary scaffoldin subunit. Cellulosomes are typically recruited to the cell surface via type II cohesin-dockerin interactions established between primary and cell-surface anchoring scaffoldin subunits. In contrast with type II interactions, type I dockerins usually display a dual binding mode that may allow increased conformational flexibility during cellulosome assembly. Acetivibrio cellulolyticus produces a highly complex cellulosome comprising an unusual adaptor scaffoldin, ScaB, which mediates the interaction between the primary scaffoldin, ScaA, through type II cohesin-dockerin interactions and the anchoring scaffoldin, ScaC, via type I cohesin-dockerin interactions. Here, we report the crystal structure of the type I ScaB dockerin in complex with a type I ScaC cohesin in two distinct orientations. The data show that the ScaB dockerin displays structural symmetry, reflected by the presence of two essentially identical binding surfaces. The complex interface is more extensive than those observed in other type I complexes, which results in an ultra-high affinity interaction (Ka ∼1012 m). A subset of ScaB dockerin residues was also identified as modulating the specificity of type I cohesin-dockerin interactions in A. cellulolyticus. This report reveals that recruitment of cellulosomes onto the cell surface may involve dockerins presenting a dual binding mode to incorporate additional flexibility into the quaternary structure of highly populated multienzyme complexes. PMID:25855788

  2. 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.

  3. Analysis of protein complexes using mass spectrometry.

    PubMed

    Gingras, Anne-Claude; Gstaiger, Matthias; Raught, Brian; Aebersold, Ruedi

    2007-08-01

    The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.

  4. p300/CREB Binding Protein-Related Protein p270 Is a Component of Mammalian SWI/SNF Complexes

    PubMed Central

    Dallas, Peter B.; Cheney, Ian Wayne; Liao, Da-Wei; Bowrin, Valerie; Byam, Whitney; Pacchione, Stephen; Kobayashi, Ryuji; Yaciuk, Peter; Moran, Elizabeth

    1998-01-01

    p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex. PMID:9584200

  5. Structural Studies of Protein-Surfactant Complexes

    SciTech Connect

    Chodankar, S. N.; Aswal, V. K.; Wagh, A. G.

    2008-03-17

    The structure of protein-surfactant complexes of two proteins bovine serum albumin (BSA) and lysozyme in presence of anionic surfactant sodium dodecyl sulfate (SDS) has been studied using small-angle neutron scattering (SANS). It is observed that these two proteins form different complex structures with the surfactant. While BSA protein undergoes unfolding on addition of surfactant, lysozyme does not show any unfolding even up to very high surfactant concentrations. The unfolding of BSA protein is caused by micelle-like aggregation of surfactant molecules in the complex. On the other hand, for lysozyme protein there is only binding of individual surfactant molecules to protein. Lysozyme in presence of higher surfactant concentrations has protein-surfactant complex structure coexisting with pure surfactant micelles.

  6. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  7. Lenalidomide inhibits proliferation of Namalwa CSN.70 cells and interferes with Gab1 phosphorylation and adaptor protein complex assembly.

    PubMed

    Gandhi, Anita K; Kang, Jian; Naziruddin, Syedah; Parton, Anastasia; Schafer, Peter H; Stirling, David I

    2006-07-01

    Lenalidomide (Revlimid, CC-5013) belongs to a line of compounds known as immunomodulatory drugs (IMiDs) that are under clinical investigation in hematopoietic and solid tumor cancers. Lenalidomide efficacy has been reported in clinical trials of multiple myeloma and myelodysplastic syndromes (MDS), particularly in MDS patients with a del 5q cytogenetic abnormality, with or without other cytogenetic abnormalities. Here we report that lenalidomide inhibits proliferation of chromosome 5 deleted hematopoietic tumor cell lines in vitro, whether from the B cell, T cell, or myeloid lineage. There was diversity in the responses of the various cell lines to lenalidomide, with one undergoing cell cycle arrest, and others undergoing apoptosis. In the most lenalidomide-sensitive chromosome 5 deleted cell line, Namalwa CSN.70, the compound induced G0/G1 cell cycle arrest, inhibited Akt and Gab1 phosphorylation, and inhibited the ability of Gab1 to associate with a receptor tyrosine kinase. Lenalidomide also enhanced AP-1 transcriptional activity in Namalwa, but not in the other cell lines tested. These studies provide evidence for the mechanism of action of lenalidomide in chromosome 5 deleted hematopoietic tumors in vitro, and may provide a better understanding of the drug's activity in clinical applications.

  8. Predictions of Protein-Protein Interfaces within Membrane Protein Complexes

    PubMed Central

    Asadabadi, Ebrahim Barzegari; Abdolmaleki, Parviz

    2013-01-01

    Background Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. PMID:23919118

  9. Co-translational assembly of protein complexes.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2015-12-01

    The interaction of biological macromolecules is a fundamental attribute of cellular life. Proteins, in particular, often form stable complexes with one another. Although the importance of protein complexes is widely recognized, we still have only a very limited understanding of the mechanisms underlying their assembly within cells. In this article, we review the available evidence for one such mechanism, namely the coupling of protein complex assembly to translation at the polysome. We discuss research showing that co-translational assembly can occur in both prokaryotic and eukaryotic organisms and can have important implications for the correct functioning of the complexes that result. Co-translational assembly can occur for both homomeric and heteromeric protein complexes and for both proteins that are translated directly into the cytoplasm and those that are translated into or across membranes. Finally, we discuss the properties of proteins that are most likely to be associated with co-translational assembly.

  10. A Protein Complex Map of Trypanosoma brucei

    PubMed Central

    Mehta, Vaibhav; Najafabadi, Hamed S.; Moshiri, Houtan; Jardim, Armando; Salavati, Reza

    2016-01-01

    The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org. PMID:26991453

  11. A Protein Complex Network of Drosophila melanogaster

    PubMed Central

    Guruharsha, K. G.; Rual, J. -F.; Zhai, B.; Mintseris, J.; Vaidya, P.; Vaidya, N.; Beekman, C.; Wong, C.; Rhee, D. Y.; Cenaj, O.; McKillip, E.; Shah, S.; Stapleton, M.; Wan, K. H.; Yu, C.; Parsa, B.; Carlson, J. W.; Chen, X.; Kapadia, B.; VijayRaghavan, K.; Gygi, S. P.; Celniker, S. E.; Obar, R. A.; Artavanis-Tsakonas, S.

    2011-01-01

    SUMMARY Determining the composition of protein complexes is an essential step towards understanding the cell as an integrated system. Using co-affinity purification coupled to mass spectrometry analysis, we examined protein associations involving nearly five thousand individual, FLAG-HA epitope-tagged Drosophila proteins. Stringent analysis of these data, based on a novel statistical framework to define individual protein-protein interactions, led to the generation of a Drosophila Protein interaction Map (DPiM) encompassing 556 protein complexes. The high quality of DPiM and its usefulness as a paradigm for metazoan proteomes is apparent from the recovery of many known complexes, significant enrichment for shared functional attributes and validation in human cells. DPiM defines potential novel members for several important protein complexes and assigns functional links to 586 protein-coding genes lacking previous experimental annotation. DPiM represents, to our knowledge, the largest metazoan protein complex map and provides a valuable resource for analysis of protein complex evolution. PMID:22036573

  12. Cutting edge: the "death" adaptor CRADD/RAIDD targets BCL10 and suppresses agonist-induced cytokine expression in T lymphocytes.

    PubMed

    Lin, Qing; Liu, Yan; Moore, Daniel J; Elizer, Sydney K; Veach, Ruth A; Hawiger, Jacek; Ruley, H Earl

    2012-03-15

    The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the "death" adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.

  13. The Canonical Inflammasome: A Macromolecular Complex Driving Inflammation.

    PubMed

    Monie, Tom P

    2017-01-01

    The inflammasome is a multi-molecular platform crucial to the induction of an inflammatory response to cellular danger. Recognition in the cytoplasm of endogenously and exogenously derived ligands initiates conformational change in sensor proteins, such as NLRP3, that permits the subsequent rapid recruitment of adaptor proteins, like ASC, and the resulting assembly of a large-scale inflammatory signalling platform. The assembly process is driven by sensor-sensor interactions as well as sensor-adaptor and adaptor-adaptor interactions. The resulting complex, which can reach diameters of around 1 micron, has a variable composition and stoichiometry. The inflammasome complex functions as a platform for the proximity induced activation of effector caspases, such as caspase-1 and caspase-8. This ultimately leads to the processing of the inflammatory cytokines pro-IL1β and pro-IL18 into their active forms, along with the cleavage of Gasdermin D, a key activator of cell death via pyroptosis.

  14. [Isolation of proteins with complex forming agents].

    PubMed

    Schwenke, K D; Raab, B; Ender, B

    1975-01-01

    Taking vegetable albumins for models, the authors report of the possibilities of isolating proteins (which cannot be precipitated isoelectrically) by using their property of forming complexes with tannin or poly-anions. The precipitation of proteins with dextran sulphate or polyphosphates, which is due to electrostatic interaction, depends on the pH value and the electrolyte content of the solution. Under appropriate experimental conditions, protein yields of 100% are achieved. By means of tannin, the proteins are completely precipitated in a wide range of pH. The protein component of the poly-anion-containing complexes is isolated by precipitation with salt or by thermal coagulation after dissolving of the complexes. The isolation of protein from the tannin complexes is preferably realized by reaction with coffeine.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-26

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

  17. Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

    PubMed Central

    Merhi, Ahmad

    2012-01-01

    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. PMID:22966204

  18. Complex lasso: new entangled motifs in proteins

    NASA Astrophysics Data System (ADS)

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-11-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules.

  19. Complex lasso: new entangled motifs in proteins

    PubMed Central

    Niemyska, Wanda; Dabrowski-Tumanski, Pawel; Kadlof, Michal; Haglund, Ellinor; Sułkowski, Piotr; Sulkowska, Joanna I.

    2016-01-01

    We identify new entangled motifs in proteins that we call complex lassos. Lassos arise in proteins with disulfide bridges (or in proteins with amide linkages), when termini of a protein backbone pierce through an auxiliary surface of minimal area, spanned on a covalent loop. We find that as much as 18% of all proteins with disulfide bridges in a non-redundant subset of PDB form complex lassos, and classify them into six distinct geometric classes, one of which resembles supercoiling known from DNA. Based on biological classification of proteins we find that lassos are much more common in viruses, plants and fungi than in other kingdoms of life. We also discuss how changes in the oxidation/reduction potential may affect the function of proteins with lassos. Lassos and associated surfaces of minimal area provide new, interesting and possessing many potential applications geometric characteristics not only of proteins, but also of other biomolecules. PMID:27874096

  20. Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors*

    PubMed Central

    Stahlschmidt, Wiebke; Robertson, Mark J.; Robinson, Phillip J.; McCluskey, Adam; Haucke, Volker

    2014-01-01

    Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane. PMID:24407285

  1. Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors.

    PubMed

    Stahlschmidt, Wiebke; Robertson, Mark J; Robinson, Phillip J; McCluskey, Adam; Haucke, Volker

    2014-02-21

    Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

  2. Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

    PubMed Central

    Martín-Villa, José Manuel; Benito-León, María; Martinez-Quiles, Narcisa

    2014-01-01

    Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization. PMID:24675776

  3. Serine residues in the LAT adaptor are essential for TCR-dependent signal transduction.

    PubMed

    Martínez-Florensa, Mario; García-Blesa, Antonio; Yélamos, José; Muñoz-Suano, Alba; Domínguez-Villar, Margarita; Valdor, Rut; Alonso, Antonio; García-Cózar, Francisco; Aparicio, Pedro; Malissen, Bernard; Aguado, Enrique

    2011-01-01

    The adaptor protein LAT has a prominent role in the transduction of intracellular signals elicited by the TCR/CD3 complex. Upon TCR engagement, LAT becomes tyrosine-phosphorylated and thereby, recruits to the membrane several proteins implicated in the activation of downstream signaling pathways. However, little is known about the role of other conserved motifs present in the LAT sequence. Here, we report that the adaptor LAT contains several conserved serine-based motifs, which are essential for proper signal transduction through the TCR. Mutation of these serine motifs in the human T cell line Jurkat prevents proper calcium influx, MAPK activation, and IL-2 production in response to TCR/CD3 stimulation. Moreover, this mutant form of LAT has a reduced ability to bind to PLC-γ1 and SLP-76, although phosphorylation of tyrosine residues 132, 171, and 191 is not decreased, raising a possible role for the serine-based motifs of LAT for the binding of important partners. The functional role of LAT serine-based motifs in signal transduction could be mediated by an effect on tyrosine phosphorylation, as their mutation significantly diminishes the phosphorylation of tyrosine residue 226. In addition, these serine motifs seem to have a regulatory role, given that upon their mutation, ZAP-70 shows enhanced phosphorylation. Therefore, the LAT serine-based motifs likely regulate signaling pathways that are essential for T cell physiology.

  4. Sub-cellular distribution of UNC-104(KIF1A) upon binding to adaptors as UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α) in C. elegans neurons.

    PubMed

    Hsu, C-C; Moncaleano, J D; Wagner, O I

    2011-03-10

    The accumulation of cargo (tau, amyloid precursor protein, neurofilaments etc.) in neurons is a hallmark of various neurodegenerative diseases while we have only little knowledge how axonal transport is regulated. Kinesin-3 UNC-104(KIF1A) is the major transporter of synaptic vesicles and recent reports suggest that a cargo itself can affect the motor's activity. Inspecting an interactome map, we identify three putative UNC-104 interactors, namely UNC-16(JIP3), DNC-1(DCTN1/Glued) and SYD-2(Liprin-α), known to be adaptors in essential neuronal protein complexes. We then employed the novel method bimolecular fluorescence complementation (BiFC) assay to visualize motor-adaptor complexes in the nervous system of living C. elegans. Interestingly, the binding of UNC-104 to each adaptor protein results in different sub-cellular distributions and has distinctive effects on the motor's motility. Specifically, if UNC-104 bound to UNC-16, the motor is primarily localized in the soma of neurons while bound to DNC-1, the motor is basically found in axonal termini. On the other hand, if UNC-104 is bound to SYD-2 we identify motor populations mostly along axons. Therefore, these three adaptors inherit different functions in steering the motor to specific sub-cellular locations in the neuron.

  5. Engineered Fv fragments as a tool for the one-step purification of integral multisubunit membrane protein complexes.

    PubMed

    Kleymann, G; Ostermeier, C; Ludwig, B; Skerra, A; Michel, H

    1995-02-01

    The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently insufficient for structural studies. To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments. cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column. The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and subcomplexes or individual subunits thereof. These membrane proteins were purified simply by combining the crude P. denitrificans membrane preparation with the E. coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidin affinity chromatography. Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag. The affinity column could thus be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.

  6. Structural Analysis of the Interaction between Dishevelled2 and Clathrin AP-2 Adaptor, A Critical Step in Noncanonical Wnt Signaling

    SciTech Connect

    Yu, Anan; Xing, Yi; Harrison, Stephen C.; Kirchhausen, Tomas

    2010-10-14

    Wnt association with its receptor, Frizzled (Fz), and recruitment by the latter of an adaptor, Dishevelled (Dvl), initiates signaling through at least two distinct pathways (canonical and noncanonical). Endocytosis and compartmentalization help determine the signaling outcome. Our previous work has shown that Dvl2 links at least one Frizzled family member (Fz4) to clathrin-mediated endocytosis by interacting with the {mu}2 subunit of the AP-2 clathrin adaptor, through both a classical endocytic tyrosine motif and a so-called DEP domain. We report here the crystal structure of a chimeric protein that mimics the Dvl2-{mu}2 complex. The DEP domain binds at one end of the elongated, C-terminal domain of {mu}2. This domain:domain interface shows that parts of the {mu}2 surface distinct from the tyrosine-motif site can help recruit specific receptors or adaptors into a clathrin coated pit. Mutation of residues at the DEP-{mu}2 contact or in the tyrosine motif reduce affinity of Dvl2 for {mu}2 and block efficient internalization of Fz4 in response to ligation by Wnt5a. The crystal structure has thus allowed us to identify the specific interaction that leads to Frizzled uptake and to downstream, noncanonical signaling events.

  7. Complementary Proteomic Analysis of Protein Complexes

    PubMed Central

    Greco, Todd M.; Miteva, Yana; Conlon, Frank L.; Cristea, Ileana M.

    2013-01-01

    Proteomic characterization of protein complexes leverages the versatile platform of liquid chromatography-tandem mass spectrometry to elucidate molecular and cellular signaling processes underlying the dynamic regulation of macromolecular assemblies. Here, we describe a complementary proteomic approach optimized for immunoisolated protein complexes. As the relative complexity, abundance, and physiochemical properties of proteins can vary significantly between samples, we have provided (1) complementary sample preparation workflows, (2) detailed steps for HPLC and mass spectrometric method development, and (3) a bioinformatic workflow that provides confident peptide/protein identification paired with unbiased functional gene ontology analysis. This protocol can also be extended for characterization of larger complexity samples from whole cell or tissue Xenopus proteomes. PMID:22956100

  8. The claudin Megatrachea protein complex.

    PubMed

    Jaspers, Martin H J; Nolde, Kai; Behr, Matthias; Joo, Seol-hee; Plessmann, Uwe; Nikolov, Miroslav; Urlaub, Henning; Schuh, Reinhard

    2012-10-26

    Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.

  9. The Fe65 adaptor protein interacts through its PID1 domain with the transcription factor CP2/LSF/LBP1.

    PubMed

    Zambrano, N; Minopoli, G; de Candia, P; Russo, T

    1998-08-07

    The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction.

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

    PubMed

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

    2015-10-13

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

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

    PubMed

    Zhong, Ming-Chao; Veillette, André

    2013-11-01

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

  12. Annotation of human cancers with EGFR signaling-associated protein complexes using proximity ligation assays

    PubMed Central

    Smith, Matthew A.; Hall, Richard; Fisher, Kate; Haake, Scott M.; Khalil, Farah; Schabath, Matthew B.; Vuaroqueaux, Vincent; Fiebig, Heinz-Herbert; Altiok, Soner; Chen, Y. Ann; Haura, Eric B.

    2015-01-01

    Strategies to measure functional signaling-associated protein complexes have the potential to augment current molecular biomarker assays, such as genotyping and expression profiling, used to annotate diseases. Aberrant activation of epidermal growth factor receptor (EGFR) signaling contributes to diverse cancers. Here, we used a proximity ligation assay (PLA) to detect EGFR in a complex with growth factor receptor-bound protein 2 (GRB2), the major signaling adaptor for EGFR. We used multiple lung cancer cell lines to develop and characterize EGFR:GRB2 PLA and correlated this assay with established biochemical measures of EGFR signaling. In a panel of patient-derived xenografts in mice, the intensity of EGFR:GRB2 PLA correlated with the reduction in tumor size in response to the EGFR inhibitor cetuximab. In tumor biopsies from three cohorts of lung cancer patients, positive EGFR:GRB2 PLA was observed in patients with and without EGFR mutations and the intensity of EGFR:GRB2 PLA was predictive of overall survival in an EGFR inhibitor-treated cohort. Thus, we established the feasibility of using PLA to measure EGFR signaling-associated protein complexes in patient-based materials, suggesting the potential for similar assays for a broader array of receptor tyrosine kinases and other key signaling molecules. PMID:25587191

  13. Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex

    PubMed Central

    1996-01-01

    Coatomer is a cytosolic protein complex that forms the coat of COP I- coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex. PMID:8858162

  14. TLR-independent control of innate immunity in Caenorhabditis elegans by the TIR domain adaptor protein TIR-1, an ortholog of human SARM.

    PubMed

    Couillault, Carole; Pujol, Nathalie; Reboul, Jérôme; Sabatier, Laurence; Guichou, Jean-François; Kohara, Yuji; Ewbank, Jonathan J

    2004-05-01

    Both plants and animals respond to infection by synthesizing compounds that directly inhibit or kill invading pathogens. We report here the identification of infection-inducible antimicrobial peptides in Caenorhabditis elegans. Expression of two of these peptides, NLP-29 and NLP-31, was differentially regulated by fungal and bacterial infection and was controlled in part by tir-1, which encodes an ortholog of SARM, a Toll-interleukin 1 receptor (TIR) domain protein. Inactivation of tir-1 by RNA interference caused increased susceptibility to infection. We identify protein partners for TIR-1 and show that the small GTPase Rab1 and the f subunit of ATP synthase participate specifically in the control of antimicrobial peptide gene expression. As the activity of tir-1 was independent of the single nematode Toll-like receptor, TIR-1 may represent a component of a previously uncharacterized, but conserved, innate immune signaling pathway.

  15. The AP-2 adaptor beta2 appendage scaffolds alternate cargo endocytosis.

    PubMed

    Keyel, Peter A; Thieman, James R; Roth, Robyn; Erkan, Elif; Everett, Eric T; Watkins, Simon C; Heuser, John E; Traub, Linton M

    2008-12-01

    The independently folded appendages of the large alpha and beta2 subunits of the endocytic adaptor protein (AP)-2 complex coordinate proper assembly and operation of endocytic components during clathrin-mediated endocytosis. The beta2 subunit appendage contains a common binding site for beta-arrestin or the autosomal recessive hypercholesterolemia (ARH) protein. To determine the importance of this interaction surface in living cells, we used small interfering RNA-based gene silencing. The effect of extinguishing beta2 subunit expression on the internalization of transferrin is considerably weaker than an AP-2 alpha subunit knockdown. We show the mild sorting defect is due to fortuitous substitution of the beta2 chain with the closely related endogenous beta1 subunit of the AP-1 adaptor complex. Simultaneous silencing of both beta1 and beta2 subunit transcripts recapitulates the strong alpha subunit RNA interference (RNAi) phenotype and results in loss of ARH from endocytic clathrin coats. An RNAi-insensitive beta2-yellow fluorescent protein (YFP) expressed in the beta1 + beta2-silenced background restores cellular AP-2 levels, robust transferrin internalization, and ARH colocalization with cell surface clathrin. The importance of the beta appendage platform subdomain over clathrin for precise deposition of ARH at clathrin assembly zones is revealed by a beta2-YFP with a disrupted ARH binding interface, which does not restore ARH colocalization with clathrin. We also show a beta-arrestin 1 mutant, which engages coated structures in the absence of any G protein-coupled receptor stimulation, colocalizes with beta2-YFP and clathrin even in the absence of an operational clathrin binding sequence. These findings argue against ARH and beta-arrestin binding to a site upon the beta2 appendage platform that is later obstructed by polymerized clathrin. We conclude that ARH and beta-arrestin depend on a privileged beta2 appendage site for proper cargo recruitment to clathrin

  16. Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

    PubMed

    Sen, Arnab; Sun, Rui; Krahn, Michael P

    2015-05-22

    The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

  17. Proteomics: bases for protein complexity understanding.

    PubMed

    Rotilio, Domenico; Della Corte, Anna; D'Imperio, Marco; Coletta, Walter; Marcone, Simone; Silvestri, Cristian; Giordano, Lucia; Di Michele, Michela; Donati, Maria Benedetta

    2012-03-01

    In the post genomic era we became aware that the genomic sequence and protein functions cannot be correlated. One gene can encode multiple protein functions mainly because of mRNA splice variants, post translational modifications (PTM) and moonlighting functions. To study the whole population of proteins present in a cell to a specific time point and under defined conditions it is necessary to investigate the proteome. Comprehensive analysis of the proteome requires the use of emerging high technologies because of the complexity and wide dynamic range of protein concentrations. Proteomics provides the tools to study protein identification and quantitation, protein-protein interactions, protein modifications and localization. The most widespread strategy for studying global protein expression employs two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowing thousands of proteins to be resolved and their expression quantified. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a high throughput technique for protein identification and characterization because of its high sensitivity, precision and accuracy. LC-MS/MS is well suited for accurate quantitation of protein expression levels, post-translational modifications and comparative and absolute quantitative analysis of peptides. Bioinformatic tools are required to elaborate the growing number of proteomic data. Here, we give an overview of the current status of the wide range of technologies that define and characterize the modern proteomics.

  18. Protein-protein interactions in the synaptonemal complex.

    PubMed Central

    Tarsounas, M; Pearlman, R E; Gasser, P J; Park, M S; Moens, P B

    1997-01-01

    In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II. Images PMID:9285814

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

    PubMed

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

    2015-12-01

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

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

    SciTech Connect

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

    2008-05-01

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

  1. Phosphorylation of the adaptor protein SH2B1β regulates its ability to enhance growth hormone-dependent macrophage motility.

    PubMed

    Su, Hsiao-Wen; Lanning, Nathan J; Morris, David L; Argetsinger, Lawrence S; Lumeng, Carey N; Carter-Su, Christin

    2013-04-15

    Previous studies have shown that growth hormone (GH) recruits the adapter protein SH2B1β to the GH-activated, GH receptor-associated tyrosine kinase JAK2, implicating SH2B1β in GH-dependent actin cytoskeleton remodeling, and suggesting that phosphorylation at serines 161 and 165 in SH2B1β releases SH2B1β from the plasma membrane. Here, we examined the role of SH2B1β in GH regulation of macrophage migration. We show that GH stimulates migration of cultured RAW264.7 macrophages, and primary cultures of peritoneal and bone marrow-derived macrophages. SH2B1β overexpression enhances, whereas SH2B1 knockdown inhibits, GH-dependent motility of RAW macrophages. At least two independent mechanisms regulate the SH2B1β-mediated changes in motility. In response to GH, tyrosines 439 and 494 in SH2B1β are phosphorylated. Mutating these tyrosines in SH2B1β decreases both basal and GH-stimulated macrophage migration. In addition, mutating the polybasic nuclear localization sequence (NLS) in SH2B1β or creating the phosphomimetics SH2B1β(S161E) or SH2B1β(S165E), all of which release SH2B1β from the plasma membrane, enhances macrophage motility. Conversely, SH2B1β(S161/165A) exhibits increased localization at the plasma membrane and decreased macrophage migration. Mutating the NLS or the nearby serine residues does not alter GH-dependent phosphorylation on tyrosines 439 and 494 in SH2B1β. Mutating tyrosines 439 and 494 does not affect localization of SH2B1β at the plasma membrane or movement of SH2B1β into focal adhesions. Taken together, these results suggest that SH2B1β enhances GH-stimulated macrophage motility via mechanisms involving phosphorylation of SH2B1β on tyrosines 439 and 494 and movement of SH2B1β out of the plasma membrane (e.g. as a result of phosphorylation of serines 161 and 165).

  2. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells

    PubMed Central

    1993-01-01

    GPI-linked protein molecules become Triton-insoluble during polarized sorting to the apical cell surface of epithelial cells. These insoluble complexes, enriched in cholesterol, glycolipids, and GPI-linked proteins, have been isolated by flotation on sucrose density gradients and are thought to contain the putative GPI-sorting machinery. As the cellular origin and molecular protein components of this complex remain unknown, we have begun to characterize these low-density insoluble complexes isolated from MDCK cells. We find that these complexes, which represent 0.4-0.8% of the plasma membrane, ultrastructurally resemble caveolae and are over 150-fold enriched in a model GPI-anchored protein and caveolin, a caveolar marker protein. However, they exclude many other plasma membrane associated molecules and organelle-specific marker enzymes, suggesting that they represent microdomains of the plasma membrane. In addition to caveolin, these insoluble complexes contain a subset of hydrophobic plasma membrane proteins and cytoplasmically-oriented signaling molecules, including: (a) GTP- binding proteins--both small and heterotrimeric; (b) annex II--an apical calcium-regulated phospholipid binding protein with a demonstrated role in exocytic fusion events; (c) c-Yes--an apically localized member of the Src family of non-receptor type protein- tyrosine kinases; and (d) an unidentified serine-kinase activity. As we demonstrate that caveolin is both a transmembrane molecule and a major phospho-acceptor component of these complexes, we propose that caveolin could function as a transmembrane adaptor molecule that couples luminal GPI-linked proteins with cytoplasmically oriented signaling molecules during GPI-membrane trafficking or GPI-mediated signal transduction events. In addition, our results have implications for understanding v- Src transformation and the actions of cholera and pertussis toxins on hetero-trimeric G proteins. PMID:8349730

  3. Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

    PubMed Central

    Tunnicliffe, Richard B.; Hautbergue, Guillaume M.; Wilson, Stuart A.; Kalra, Priti; Golovanov, Alexander P.

    2014-01-01

    The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions. PMID:24550725

  4. Quantification of Detergents Complexed with Membrane Proteins

    PubMed Central

    Chaptal, Vincent; Delolme, Frédéric; Kilburg, Arnaud; Magnard, Sandrine; Montigny, Cédric; Picard, Martin; Prier, Charlène; Monticelli, Luca; Bornert, Olivier; Agez, Morgane; Ravaud, Stéphanie; Orelle, Cédric; Wagner, Renaud; Jawhari, Anass; Broutin, Isabelle; Pebay-Peyroula, Eva; Jault, Jean-Michel; Kaback, H. Ronald; le Maire, Marc; Falson, Pierre

    2017-01-01

    Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies. PMID:28176812

  5. Peroxisome protein import: a complex journey

    PubMed Central

    Baker, Alison; Hogg, Thomas Lanyon; Warriner, Stuart L.

    2016-01-01

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor–cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. PMID:27284042

  6. New Anthocyanin-Human Salivary Protein Complexes.

    PubMed

    Ferrer-Gallego, Raúl; Soares, Susana; Mateus, Nuno; Rivas-Gonzalo, Julián; Escribano-Bailón, M Teresa; de Freitas, Victor

    2015-08-04

    The interaction between phenolic compounds and salivary proteins is considered the basis of the poorly understood phenomenon of astringency. Furthermore, this interaction is an important factor in relation to their bioavailability. In this work, interactions between anthocyanin and human salivary protein fraction were studied by mass spectrometry (MALDI-TOF-MS and FIA-ESI-MS) and saturation-transfer difference (STD) NMR spectroscopy. Anthocyanins were able to interact with saliva proteins. The dissociation constant (KD) between malvidin 3-glucoside and salivary proline-rich proteins was 1.92 mM for the hemiketal form (pH 3.4) and 1.83 mM for the flavylium cation (pH 1.0). New soluble complexes between these salivary proteins and malvidin 3-glucoside were identified for the first time.

  7. Adaptor assembly for coupling turbine blades to rotor disks

    SciTech Connect

    Garcia-Crespo, Andres Jose; Delvaux, John McConnell

    2014-09-23

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

  8. Assembly reflects evolution of protein complexes.

    PubMed

    Levy, Emmanuel D; Boeri Erba, Elisabetta; Robinson, Carol V; Teichmann, Sarah A

    2008-06-26

    A homomer is formed by self-interacting copies of a protein unit. This is functionally important, as in allostery, and structurally crucial because mis-assembly of homomers is implicated in disease. Homomers are widespread, with 50-70% of proteins with a known quaternary state assembling into such structures. Despite their prevalence, their role in the evolution of cellular machinery and the potential for their use in the design of new molecular machines, little is known about the mechanisms that drive formation of homomers at the level of evolution and assembly in the cell. Here we present an analysis of over 5,000 unique atomic structures and show that the quaternary structure of homomers is conserved in over 70% of protein pairs sharing as little as 30% sequence identity. Where quaternary structure is not conserved among the members of a protein family, a detailed investigation revealed well-defined evolutionary pathways by which proteins transit between different quaternary structure types. Furthermore, we show by perturbing subunit interfaces within complexes and by mass spectrometry analysis, that the (dis)assembly pathway mimics the evolutionary pathway. These data represent a molecular analogy to Haeckel's evolutionary paradigm of embryonic development, where an intermediate in the assembly of a complex represents a form that appeared in its own evolutionary history. Our model of self-assembly allows reliable prediction of evolution and assembly of a complex solely from its crystal structure.

  9. Hsp70 Protein Complexes as Drug Targets

    PubMed Central

    Assimon, Victoria A.; Gillies, Anne T.; Rauch, Jennifer N.; Gestwicki, Jason E.

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70’s interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, such as pro-folding, pro-degradation and pro-trafficking. Thus, a promising strategy may be to block protein-protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to those goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology. PMID:22920901

  10. Hsp70 protein complexes as drug targets.

    PubMed

    Assimon, Victoria A; Gillies, Anne T; Rauch, Jennifer N; Gestwicki, Jason E

    2013-01-01

    Heat shock protein 70 (Hsp70) plays critical roles in proteostasis and is an emerging target for multiple diseases. However, competitive inhibition of the enzymatic activity of Hsp70 has proven challenging and, in some cases, may not be the most productive way to redirect Hsp70 function. Another approach is to inhibit Hsp70's interactions with important co-chaperones, such as J proteins, nucleotide exchange factors (NEFs) and tetratricopeptide repeat (TPR) domain-containing proteins. These co-chaperones normally bind Hsp70 and guide its many diverse cellular activities. Complexes between Hsp70 and co-chaperones have been shown to have specific functions, including roles in pro-folding, pro-degradation and pro-trafficking pathways. Thus, a promising strategy may be to block protein- protein interactions between Hsp70 and its co-chaperones or to target allosteric sites that disrupt these contacts. Such an approach might shift the balance of Hsp70 complexes and re-shape the proteome and it has the potential to restore healthy proteostasis. In this review, we discuss specific challenges and opportunities related to these goals. By pursuing Hsp70 complexes as drug targets, we might not only develop new leads for therapeutic development, but also discover new chemical probes for use in understanding Hsp70 biology.

  11. Characterization of protein complexes using targeted proteomics.

    PubMed

    Gomez, Yassel Ramos; Gallien, Sebastien; Huerta, Vivian; van Oostrum, Jan; Domon, Bruno; Gonzalez, Luis Javier

    2014-01-01

    Biological systems are not only controlled by the abundance of individual proteins, but also by the formation of complexes and the dynamics of protein-protein interactions. The identification of the components of protein complexes can be obtained by shotgun proteomics using affinity purification coupled to mass spectrometry. Such studies include the analyses of several samples and experimental controls in order to discriminate true specific interactions from unspecific interactions and contaminants. However, shotgun proteomics have limited quantification capabilities for low abundant proteins on large sample sets due to the undersampling and the stochastic precursor ion selection. In this context, targeted proteomics constitutes a powerful analytical tool to systematically detect and quantify peptides in multiple samples, for instance those obtained from affinity purification experiments. Hypothesis-driven strategies have mainly relied on the selected reaction monitoring (SRM) technique performed on triple quadrupole instruments, which enables highly selective and sensitive measurements of peptides, acting as surrogates of the pre-selected proteins, over a wide range of concentrations. More recently, novel quantitative methods based on high resolution instruments, such as the parallel reaction monitoring (PRM) technique implemented on the quadrupole-orbitrap instrument, have arisen and provided alternatives to perform quantitative analyses with enhanced selectivity.The application of targeted proteomics to protein-protein interaction experiments from plasma and other physiological fluid samples and the inclusion of parallel reaction monitoring (PRM), combined with other recent technology developments opens a vast area for clinical application of proteomics. It is anticipated that it will reveal valuable information about specific, individual, responses against drugs, exogenous proteins or pathogens.

  12. Interaction graph mining for protein complexes using local clique merging.

    PubMed

    Li, Xiao-Li; Tan, Soon-Heng; Foo, Chuan-Sheng; Ng, See-Kiong

    2005-01-01

    While recent technological advances have made available large datasets of experimentally-detected pairwise protein-protein interactions, there is still a lack of experimentally-determined protein complex data. To make up for this lack of protein complex data, we explore the mining of existing protein interaction graphs for protein complexes. This paper proposes a novel graph mining algorithm to detect the dense neighborhoods (highly connected regions) in an interaction graph which may correspond to protein complexes. Our algorithm first locates local cliques for each graph vertex (protein) and then merge the detected local cliques according to their affinity to form maximal dense regions. We present experimental results with yeast protein interaction data to demonstrate the effectiveness of our proposed method. Compared with other existing techniques, our predicted complexes can match or overlap significantly better with the known protein complexes in the MIPS benchmark database. Novel protein complexes were also predicted to help biologists in their search for new protein complexes.

  13. A single common portal for clathrin-mediated endocytosis of distinct cargo governed by cargo-selective adaptors.

    PubMed

    Keyel, Peter A; Mishra, Sanjay K; Roth, Robyn; Heuser, John E; Watkins, Simon C; Traub, Linton M

    2006-10-01

    Sorting of transmembrane cargo into clathrin-coated vesicles requires endocytic adaptors, yet RNA interference (RNAi)-mediated gene silencing of the AP-2 adaptor complex only disrupts internalization of a subset of clathrin-dependent cargo. This suggests alternate clathrin-associated sorting proteins participate in cargo capture at the cell surface, and a provocative recent proposal is that discrete endocytic cargo are sorted into compositionally and functionally distinct clathrin coats. We show here that the FXNPXY-type internalization signal within cytosolic domain of the LDL receptor is recognized redundantly by two phosphotyrosine-binding domain proteins, Dab2 and ARH; diminishing both proteins by RNAi leads to conspicuous LDL receptor accumulation at the cell surface. AP-2-dependent uptake of transferrin ensues relatively normally in the absence of Dab2 and ARH, clearly revealing delegation of sorting operations at the bud site. AP-2, Dab2, ARH, transferrin, and LDL receptors are all present within the vast majority of clathrin structures at the surface, challenging the general existence of specialized clathrin coats for segregated internalization of constitutively internalized cargo. However, Dab2 expression is exceptionally low in hepatocytes, likely accounting for the pathological hypercholesterolemia that accompanies ARH loss.

  14. 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)

  15. Dynamic complex formation between HD-GYP, GGDEF and PilZ domain proteins regulates motility in Xanthomonas campestris.

    PubMed

    Ryan, Robert P; McCarthy, Yvonne; Kiely, Patrick A; O'Connor, Rosemary; Farah, Chuck S; Armitage, Judith P; Dow, J Maxwell

    2012-11-01

    RpfG is a member of a class of wide spread bacterial two-component regulators with an HD-GYP cyclic di-GMP phosphodiesterase domain. In the plant pathogen Xanthomonas campestris, RpfG together with the sensor kinase RpfC regulates multiple factors as a response to the cell-to-cell Diffusible Signalling Factor (DSF). A dynamic physical interaction of RpfG with two diguanylate cyclase (GGDEF) domain proteins controls motility. Here we show that, contrary to expectation, regulation of motility by the GGDEF domain proteins does not depend upon their cyclic di-GMP synthetic activity. Furthermore we show that the complex of RpfG and GGDEF domain proteins recruits a specific PilZ domain 'adaptor' protein, and this complex then interacts with the pilus motor proteins PilU and PiIT. The results support a model in which DSF signalling influences motility through the highly regulated dynamic interaction of proteins that affect pilus action. A specific motif that we identify to be required for HD-GYP domain interaction is conserved in a number of GGDEF domain proteins, suggesting that regulation via interdomain interactions is of broad relevance.

  16. BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes

    PubMed Central

    Di Pietro, Santiago M.; Falcón-Pérez, Juan M.; Tenza, Danièle; Setty, Subba R.G.; Marks, Michael S.; Raposo, Graça

    2006-01-01

    The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery. PMID:16837549

  17. BLOC-1 interacts with BLOC-2 and the AP-3 complex to facilitate protein trafficking on endosomes.

    PubMed

    Di Pietro, Santiago M; Falcón-Pérez, Juan M; Tenza, Danièle; Setty, Subba R G; Marks, Michael S; Raposo, Graça; Dell'Angelica, Esteban C

    2006-09-01

    The adaptor protein (AP)-3 complex is a component of the cellular machinery that controls protein sorting from endosomes to lysosomes and specialized related organelles such as melanosomes. Mutations in an AP-3 subunit underlie a form of Hermansky-Pudlak syndrome (HPS), a disorder characterized by abnormalities in lysosome-related organelles. HPS in humans can also be caused by mutations in genes encoding subunits of three complexes of unclear function, named biogenesis of lysosome-related organelles complex (BLOC)-1, -2, and -3. Here, we report that BLOC-1 interacts physically and functionally with AP-3 to facilitate the trafficking of a known AP-3 cargo, CD63, and of tyrosinase-related protein 1 (Tyrp1), a melanosomal membrane protein previously thought to traffic only independently of AP-3. BLOC-1 also interacts with BLOC-2 to facilitate Tyrp1 trafficking by a mechanism apparently independent of AP-3 function. Both BLOC-1 and -2 localize mainly to early endosome-associated tubules as determined by immunoelectron microscopy. These findings support the idea that BLOC-1 and -2 represent hitherto unknown components of the endosomal protein trafficking machinery.

  18. Thermodynamics of interfacial changes in a protein-protein complex.

    PubMed

    Das, Amit; Chakrabarti, Jaydeb; Ghosh, Mahua

    2014-03-04

    Recent experiments with biomacromolecular complexes suggest that structural modifications at the interfaces are vital for stability of the complexes and the functions of the biomacromolecules. Although several qualitative aspects about such interfaces are known from structural data, quantification of the interfacial changes is lacking. In this work, we study the thermodynamic changes at the interface in the complex between an enzyme, Nuclease A (NucA), and a specific inhibitor protein, NuiA. We calculate the conformational free energy and conformational entropy costs from histograms of the dihedral angles generated from all-atom molecular dynamics simulations on the complex and the free proteins. We extract the conformational thermodynamic parameters for changes in the tertiary structure of NuiA. We show that the binding is dominated by the interfacial changes, where the basic residues of NucA and acidic residues of NuiA are highly ordered and stabilized via strong electrostatic interactions. Our results correlate well with known information from structural studies. The tight interfacial structure is reflected in the significant changes in the structure and dynamics of the water molecules at the enzyme-inhibitor interface. The interfacial water molecules contribute significantly to the entropy loss for the overall complexation.

  19. Structural Determinants of Human FANCF Protein That Function in the Assembly of a DNA Damage Signaling Complex

    SciTech Connect

    Kowal,P.; Gurtan, A.; Stuckert, P.; D'Andrea, A.; Ellenberger, T.

    2007-01-01

    Fanconi anemia (FA) is a rare autosomal recessive and X-linked chromosomal instability disorder. At least eight FA proteins (FANCA, B, C, E, F, G, L, and M) form a nuclear core complex required for monoubiquitination of a downstream protein, FANCD2. The human FANCF protein reportedly functions as a molecular adaptor within the FA nuclear complex, bridging between the subcomplexes A:G and C:E. Our x-ray crystallographic studies of the C-terminal domain of FANCF reveal a helical repeat structure similar to the Cand1 regulator of the Cul1-Rbx1-Skp1-Fbox(Skp2) ubiquitin ligase complex. Two C-terminal loops of FANCF are essential for monoubiquitination of FANCD2 and normal cellular resistance to the DNA cross-linking agent mitomycin C. FANCF mutants bearing amino acid substitutions in this C-terminal surface fail to interact with other components of the FA complex, indicating that this surface is critical for the proper assembly of the FA core complex.

  20. 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,…

  1. 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...

  2. 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...

  3. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  4. 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...

  5. 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...

  6. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  7. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  8. 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...

  9. 21 CFR 870.2350 - Electrocardiograph lead switching adaptor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  10. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion.

    PubMed

    Ekiert, Damian C; Cox, Jeffery S

    2014-10-14

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Moreover, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.

  11. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    DOE PAGES

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-10-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here in this paper, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), whichmore » adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Furthermore, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.« less

  12. DAPP1: a dual adaptor for phosphotyrosine and 3-phosphoinositides.

    PubMed

    Dowler, S; Currie, R A; Downes, C P; Alessi, D R

    1999-08-15

    We have identified a novel 280 amino acid protein which contains a putative myristoylation site at its N-terminus followed by an Src homology (SH2) domain and a pleckstrin homology (PH) domain at its C-terminus. It has been termed dual adaptor for phosphotyrosine and 3-phosphoinositides (DAPP1). DAPP1 is widely expressed and exhibits high-affinity interactions with PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), but not with other phospholipids tested. These observations predict that DAPP1 will interact with both tyrosine phosphorylated proteins and 3-phosphoinositides and may therefore play a role in regulating the location and/or activity of such proteins(s) in response to agonists that elevate PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

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

    PubMed

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

    2011-04-01

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

  14. The myxoma virus m-t5 ankyrin repeat host range protein is a novel adaptor that coordinately links the cellular signaling pathways mediated by Akt and Skp1 in virus-infected cells.

    PubMed

    Werden, Steven J; Lanchbury, Jerry; Shattuck, Donna; Neff, Chris; Dufford, Max; McFadden, Grant

    2009-12-01

    Most poxviruses express multiple proteins containing ankyrin (ANK) repeats accounting for a large superfamily of related but unique determinants of poxviral tropism. Recently, select members of this novel family of poxvirus proteins have drawn considerable attention for their potential roles in modulating intracellular signaling networks during viral infection. The rabbit-specific poxvirus, myxoma virus (MYXV), encodes four unique ANK repeat proteins, termed M-T5, M148, M149, and M150, all of which include a carboxy-terminal PRANC domain which closely resembles a cellular protein motif called the F-box domain. Here, we show that each MYXV-encoded ANK repeat protein, including M-T5, interacts directly with the Skp1 component of the host SCF ubiquitin ligase complex, and that the binding of M-T5 to cullin 1 is indirect via binding to Skp1 in the host SCF complex. To understand the significance of these virus-host protein interactions, the various binding domains of M-T5 were mapped. The N-terminal ANK repeats I and II were identified as being important for interaction with Akt, whereas the C-terminal PRANC/F-box-like domain was essential for binding to Skp1. We also report that M-T5 can bind Akt and the host SCF complex (via Skp1) simultaneously in MYXV-infected cells. Finally, we report that M-T5 specifically mediates the relocalization of Akt from the nucleus to the cytoplasm during infection with the wild-type MYXV, but not the M-T5 knockout version of the virus. These results indicate that ANK/PRANC proteins play a critical role in reprogramming disparate cellular signaling cascades to establish a new cellular environment more favorable for virus replication.

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

    SciTech Connect

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

    2010-10-28

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

  16. Analysis of DNA-protein complexes induced by chemical carcinogens

    SciTech Connect

    Costa, M. )

    1990-11-01

    DNA-protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis-platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA-protein complex. An antiserum which was raised to chromate-induced DNA-protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA-protein complexes formed by cis-platinum, UV, formaldehyde, and chromate. Other work in this area, involving studying whether DNA-protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA-protein complexes, the stability and repair of these lesions, and characterization of DNA-protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA-protein complexes and these findings are also reviewed.

  17. The LIM domain protein nTRIP6 recruits the mediator complex to AP-1-regulated promoters.

    PubMed

    Diefenbacher, Markus E; Reich, Daniela; Dahley, Oliver; Kemler, Denise; Litfin, Margarethe; Herrlich, Peter; Kassel, Olivier

    2014-01-01

    Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters.

  18. Neuronal Roles of the Bicaudal D Family of Motor Adaptors.

    PubMed

    Budzinska, M; Wicher, K B; Terenzio, M

    2017-01-01

    All cell types rely on active intracellular cargo transport to shuttle essential cellular components such as proteins, lipids, RNA, and even organelles from the center to the periphery and vice versa. Additionally, several signaling pathways take advantage of intracellular transport to propagate their signals by moving activated receptors and protein effectors to specific locations inside the cell. Neurons particularly, being a very polarized cell type, are highly dependent on molecular motors for the anterograde and retrograde delivery of essential cellular components and signaling molecules. For these reasons, motor adaptor proteins have been extensively investigated in regard to their role in physiology and pathology of the nervous system. In this chapter, we will concentrate on a family of motor adaptor proteins, Bicaudal D (BICD), and their function in the context of the nervous system. BicD was originally described as essential for the correct localization of maternal mRNAs in Drosophila's oocyte and a regulator of the Golgi to ER retrograde transport in mammalian cells. Both mammalian BICD1 and BICD2 are highly expressed in the nervous system during development, and their importance in neuronal homeostasis has been recently under scrutiny. Several mutations in BICD2 have been linked to the development of neuromuscular diseases, and BICD2 knockout (KO) mice display migration defects of the radial cerebellar granule cells. More in line with the overall topic of this book, BICD1 was identified as a novel regulator of neurotrophin (NT) signaling as its deletion leads to defective sorting of ligand-activated NT receptors with dramatic consequences on the NT-mediated signaling pathway.

  19. Engineering of complex protein sialylation in plants

    PubMed Central

    Kallolimath, Somanath; Castilho, Alexandra; Strasser, Richard; Grünwald-Gruber, Clemens; Altmann, Friedrich; Strubl, Sebastian; Galuska, Christina Elisabeth; Zlatina, Kristina; Galuska, Sebastian Peter; Werner, Stefan; Thiesler, Hauke; Werneburg, Sebastian; Hildebrandt, Herbert; Gerardy-Schahn, Rita; Steinkellner, Herta

    2016-01-01

    Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions. PMID:27444013

  20. AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster.

    PubMed

    Kametaka, Satoshi; Kametaka, Ai; Yonekura, Shinichi; Haruta, Mineyuki; Takenoshita, Seiichi; Goto, Satoshi; Waguri, Satoshi

    2012-02-01

    Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

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

    PubMed

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

    2013-01-01

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

  2. Detecting overlapping protein complexes by rough-fuzzy clustering in protein-protein interaction networks.

    PubMed

    Wu, Hao; Gao, Lin; Dong, Jihua; Yang, Xiaofei

    2014-01-01

    In this paper, we present a novel rough-fuzzy clustering (RFC) method to detect overlapping protein complexes in protein-protein interaction (PPI) networks. RFC focuses on fuzzy relation model rather than graph model by integrating fuzzy sets and rough sets, employs the upper and lower approximations of rough sets to deal with overlapping complexes, and calculates the number of complexes automatically. Fuzzy relation between proteins is established and then transformed into fuzzy equivalence relation. Non-overlapping complexes correspond to equivalence classes satisfying certain equivalence relation. To obtain overlapping complexes, we calculate the similarity between one protein and each complex, and then determine whether the protein belongs to one or multiple complexes by computing the ratio of each similarity to maximum similarity. To validate RFC quantitatively, we test it in Gavin, Collins, Krogan and BioGRID datasets. Experiment results show that there is a good correspondence to reference complexes in MIPS and SGD databases. Then we compare RFC with several previous methods, including ClusterONE, CMC, MCL, GCE, OSLOM and CFinder. Results show the precision, sensitivity and separation are 32.4%, 42.9% and 81.9% higher than mean of the five methods in four weighted networks, and are 0.5%, 11.2% and 66.1% higher than mean of the six methods in five unweighted networks. Our method RFC works well for protein complexes detection and provides a new insight of network division, and it can also be applied to identify overlapping community structure in social networks and LFR benchmark networks.

  3. Adaptor assembly for coupling turbine blades to rotor disks

    SciTech Connect

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

    2014-06-03

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

  4. 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.

  5. Construction of ontology augmented networks for protein complex prediction.

    PubMed

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian

    2013-01-01

    Protein complexes are of great importance in understanding the principles of cellular organization and function. The increase in available protein-protein interaction data, gene ontology and other resources make it possible to develop computational methods for protein complex prediction. Most existing methods focus mainly on the topological structure of protein-protein interaction networks, and largely ignore the gene ontology annotation information. In this article, we constructed ontology augmented networks with protein-protein interaction data and gene ontology, which effectively unified the topological structure of protein-protein interaction networks and the similarity of gene ontology annotations into unified distance measures. After constructing ontology augmented networks, a novel method (clustering based on ontology augmented networks) was proposed to predict protein complexes, which was capable of taking into account the topological structure of the protein-protein interaction network, as well as the similarity of gene ontology annotations. Our method was applied to two different yeast protein-protein interaction datasets and predicted many well-known complexes. The experimental results showed that (i) ontology augmented networks and the unified distance measure can effectively combine the structure closeness and gene ontology annotation similarity; (ii) our method is valuable in predicting protein complexes and has higher F1 and accuracy compared to other competing methods.

  6. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

    PubMed

    Lee, Hyeon-Cheol; Portnoff, Alyse D; Rocco, Mark A; DeLisa, Matthew P

    2014-12-22

    The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

  7. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism

    PubMed Central

    Lee, Hyeon-Cheol; Portnoff, Alyse D.; Rocco, Mark A.; DeLisa, Matthew P.

    2014-01-01

    The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary “hitchhiker” mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria. PMID:25531212

  8. Comparative distribution of protein components of the A20 ubiquitin-editing complex in normal human brain

    PubMed Central

    Pranski, Elaine L.; Van Sanford, Carson D.; Dalal, Nirjari V.; Orr, Adam L.; Karmali, Dipan; Cooper, Deborah S.; Costa, Nicole; Heilman, Craig J.; Gearing, Marla; Lah, James J.; Levey, Allan I.; Betarbet, Ranjita S.

    2012-01-01

    Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration. PMID:22634524

  9. Separation of membrane protein complexes by native LDS-PAGE.

    PubMed

    Arnold, Janine; Shapiguzov, Alexey; Fucile, Geoffrey; Rochaix, Jean-David; Goldschmidt-Clermont, Michel; Eichacker, Lutz Andreas

    2014-01-01

    Gel electrophoresis has become one of the most important methods for the analysis of proteins and protein complexes in a molecular weight range of 1-10(7) kDa. The separation of membrane protein complexes remained challenging to standardize until the demonstration of Blue Native PAGE in 1991 [1] and Clear Native PAGE in 1994 [2]. We present a robust protocol for high-resolution separation of photosynthetic complexes from Arabidopsis thaliana using lithium dodecyl sulfate as anion in a modified Blue Native PAGE (LDS-PAGE). Here, non-covalently bound chlorophyll is used as a sensitive probe to characterize the assembly/biogenesis of the pigment-protein complexes essential for photosynthesis. The high fluorescence yield recorded from chlorophyll-binding protein complexes can also be used to establish the separation of native protein complexes as an electrophoretic standard.

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

    PubMed

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

    2015-04-20

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

  11. SCIMP is a transmembrane non-TIR TLR adaptor that promotes proinflammatory cytokine production from macrophages

    PubMed Central

    Luo, Lin; Bokil, Nilesh J.; Wall, Adam A.; Kapetanovic, Ronan; Lansdaal, Natalie M.; Marceline, Faustine; Burgess, Belinda J.; Tong, Samuel J.; Guo, Zhong; Alexandrov, Kirill; Ross, Ian L.; Hibbs, Margaret L.; Stow, Jennifer L.; Sweet, Matthew J.

    2017-01-01

    Danger signals activate Toll-like receptors (TLRs), thereby initiating inflammatory responses. Canonical TLR signalling, via Toll/Interleukin-1 receptor domain (TIR)-containing adaptors and proinflammatory transcription factors such as NF-κB, occurs in many cell types; however, additional mechanisms are required for specificity of inflammatory responses in innate immune cells. Here we show that SCIMP, an immune-restricted, transmembrane adaptor protein (TRAP), promotes selective proinflammatory cytokine responses by direct modulation of TLR4. SCIMP is a non-TIR-containing adaptor, binding directly to the TLR4-TIR domain in response to lipopolysaccharide. In macrophages, SCIMP is constitutively associated with the Lyn tyrosine kinase, is required for tyrosine phosphorylation of TLR4, and facilitates TLR-inducible production of the proinflammatory cytokines IL-6 and IL-12p40. Point mutations in SCIMP abrogating TLR4 binding also prevent SCIMP-mediated cytokine production. SCIMP is, therefore, an immune-specific TLR adaptor that shapes host defence and inflammation. PMID:28098138

  12. The giardial ENTH protein participates in lysosomal protein trafficking and endocytosis.

    PubMed

    Feliziani, Constanza; Zamponi, Nahuel; Gottig, Natalia; Rópolo, Andrea S; Lanfredi-Rangel, Adriana; Touz, Maria C

    2015-03-01

    In the protozoa parasite Giardia lamblia, endocytosis and lysosomal protein trafficking are vital parasite-specific processes that involve the action of the adaptor complexes AP-1 and AP-2 and clathrin. In this work, we have identified a single gene in Giardia encoding a protein containing an ENTH domain that defines monomeric adaptor proteins of the epsin family. This domain is present in the epsin or epsin-related (epsinR) adaptor proteins, which are implicated in endocytosis and Golgi-to-endosome protein trafficking, respectively, in other eukaryotic cells. We found that GlENTHp (for G. lamblia ENTH protein) localized in the cytosol, strongly interacted with PI3,4,5P3, was associated with the alpha subunit of AP-2, clathrin and ubiquitin and was involved in receptor-mediated endocytosis. It also bonded PI4P, the gamma subunit of AP-1 and was implicated in ER-to-PV trafficking. Alteration of the GlENTHp function severely affected trophozoite growth showing an unusual accumulation of dense material in the lysosome-like peripheral vacuoles (PVs), indicating that GlENTHp might be implicated in the maintenance of PV homeostasis. In this study, we showed evidence suggesting that GlENTHp might function as a monomeric adaptor protein supporting the findings of other group indicating that GlENTHp might be placed at the beginning of the ENTH family.

  13. Structural Basis for Recruitment of Mitochondrial Fission Complexes By Fis1

    SciTech Connect

    Zhang, Y.; Chan, D.C.

    2009-06-04

    Mitochondrial fission controls mitochondrial shape and physiology, including mitochondrial remodeling in apoptosis. During assembly of the yeast mitochondrial fission complex, the outer membrane protein Fis1 recruits the dynamin-related GTPase Dnm1 to mitochondria. Fis1 contains a tetratricopeptide repeat (TPR) domain and interacts with Dnm1 via the molecular adaptors Mdv1 and Caf4. By using crystallographic analysis of adaptor-Fis1 complexes, we show that these adaptors use two helices to bind to both the concave and convex surfaces of the Fis1 TPR domain. Fis1 therefore contains two interaction interfaces, a binding mode that, to our knowledge, has not been observed previously for TPR domains. Genetic and biochemical studies indicate that both binding interfaces are important for binding of Mdv1 and Caf4 to Fis1 and for mitochondrial fission activity in vivo. Our results reveal how Fis1 recruits the mitochondrial fission complex and will facilitate efforts to manipulate mitochondrial fission.

  14. Structure, dynamics, assembly, and evolution of protein complexes.

    PubMed

    Marsh, Joseph A; Teichmann, Sarah A

    2015-01-01

    The assembly of individual proteins into functional complexes is fundamental to nearly all biological processes. In recent decades, many thousands of homomeric and heteromeric protein complex structures have been determined, greatly improving our understanding of the fundamental principles that control symmetric and asymmetric quaternary structure organization. Furthermore, our conception of protein complexes has moved beyond static representations to include dynamic aspects of quaternary structure, including conformational changes upon binding, multistep ordered assembly pathways, and structural fluctuations occurring within fully assembled complexes. Finally, major advances have been made in our understanding of protein complex evolution, both in reconstructing evolutionary histories of specific complexes and in elucidating general mechanisms that explain how quaternary structure tends to evolve. The evolution of quaternary structure occurs via changes in self-assembly state or through the gain or loss of protein subunits, and these processes can be driven by both adaptive and nonadaptive influences.

  15. Hysteresis as a Marker for Complex, Overlapping Landscapes in Proteins

    PubMed Central

    Andrews, Benjamin T.; Capraro, Dominique T.; Sulkowska, Joanna I.; Onuchic, José N.; Jennings, Patricia A.

    2013-01-01

    Topologically complex proteins fold by multiple routes as a result of hard-to-fold regions of the proteins. Oftentimes these regions are introduced into the protein scaffold for function and increase frustration in the otherwise smooth-funneled landscape. Interestingly, while functional regions add complexity to folding landscapes, they may also contribute to a unique behavior referred to as hysteresis. While hysteresis is predicted to be rare, it is observed in various proteins, including proteins containing a unique peptide cyclization to form a fluorescent chromophore as well as proteins containing a knotted topology in their native fold. Here, hysteresis is demonstrated to be a consequence of the decoupling of unfolding events from the isomerization or hula-twist of a chromophore in one protein and the untying of the knot in a second protein system. The question now is- can hysteresis be a marker for the interplay of landscapes where complex folding and functional regions overlap? PMID:23525263

  16. A proteomic strategy for global analysis of plant protein complexes.

    PubMed

    Aryal, Uma K; Xiong, Yi; McBride, Zachary; Kihara, Daisuke; Xie, Jun; Hall, Mark C; Szymanski, Daniel B

    2014-10-01

    Global analyses of protein complex assembly, composition, and location are needed to fully understand how cells coordinate diverse metabolic, mechanical, and developmental activities. The most common methods for proteome-wide analysis of protein complexes rely on affinity purification-mass spectrometry or yeast two-hybrid approaches. These methods are time consuming and are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. Here, we describe the proof of concept for a method allowing simultaneous global analysis of endogenous protein complexes that begins with intact leaves and combines chromatographic separation of extracts from subcellular fractions with quantitative label-free protein abundance profiling by liquid chromatography-coupled mass spectrometry. Applying this approach to the crude cytosolic fraction of Arabidopsis thaliana leaves using size exclusion chromatography, we identified hundreds of cytosolic proteins that appeared to exist as components of stable protein complexes. The reliability of the method was validated by protein immunoblot analysis and comparisons with published size exclusion chromatography data and the masses of known complexes. The method can be implemented with appropriate instrumentation, is applicable to any biological system, and has the potential to be further developed to characterize the composition of protein complexes and measure the dynamics of protein complex localization and assembly under different conditions.

  17. Design and characterization of complex protein films

    NASA Astrophysics Data System (ADS)

    Bui, Holt P.

    Once a biomaterial is implanted into biological system, a layer of protein is immediately deposited on the surface of that material. The newly formed protein film will dictate how the implanted material will interact with the surrounding biological environment and lead to either the acceptance or rejection of the biomaterial. One method to enhance performance involves the activation the surface of the biomaterial with one or more proteins to direct specific interactions with the host environment. The focus of my dissertation was to develop and characterize model biomaterials surfaces that are activated with one or more proteins to help understand how the protein films may affect biological processes and a biomaterial's performance. One model system consisted of a patterned film of two proteins on a gold surface. Characterization of this protein pattern indicated that patterning protein films with a focused ion beam produced protein patterns with high biological contrast and high spatial control. The second model protein film involved the adsorption of fibronectin on surfaces with different surface energies. The characterization of the adsorbed fibronectin films suggest that fibronectin adsorbed on a hydrophilic surface is in an orientation that projects hydrophilic amino acid residues towards surface of the protein and dehydration causes reorientation to project hydrophobic amino acids towards the surface. In contrast, fibronectin is adsorbed onto a hydrophobic surface in a manner that resulted in dehydration and denaturation during the adsorption process. The last model protein film studied in this work consisted of fibronectin patterned in a manner so that the film consisted of spatially controlled domains of fibronectin adsorbed onto a hydrophilic surface as well as a hydrophobic surface. Lateral characterization of this pattern demonstrated a difference in secondary structure of fibronectin adsorbed on the two domains with varying surface energies.

  18. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae.

    PubMed

    Krogan, Nevan J; Cagney, Gerard; Yu, Haiyuan; Zhong, Gouqing; Guo, Xinghua; Ignatchenko, Alexandr; Li, Joyce; Pu, Shuye; Datta, Nira; Tikuisis, Aaron P; Punna, Thanuja; Peregrín-Alvarez, José M; Shales, Michael; Zhang, Xin; Davey, Michael; Robinson, Mark D; Paccanaro, Alberto; Bray, James E; Sheung, Anthony; Beattie, Bryan; Richards, Dawn P; Canadien, Veronica; Lalev, Atanas; Mena, Frank; Wong, Peter; Starostine, Andrei; Canete, Myra M; Vlasblom, James; Wu, Samuel; Orsi, Chris; Collins, Sean R; Chandran, Shamanta; Haw, Robin; Rilstone, Jennifer J; Gandi, Kiran; Thompson, Natalie J; Musso, Gabe; St Onge, Peter; Ghanny, Shaun; Lam, Mandy H Y; Butland, Gareth; Altaf-Ul, Amin M; Kanaya, Shigehiko; Shilatifard, Ali; O'Shea, Erin; Weissman, Jonathan S; Ingles, C James; Hughes, Timothy R; Parkinson, John; Gerstein, Mark; Wodak, Shoshana J; Emili, Andrew; Greenblatt, Jack F

    2006-03-30

    Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

  19. Principles of assembly reveal a periodic table of protein complexes.

    PubMed

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering.

  20. Recording information on protein complexes in an information management system.

    PubMed

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described.

  1. Multi-LZerD: Multiple protein docking for asymmetric complexes

    PubMed Central

    Esquivel-Rodríguez, Juan; Yang, Yifeng David; Kihara, Daisuke

    2012-01-01

    The tertiary structures of protein complexes provide a crucial insight about the molecular mechanisms that regulate their functions and assembly. However, solving protein complex structures by experimental methods is often more difficult than single protein structures. Here, we have developed a novel computational multiple protein docking algorithm, Multi-LZerD, that builds models of multimeric complexes by effectively reusing pairwise docking predictions of component proteins. A genetic algorithm is applied to explore the conformational space followed by a structure refinement procedure. Benchmark on eleven hetero-multimeric complexes resulted in near native conformations for all but one of them (a root mean square deviation smaller than 2.5Å). We also show that our method copes with unbound docking cases well, outperforming the methodology that can be directly compared to our approach. Multi-LZerD was able to predict near native structures for multimeric complexes of various topologies. PMID:22488467

  2. Native Mass Spectrometry of Photosynthetic Pigment-Protein Complexes

    PubMed Central

    Zhang, Hao; Cui, Weidong; Gross, Michael L.; Blankenship, Robert E.

    2013-01-01

    Native mass spectrometry, or as is sometimes called “native electrospray (ESI)” allows proteins in their native or near-native protein in solution to be introduced into gas phase and interrogated by MS. This approach is now a powerful tool to investigate protein complexes. This article reviews the background of native MS of protein complexes and describes its strengths, taking photosynthetic pigment-protein complexes as examples. Native MS can be utilized in combination with other MS-based approaches to obtain complementary information to that provided by tools such as X-ray crystallography and NMR spectroscopy to understand the structure-function relationships of protein complexes. When additional information beyond that provided by native MS is required, other MS-based strategies can be successfully applied to augment the results of native MS. PMID:23337874

  3. Advances in protein complex analysis using mass spectrometry

    PubMed Central

    Gingras, Anne-Claude; Aebersold, Ruedi; Raught, Brian

    2005-01-01

    Proteins often function as components of larger complexes to perform a specific function, and formation of these complexes may be regulated. For example, intracellular signalling events often require transient and/or regulated protein–protein interactions for propagation, and protein binding to a specific DNA sequence, RNA molecule or metabolite is often regulated to modulate a particular cellular function. Thus, characterizing protein complexes can offer important insights into protein function. This review describes recent important advances in mass spectrometry (MS)-based techniques for the analysis of protein complexes. Following brief descriptions of how proteins are identified using MS, and general protein complex purification approaches, we address two of the most important issues in these types of studies: specificity and background protein contaminants. Two basic strategies for increasing specificity and decreasing background are presented: whereas (1) tandem affinity purification (TAP) of tagged proteins of interest can dramatically improve the signal-to-noise ratio via the generation of cleaner samples, (2) stable isotopic labelling of proteins may be used to discriminate between contaminants and bona fide binding partners using quantitative MS techniques. Examples, as well as advantages and disadvantages of each approach, are presented. PMID:15611014

  4. Luminogenic "clickable" lanthanide complexes for protein labeling.

    PubMed

    Candelon, Nicolas; Hădade, Niculina D; Matache, Mihaela; Canet, Jean-Louis; Cisnetti, Federico; Funeriu, Daniel P; Nauton, Lionel; Gautier, Arnaud

    2013-10-14

    Development of lanthanide-based luminescent "switch-on" systems via azide-alkyne [3+2] cycloaddition is described. We used these for non-specific protein labeling and as tags for specific and selective activity-based protein labeling.

  5. Multiscale Model for the Assembly Kinetics of Protein Complexes.

    PubMed

    Xie, Zhong-Ru; Chen, Jiawen; Wu, Yinghao

    2016-02-04

    The assembly of proteins into high-order complexes is a general mechanism for these biomolecules to implement their versatile functions in cells. Natural evolution has developed various assembling pathways for specific protein complexes to maintain their stability and proper activities. Previous studies have provided numerous examples of the misassembly of protein complexes leading to severe biological consequences. Although the research focusing on protein complexes has started to move beyond the static representation of quaternary structures to the dynamic aspect of their assembly, the current understanding of the assembly mechanism of protein complexes is still largely limited. To tackle this problem, we developed a new multiscale modeling framework. This framework combines a lower-resolution rigid-body-based simulation with a higher-resolution Cα-based simulation method so that protein complexes can be assembled with both structural details and computational efficiency. We applied this model to a homotrimer and a heterotetramer as simple test systems. Consistent with experimental observations, our simulations indicated very different kinetics between protein oligomerization and dimerization. The formation of protein oligomers is a multistep process that is much slower than dimerization but thermodynamically more stable. Moreover, we showed that even the same protein quaternary structure can have very diverse assembly pathways under different binding constants between subunits, which is important for regulating the functions of protein complexes. Finally, we revealed that the binding between subunits in a complex can be synergistically strengthened during assembly without considering allosteric regulation or conformational changes. Therefore, our model provides a useful tool to understand the general principles of protein complex assembly.

  6. Protein-protein interactions in complex cosolvent solutions.

    PubMed

    Javid, Nadeem; Vogtt, Karsten; Krywka, Chris; Tolan, Metin; Winter, Roland

    2007-04-02

    The effects of various kosmotropic and chaotropic cosolvents and salts on the intermolecular interaction potential of positively charged lysozyme is evaluated at varying protein concentrations by using synchrotron small-angle X-ray scattering in combination with liquid-state theoretical approaches. The experimentally derived static structure factors S(Q) obtained without and with added cosolvents and salts are analysed with a statistical mechanical model based on the Derjaguin-Landau-Verwey-Overbeek (DLVO) potential, which accounts for repulsive and attractive interactions between the protein molecules. Different cosolvents and salts influence the interactions between protein molecules differently as a result of changes in the hydration level or solvation, in charge screening, specific adsorption of the additives at the protein surface, or increased hydrophobic interactions. Intermolecular interaction effects are significant above protein concentrations of 1 wt %, and with increasing protein concentration, the repulsive nature of the intermolecular pair potential V(r) increases markedly. Kosmotropic cosolvents like glycerol and sucrose exhibit strong concentration-dependent effects on the interaction potential, leading to an increase of repulsive forces between the protein molecules at low to medium high osmolyte concentrations. Addition of trifluoroethanol exhibits a multiphasic effect on V(r) when changing its concentration. Salts like sodium chloride and potassium sulfate exhibit strong concentration-dependent changes of the interaction potential due to charge screening of the positively charged protein molecules. Guanidinium chloride (GdmCl) at low concentrations exhibits a similar charge-screening effect, resulting in increased attractive interactions between the protein molecules. At higher GdmCl concentrations, V(r) becomes more repulsive in nature due to the presence of high concentrations of Gdm(+) ions binding to the protein molecules. Our findings also

  7. Cerebral cavernous malformation proteins at a glance.

    PubMed

    Draheim, Kyle M; Fisher, Oriana S; Boggon, Titus J; Calderwood, David A

    2014-02-15

    Loss-of-function mutations in genes encoding KRIT1 (also known as CCM1), CCM2 (also known as OSM and malcavernin) or PDCD10 (also known as CCM3) cause cerebral cavernous malformations (CCMs). These abnormalities are characterized by dilated leaky blood vessels, especially in the neurovasculature, that result in increased risk of stroke, focal neurological defects and seizures. The three CCM proteins can exist in a trimeric complex, and each of these essential multi-domain adaptor proteins also interacts with a range of signaling, cytoskeletal and adaptor proteins, presumably accounting for their roles in a range of basic cellular processes including cell adhesion, migration, polarity and apoptosis. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of current models of CCM protein function focusing on how known protein-protein interactions might contribute to cellular phenotypes and highlighting gaps in our current understanding.

  8. Identifying protein complexes in protein-protein interaction networks by using clique seeds and graph entropy.

    PubMed

    Chen, Bolin; Shi, Jinhong; Zhang, Shenggui; Wu, Fang-Xiang

    2013-01-01

    The identification of protein complexes plays a key role in understanding major cellular processes and biological functions. Various computational algorithms have been proposed to identify protein complexes from protein-protein interaction (PPI) networks. In this paper, we first introduce a new seed-selection strategy for seed-growth style algorithms. Cliques rather than individual vertices are employed as initial seeds. After that, a result-modification approach is proposed based on this seed-selection strategy. Predictions generated by higher order clique seeds are employed to modify results that are generated by lower order ones. The performance of this seed-selection strategy and the result-modification approach are tested by using the entropy-based algorithm, which is currently the best seed-growth style algorithm to detect protein complexes from PPI networks. In addition, we investigate four pairs of strategies for this algorithm in order to improve its accuracy. The numerical experiments are conducted on a Saccharomyces cerevisiae PPI network. The group of best predictions consists of 1711 clusters, with the average f-score at 0.68 after removing all similar and redundant clusters. We conclude that higher order clique seeds can generate predictions with higher accuracy and that our improved entropy-based algorithm outputs more reasonable predictions than the original one.

  9. How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4.

    PubMed

    Ren, Xuefeng; Park, Sang Yoon; Bonifacino, Juan S; Hurley, James H

    2014-01-01

    The Nef protein of HIV-1 downregulates the cell surface co-receptor CD4 by hijacking the clathrin adaptor complex AP-2. The structural basis for the hijacking of AP-2 by Nef is revealed by a 2.9 Å crystal structure of Nef bound to the α and σ2 subunits of AP-2. Nef binds to AP-2 via its central loop (residues 149-179) and its core. The determinants for Nef binding include residues that directly contact AP-2 and others that stabilize the binding-competent conformation of the central loop. Residues involved in both direct and indirect interactions are required for the binding of Nef to AP-2 and for downregulation of CD4. These results lead to a model for the docking of the full AP-2 tetramer to membranes as bound to Nef, such that the cytosolic tail of CD4 is situated to interact with its binding site on Nef. DOI: http://dx.doi.org/10.7554/eLife.01754.001.

  10. Measurement of protein-ligand complex formation.

    PubMed

    Lowe, Peter N; Vaughan, Cara K; Daviter, Tina

    2013-01-01

    Experimental approaches to detect, measure, and quantify protein-ligand binding, along with their theoretical bases, are described. A range of methods for detection of protein-ligand interactions is summarized. Specific protocols are provided for a nonequilibrium procedure pull-down assay, for an equilibrium direct binding method and its modification into a competition-based measurement and for steady-state measurements based on the effects of ligands on enzyme catalysis.

  11. Recording information on protein complexes in an information management system

    PubMed Central

    Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

    2011-01-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682

  12. Conservation of Telomere protein complexes: Shuffling through Evolution

    PubMed Central

    Linger, Benjamin R.; Price, Carolyn M.

    2009-01-01

    The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology. PMID:19839711

  13. The retromer complex - endosomal protein recycling and beyond.

    PubMed

    Seaman, Matthew N J

    2012-10-15

    The retromer complex is a vital element of the endosomal protein sorting machinery that is conserved across all eukaryotes. Retromer is most closely associated with the endosome-to-Golgi retrieval pathway and is necessary to maintain an active pool of hydrolase receptors in the trans-Golgi network. Recent progress in studies of retromer have identified new retromer-interacting proteins, including the WASH complex and cargo such as the Wntless/MIG-14 protein, which now extends the role of retromer beyond the endosome-to-Golgi pathway and has revealed that retromer is required for aspects of endosome-to-plasma membrane sorting and regulation of signalling events. The interactions between the retromer complex and other macromolecular protein complexes now show how endosomal protein sorting is coordinated with actin assembly and movement along microtubules, and place retromer squarely at the centre of a complex set of protein machinery that governs endosomal protein sorting. Dysregulation of retromer-mediated endosomal protein sorting leads to various pathologies, including neurodegenerative diseases such as Alzheimer disease and spastic paraplegia and the mechanisms underlying these pathologies are starting to be understood. In this Commentary, I will highlight recent advances in the understanding of retromer-mediated endosomal protein sorting and discuss how retromer contributes to a diverse set of physiological processes.

  14. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  15. Mass Spectrometry of Protein Complexes: From Origins to Applications

    NASA Astrophysics Data System (ADS)

    Mehmood, Shahid; Allison, Timothy M.; Robinson, Carol V.

    2015-04-01

    Now routine is the ability to investigate soluble and membrane protein complexes in the gas phase of a mass spectrometer while preserving folded structure and ligand-binding properties. Several recent transformative developments have occurred to arrive at this point. These include advances in mass spectrometry instrumentation, particularly with respect to resolution; the ability to study intact membrane protein complexes released from detergent micelles; and the use of protein unfolding in the gas phase to obtain stability parameters. Together, these discoveries are providing unprecedented information on the compositional heterogeneity of biomacromolecules, the unfolding trajectories of multidomain proteins, and the stability imparted by ligand binding to both soluble and membrane-embedded protein complexes. We review these recent breakthroughs, highlighting the challenges that had to be overcome and the physicochemical insight that can now be gained from studying proteins and their assemblies in the gas phase.

  16. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

    PubMed

    Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-12-28

    Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function.

  17. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression

    PubMed Central

    Mir, Riyaz A.; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A.; Ammons, Shalis A.; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B.; Qiu, Fang; Band, Hamid

    2015-01-01

    Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. PMID:26711270

  18. Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

    PubMed Central

    Fink, Gero; Löwe, Jan

    2015-01-01

    Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton. PMID:25825718

  19. Computational approaches for detecting protein complexes from protein interaction networks: a survey

    PubMed Central

    2010-01-01

    Background Most proteins form macromolecular complexes to perform their biological functions. However, experimentally determined protein complex data, especially of those involving more than two protein partners, are relatively limited in the current state-of-the-art high-throughput experimental techniques. Nevertheless, many techniques (such as yeast-two-hybrid) have enabled systematic screening of pairwise protein-protein interactions en masse. Thus computational approaches for detecting protein complexes from protein interaction data are useful complements to the limited experimental methods. They can be used together with the experimental methods for mapping the interactions of proteins to understand how different proteins are organized into higher-level substructures to perform various cellular functions. Results Given the abundance of pairwise protein interaction data from high-throughput genome-wide experimental screenings, a protein interaction network can be constructed from protein interaction data by considering individual proteins as the nodes, and the existence of a physical interaction between a pair of proteins as a link. This binary protein interaction graph can then be used for detecting protein complexes using graph clustering techniques. In this paper, we review and evaluate the state-of-the-art techniques for computational detection of protein complexes, and discuss some promising research directions in this field. Conclusions Experimental results with yeast protein interaction data show that the interaction subgraphs discovered by various computational methods matched well with actual protein complexes. In addition, the computational approaches have also improved in performance over the years. Further improvements could be achieved if the quality of the underlying protein interaction data can be considered adequately to minimize the undesirable effects from the irrelevant and noisy sources, and the various biological evidences can be better

  20. Strategies for crystallization of large membrane protein complexes

    NASA Astrophysics Data System (ADS)

    Yoshikawa, Shinya; Shinzawa-Itoh, Kyoko; Ueda, Hidefumi; Tsukihara, Tomitake; Fukumoto, Yoshihisa; Kubota, Tomomi; Kawamoto, Masahide; Fukuyama, Keiichi; Matsubara, Hiroshi

    1992-08-01

    Crystalline cytochrome c oxidase and ubiquinol: cytochrome c oxidoreductase which diffracted X-rays at 7-8A˚resolution were obtained from bovine heart mitochondria. The methods for the purification and crystallization of these enzymes indicate that large membrane protein complexes are easier to purify and crystallize than smaller homologous membrane protein complexes, because the former have more hydrophilic surface than the latter. Bulky and polydispersed detergents such as Brij-35 and Tween 20 attached to the isolated complex are not always obstructive to crystallization if they are effective for stabilizing the complexes.

  1. Quantitative proteomics analysis of signalosome dynamics in primary T cells identifies the surface receptor CD6 as a Lat adaptor-independent TCR signaling hub.

    PubMed

    Roncagalli, Romain; Hauri, Simon; Fiore, Fréderic; Liang, Yinming; Chen, Zhi; Sansoni, Amandine; Kanduri, Kartiek; Joly, Rachel; Malzac, Aurélie; Lähdesmäki, Harri; Lahesmaa, Riitta; Yamasaki, Sho; Saito, Takashi; Malissen, Marie; Aebersold, Ruedi; Gstaiger, Matthias; Malissen, Bernard

    2014-04-01

    T cell antigen receptor (TCR)-mediated activation of T cells requires the interaction of dozens of proteins. Here we used quantitative mass spectrometry and activated primary CD4(+) T cells from mice in which a tag for affinity purification was knocked into several genes to determine the composition and dynamics of multiprotein complexes that formed around the kinase Zap70 and the adaptors Lat and SLP-76. Most of the 112 high-confidence time-resolved protein interactions we observed were previously unknown. The surface receptor CD6 was able to initiate its own signaling pathway by recruiting SLP-76 and the guanine nucleotide-exchange factor Vav1 regardless of the presence of Lat. Our findings provide a more complete model of TCR signaling in which CD6 constitutes a signaling hub that contributes to the diversification of TCR signaling.

  2. Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion

    SciTech Connect

    Ekiert, Damian C.; Cox, Jeffery S.

    2014-10-01

    Nearly 10% of the coding capacity of the Mycobacterium tuberculosis genome is devoted to two highly expanded and enigmatic protein families called PE and PPE, some of which are important virulence/immunogenicity factors and are secreted during infection via a unique alternative secretory system termed "type VII." How PE-PPE proteins function during infection and how they are translocated to the bacterial surface through the five distinct type VII secretion systems [ESAT-6 secretion system (ESX)] of M. tuberculosis is poorly understood. Here in this paper, we report the crystal structure of a PE-PPE heterodimer bound to ESX secretion-associated protein G (EspG), which adopts a novel fold. This PE-PPE-EspG complex, along with structures of two additional EspGs, suggests that EspG acts as an adaptor that recognizes specific PE-PPE protein complexes via extensive interactions with PPE domains, and delivers them to ESX machinery for secretion. Surprisingly, secretion of most PE-PPE proteins in M. tuberculosis is likely mediated by EspG from the ESX-5 system, underscoring the importance of ESX-5 in mycobacterial pathogenesis. Furthermore, our results indicate that PE-PPE domains function as cis-acting targeting sequences that are read out by EspGs, revealing the molecular specificity for secretion through distinct ESX pathways.

  3. Sizing Large Proteins and Protein Complexes by Electrospray Ionization Mass Spectrometry and Ion Mobility

    PubMed Central

    Kaddis, Catherine S.; Lomeli, Shirley H.; Yin, Sheng; Berhane, Beniam; Apostol, Marcin I.; Kickhoefer, Valerie A.; Rome, Leonard H.; Loo, Joseph A.

    2009-01-01

    Mass spectrometry (MS) and ion mobility with electrospray ionization (ESI) have the capability to measure and detect large noncovalent protein-ligand and protein-protein complexes. Using an ion mobility method termed GEMMA (Gas-Phase Electrophoretic Mobility Molecular Analysis), protein particles representing a range of sizes can be separated by their electrophoretic mobility in air. Highly charged particles produced from a protein complex solution using electrospray can be manipulated to produce singly charged ions which can be separated and quantified by their electrophoretic mobility. Results from ESI-GEMMA analysis from our laboratory and others were compared to other experimental and theoretically determined parameters, such as molecular mass and cryoelectron microscopy and x-ray crystal structure dimensions. There is a strong correlation between the electrophoretic mobility diameter determined from GEMMA analysis and the molecular mass for protein complexes up to 12 MDa, including the 93 kDa enolase dimer, the 480 kDa ferritin 24-mer complex, the 4.6 MDa cowpea chlorotic mottle virus (CCMV), and the 9 MDa MVP-vault assembly. ESI-GEMMA is used to differentiate a number of similarly sized vault complexes that are composed of different N-terminal protein tags on the MVP subunit. The average effective density of the proteins and protein complexes studied was 0.6 g/cm3. Moreover, there is evidence that proteins and protein complexes collapse or become more compact in the gas phase in the absence of water. PMID:17434746

  4. Nanoparticle-protein complexes mimicking corona formation in ocular environment.

    PubMed

    Jo, Dong Hyun; Kim, Jin Hyoung; Son, Jin Gyeong; Dan, Ki Soon; Song, Sang Hoon; Lee, Tae Geol; Kim, Jeong Hun

    2016-12-01

    Nanoparticles adsorb biomolecules to form corona upon entering the biological environment. In this study, tissue-specific corona formation is provided as a way of controlling protein interaction with nanoparticles in vivo. In the vitreous, the composition of the corona was determined by the electrostatic and hydrophobic properties of the associated proteins, regardless of the material (gold and silica) or size (20- and 100-nm diameter) of the nanoparticles. To control protein adsorption, we pre-incubate 20-nm gold nanoparticles with 5 selectively enriched proteins from the corona, formed in the vitreous, to produce nanoparticle-protein complexes. Compared to bare nanoparticles, nanoparticle-protein complexes demonstrate improved binding to vascular endothelial growth factor (VEGF) in the vitreous. Furthermore, nanoparticle-protein complexes retain in vitro anti-angiogenic properties of bare nanoparticles. In particular, priming the nanoparticles (gold and silica) with tissue-specific corona proteins allows nanoparticle-protein complexes to exert better in vivo therapeutic effects by higher binding to VEGF than bare nanoparticles. These results suggest that controlled corona formation that mimics in vivo processes may be useful in the therapeutic use of nanomaterials in local environment.

  5. Unwinding protein complexes in ALTernative telomere maintenance.

    PubMed

    Bhattacharyya, Saumitri; Sandy, April; Groden, Joanna

    2010-01-01

    Telomeres are composed of specialized chromatin that includes DNA repair/recombination proteins, telomere DNA-binding proteins and a number of three dimensional nucleic acid structures including G-quartets and D-loops. A number of studies suggest that the BLM and WRN recQ-like helicases play important roles in recombination-mediated mechanisms of telomere elongation or Alternative Lengthening of Telomeres (ALT), processes that maintain/elongate telomeres in the absence of telomerase. BLM and WRN localize within ALT-associated nuclear bodies in telomerase-negative immortalized cell lines and interact with the telomere-specific proteins POT1, TRF1 and TRF2. Helicase activity is modulated by these interactions. BLM functions in DNA double-strand break repair processes such as non-homologous end joining, homologous recombination-mediated repair, resolution of stalled replication forks and synthesis-dependent strand annealing, although its precise functions at the telomeres are speculative. WRN also functions in DNA replication, recombination and repair, and in addition to its helicase domain, includes an exonuclease domain not found in other recQ-like helicases. The biochemical properties of BLM and WRN are, therefore, important in biological processes other than DNA replication, recombination and repair. In this review, we discuss some previous and recent findings of human rec-Q-like helicases and their role in telomere elongation during ALT processes.

  6. From quantitative protein complex analysis to disease mechanism.

    PubMed

    Texier, Y; Kinkl, N; Boldt, K; Ueffing, M

    2012-12-15

    Interest in the field of cilia biology and cilia-associated diseases - ciliopathies - has strongly increased over the last few years. Proteomic technologies, especially protein complex analysis by affinity purification-based methods, have been used to decipher various basic but also disease-associated mechanisms. This review focusses on some selected recent studies using affinity purification-based protein complex analysis, thereby exemplifying the great possibilities this technology offers.

  7. Mass Spectrometry of Protein-Ligand Complexes: Enhanced Gas Phase Stability of Ribonuclease-Nucleotide Complexes

    PubMed Central

    Yin, Sheng; Xie, Yongming; Loo, Joseph A.

    2008-01-01

    Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas phase stabilities of noncovalent complexes that can result. PMID:18565758

  8. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ...) MEDICAL DEVICES CARDIOVASCULAR DEVICES Cardiovascular Prosthetic Devices § 870.3620 Pacemaker lead adaptor... entitled “Guidance for the Submission of Research and Marketing Applications for Permanent Pacemaker...

  9. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    SciTech Connect

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster is relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.

  10. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    DOE PAGES

    Bordner, Andrew J.; Gorin, Andrey A.

    2008-05-12

    Here, protein-protein interactions are ubiquitous and essential for cellular processes. High-resolution X-ray crystallographic structures of protein complexes can elucidate the details of their function and provide a basis for many computational and experimental approaches. Here we demonstrate that existing annotations of protein complexes, including those provided by the Protein Data Bank (PDB) itself, contain a significant fraction of incorrect annotations. Results: We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1) comprehensively collecting all protein-protein interfaces; (2) clustering similar protein-protein interfaces together; (3) estimating the probability that each cluster ismore » relevant based on a diverse set of properties; and (4) finally combining these scores for each entry in order to predict the complex structure. Unlike previous annotation methods, consistent prediction of complexes with identical or almost identical protein content is insured. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions.« less

  11. Adenosine deaminase complexing protein in cancer studies.

    PubMed

    Ten Kate, J; Dinjens, W N; Meera Khan, P; Bosman, F T

    1986-01-01

    ADCP is a dimeric glycoprotein of about 200KD, for which the physiological role is still obscure. This protein occurs mainly in a membrane bound form in various human tissues. In this paper we review the current literature on ADCP in cancer studies. Soluble ADCP was described to be consistently decreased or absent in cancers of lung, liver, kidney and colon. These findings could not be confirmed by immunohistochemical and quantitative biochemical studies in a series of colorectal-, prostatic-, and renal carcinomas. Only in a third of these tumors a decrease could be demonstrated, whereas in the other cases unaltered or even increased amounts were observed. However, in virally transformed human fibroblasts a consistent decrease or complete absence of ADCP was seen, while primary fibroblasts were found to contain high amounts of this protein. Recently, the use of ADCP as a differentiation marker in colonic cancer has been advocated. Furthermore the presence of ADCP in the serum of renal adenocarcinoma patients was found to be indicative of a better chance of five year survival. These studies suggest that ADCP may be a differentiation marker useful for immunohistochemical characterization of colonic and renal carcinomas as well as a serum marker useful for follow-up studies of these types of cancer, analogous to CEA. Finally, ADCP has been found to be selectively expressed by certain T-cell subsets and henceforth may be useful in the studies on leukemias.

  12. Biochemical isolation of Argonaute protein complexes by Ago-APP

    PubMed Central

    Hauptmann, Judith; Schraivogel, Daniel; Bruckmann, Astrid; Manickavel, Sudhir; Jakob, Leonhard; Eichner, Norbert; Pfaff, Janina; Urban, Marc; Sprunck, Stefanie; Hafner, Markus; Tuschl, Thomas; Deutzmann, Rainer; Meister, Gunter

    2015-01-01

    During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as “Ago protein Affinity Purification by Peptides“ (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells. PMID:26351695

  13. VPS37 isoforms differentially modulate the ternary complex formation of ALIX, ALG-2, and ESCRT-I.

    PubMed

    Okumura, Mayumi; Katsuyama, Angela M; Shibata, Hideki; Maki, Masatoshi

    2013-01-01

    The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca²⁺-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca²⁺-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.

  14. Luminescent iridium(III) complexes as novel protein staining agents.

    PubMed

    Jia, Junli; Fei, Hao; Zhou, Ming

    2012-05-01

    This article reports a new class of luminescent metal complexes, biscyclometalated iridium(III) complexes with an ancillary bathophenanthroline disulfonate ligand, for staining protein bands that are separated by electrophoresis. The performances of these novel staining agents have been studied in comparison with tris(bathophenanthroline disulfonate) ruthenium(II) tetrasodium salt (i.e. RuBPS) using a commercially available imaging system. The staining agents showed different limits of detection, linear dynamic ranges, and protein-to-protein variations. The overall performances of all three stains were found to be better than or equivalent to RuBPS under the experimental conditions.

  15. Protein Complex Production from the Drug Discovery Standpoint.

    PubMed

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  16. EGFR-targeted mAb therapy modulates autophagy in head and neck squamous cell carcinoma through NLRX1-TUFM protein complex.

    PubMed

    Lei, Y; Kansy, B A; Li, J; Cong, L; Liu, Y; Trivedi, S; Wen, H; Ting, J P-Y; Ouyang, H; Ferris, R L

    2016-09-08

    Epidermal growth factor receptor (EGFR)-targeted therapy in head and neck squamous cell carcinoma (HNSCC) patients frequently results in tumor resistance to treatment. Autophagy is an emerging underlying resistance mechanism, however, the molecular autophagy machinery in HNSCC cells and potential biomarkers of patient response to EGFR-targeted therapy remain insufficiently characterized. Here we show that the EGFR blocking with cetuximab leads to varied autophagic responses, which modulate cancer cell susceptibility to EGFR inhibition. Inhibition of autophagy sensitizes HNSCC cells to EGFR blockade. Importantly, we identify a novel signaling hub centering on the NLRX1 (nucleotide-binding, lots of leucine-rich repeats-containing protein member X1)-TUFM (Tu translation elongation factor mitochondrial) protein complex, promoting autophagic flux. Defects in the expression of either NLRX1 or TUFM result in compromised autophagy when treated with EGFR inhibitors. As a previously undefined autophagy-promoting mechanism, we found that TUFM serves as a novel anchorage site, recruiting Beclin-1 to mitochondria, promoting its polyubiquitination, and interfering with its interaction with Rubicon. This protein complex is also essential for endoplasmic reticulum stress signaling induction, possibly as an additional mechanism to promote autophagy. Utilizing tumor specimens from a novel neoadjuvant clinical trial, we show that increased expression of the autophagy adaptor protein, SQSTM1/p62, is associated with poor response to cetuximab therapy. These findings expand our understanding of the components involved in HNSCC autophagy machinery that responds to EGFR inhibitors, and suggest potential combinatorial approaches to enhance its therapeutic efficacy.

  17. Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

    PubMed Central

    Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

    2013-01-01

    Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423

  18. Proteins associated with RNase E in a multicomponent ribonucleolytic complex.

    PubMed Central

    Miczak, A; Kaberdin, V R; Wei, C L; Lin-Chao, S

    1996-01-01

    The Escherichia coli endoribonuclease RNase E is essential for RNA processing and degradation. Earlier work provided evidence that RNase E exists intracellularly as part of a multicomponent complex and that one of the components of this complex is a 3'-to-5' exoribonuclease, polynucleotide phosphorylase (EC 2.7.7.8). To isolate and identify other components of the RNase E complex, FLAG-epitope-tagged RNase E (FLAG-Rne) fusion protein was purified on a monoclonal antibody-conjugated agarose column. The FLAG-Rne fusion protein, eluted by competition with the synthetic FLAG peptide, was found to be associated with other proteins. N-terminal sequencing of these proteins revealed the presence in the RNase E complex not only of polynucleotide phosphorylase but also of DnaK, RNA helicase, and enolase (EC 4.2.1.11). Another protein associated only with epitope-tagged temperature-sensitive (Rne-3071) mutant RNase E but not with the wild-type enzyme is GroEL. The FLAG-Rne complex has RNase E activity in vivo and in vitro. The relative amount of proteins associated with wild-type and Rne-3071 expressed at an elevated temperature differed. Images Fig. 1 Fig. 2 PMID:8632981

  19. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes

    PubMed Central

    Luo, Jiawei; Qi, Yi

    2015-01-01

    Background Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins. Method In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC), based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID), of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification. Results Experimental results based on three different PPI(protein-protein interaction) networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC). Conclusions LIDC is more effective for the prediction of essential proteins than other recently developed methods. PMID:26125187

  20. Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes

    SciTech Connect

    Martinovic, Suzana; Pasa-Tolic, Liljiana; Smith, Richard D.

    2004-10-01

    Complex proteome samples require efficient separation and detection methods in order to characterize their protein components. On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization (ESI) mass spectrometry (MS) is shown as an effective method to analyze complex protein mixtures. Our experience with several microorganisms allowed us to establish successful experimental protocol. Here we use the example of E. coli whole cell lysate for the CIEF separation and MS detection on the intact protein level. The protocol was further adapted for the analysis of the mixture of non-covalent complexes on the intact complex level.

  1. Global landscape of HIV-human protein complexes.

    PubMed

    Jäger, Stefanie; Cimermancic, Peter; Gulbahce, Natali; Johnson, Jeffrey R; McGovern, Kathryn E; Clarke, Starlynn C; Shales, Michael; Mercenne, Gaelle; Pache, Lars; Li, Kathy; Hernandez, Hilda; Jang, Gwendolyn M; Roth, Shoshannah L; Akiva, Eyal; Marlett, John; Stephens, Melanie; D'Orso, Iván; Fernandes, Jason; Fahey, Marie; Mahon, Cathal; O'Donoghue, Anthony J; Todorovic, Aleksandar; Morris, John H; Maltby, David A; Alber, Tom; Cagney, Gerard; Bushman, Frederic D; Young, John A; Chanda, Sumit K; Sundquist, Wesley I; Kortemme, Tanja; Hernandez, Ryan D; Craik, Charles S; Burlingame, Alma; Sali, Andrej; Frankel, Alan D; Krogan, Nevan J

    2011-12-21

    Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat). Using a quantitative scoring system that we call MiST, we identified with high confidence 497 HIV-human protein-protein interactions involving 435 individual human proteins, with ∼40% of the interactions being identified in both cell types. We found that the host proteins hijacked by HIV, especially those found interacting in both cell types, are highly conserved across primates. We uncovered a number of host complexes targeted by viral proteins, including the finding that HIV protease cleaves eIF3d, a subunit of eukaryotic translation initiation factor 3. This host protein is one of eleven identified in this analysis that act to inhibit HIV replication. This data set facilitates a more comprehensive and detailed understanding of how the host machinery is manipulated during the course of HIV infection.

  2. Negative Ions Enhance Survival of Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Liko, Idlir; Hopper, Jonathan T. S.; Allison, Timothy M.; Benesch, Justin L. P.; Robinson, Carol V.

    2016-06-01

    Membrane protein complexes are commonly introduced to the mass spectrometer solubilized in detergent micelles. The collisional activation used to remove the detergent, however, often causes protein unfolding and dissociation. As in the case for soluble proteins, electrospray in the positive ion mode is most commonly used for the study of membrane proteins. Here we show several distinct advantages of employing the negative ion mode. Negative polarity can yield lower average charge states for membrane proteins solubilized in saccharide detergents, with enhanced peak resolution and reduced adduct formation. Most importantly, we demonstrate that negative ion mode electrospray ionization (ESI) minimizes subunit dissociation in the gas phase, allowing access to biologically relevant oligomeric states. Together, these properties mean that intact membrane protein ions can be generated in a greater range of solubilizing detergents. The formation of negative ions, therefore, greatly expands the possibilities of using mass spectrometry on this intractable class of protein.

  3. Chimeric Protein Complexes in Hybrid Species Generate Novel Phenotypes

    PubMed Central

    Piatkowska, Elzbieta M.; Naseeb, Samina; Knight, David; Delneri, Daniela

    2013-01-01

    Hybridization between species is an important mechanism for the origin of novel lineages and adaptation to new environments. Increased allelic variation and modification of the transcriptional network are the two recognized forces currently deemed to be responsible for the phenotypic properties seen in hybrids. However, since the majority of the biological functions in a cell are carried out by protein complexes, inter-specific protein assemblies therefore represent another important source of natural variation upon which evolutionary forces can act. Here we studied the composition of six protein complexes in two different Saccharomyces “sensu stricto” hybrids, to understand whether chimeric interactions can be freely formed in the cell in spite of species-specific co-evolutionary forces, and whether the different types of complexes cause a change in hybrid fitness. The protein assemblies were isolated from the hybrids via affinity chromatography and identified via mass spectrometry. We found evidence of spontaneous chimericity for four of the six protein assemblies tested and we showed that different types of complexes can cause a variety of phenotypes in selected environments. In the case of TRP2/TRP3 complex, the effect of such chimeric formation resulted in the fitness advantage of the hybrid in an environment lacking tryptophan, while only one type of parental combination of the MBF complex allowed the hybrid to grow under respiratory conditions. These phenotypes were dependent on both genetic and environmental backgrounds. This study provides empirical evidence that chimeric protein complexes can freely assemble in cells and reveals a new mechanism to generate phenotypic novelty and plasticity in hybrids to complement the genomic innovation resulting from gene duplication. The ability to exchange orthologous members has also important implications for the adaptation and subsequent genome evolution of the hybrids in terms of pattern of gene loss. PMID

  4. Machine Learning Approaches for Predicting Protein Complex Similarity.

    PubMed

    Farhoodi, Roshanak; Akbal-Delibas, Bahar; Haspel, Nurit

    2017-01-01

    Discriminating native-like structures from false positives with high accuracy is one of the biggest challenges in protein-protein docking. While there is an agreement on the existence of a relationship between various favorable intermolecular interactions (e.g., Van der Waals, electrostatic, and desolvation forces) and the similarity of a conformation to its native structure, the precise nature of this relationship is not known. Existing protein-protein docking methods typically formulate this relationship as a weighted sum of selected terms and calibrate their weights by using a training set to evaluate and rank candidate complexes. Despite improvements in the predictive power of recent docking methods, producing a large number of false positives by even state-of-the-art methods often leads to failure in predicting the correct binding of many complexes. With the aid of machine learning methods, we tested several approaches that not only rank candidate structures relative to each other but also predict how similar each candidate is to the native conformation. We trained a two-layer neural network, a multilayer neural network, and a network of Restricted Boltzmann Machines against extensive data sets of unbound complexes generated by RosettaDock and PyDock. We validated these methods with a set of refinement candidate structures. We were able to predict the root mean squared deviations (RMSDs) of protein complexes with a very small, often less than 1.5 Å, error margin when trained with structures that have RMSD values of up to 7 Å. In our most recent experiments with the protein samples having RMSD values up to 27 Å, the average prediction error was still relatively small, attesting to the potential of our approach in predicting the correct binding of protein-protein complexes.

  5. β-Barrel membrane protein assembly by the Bam complex.

    PubMed

    Hagan, Christine L; Silhavy, Thomas J; Kahne, Daniel

    2011-01-01

    β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

  6. Protein-complex structure completion using IPCAS (Iterative Protein Crystal structure Automatic Solution).

    PubMed

    Zhang, Weizhe; Zhang, Hongmin; Zhang, Tao; Fan, Haifu; Hao, Quan

    2015-07-01

    Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite, IPCAS (Iterative Protein Crystal structure Automatic Solution). The IPCAS iteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to use IPCAS to build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case, IPCAS was able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

  7. Emergence of Complexity in Protein Functions and Metabolic Networks

    NASA Technical Reports Server (NTRS)

    Pohorille, Andzej

    2009-01-01

    In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis of very large libraries of random amino acid sequences and subsequently subjecting them to in vitro evolution. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions, important clues have been uncovered. Considerable progress has been also achieved in understanding the origins of membrane proteins. We will address this issue in the example of ion channels - proteins that mediate transport of ions across cell walls. Remarkably, despite overall complexity of these proteins in contemporary cells, their structural motifs are quite simple, with -helices being most common. By combining results of experimental and computer simulation studies on synthetic models and simple, natural channels, I will show that, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during

  8. Subcellular localization of RNA degrading proteins and protein complexes in prokaryotes.

    PubMed

    Evguenieva-Hackenberg, Elena; Roppelt, Verena; Lassek, Christian; Klug, Gabriele

    2011-01-01

    The archaeal exosome is a prokaryotic protein complex with RNA processing and degrading activities. Recently it was shown that the exosome is localized at the periphery of the cell in the thermoacidophilic archaeon Sulfolobus solfataricus. This localization is most likely mediated by the archaeal DnaG protein and depends on (direct or indirect) hydrophobic interactions with the membrane. A localization of RNA degrading proteins and protein complexes was also demonstrated in several bacteria. In bacteria a subcellular localization was also shown for substrates of these proteins and protein complexes, i.e. chromosomally encoded mRNAs and a small RNA. Thus, despite the missing compartmentalization, a spatial organization of RNA processing and degradation exists in prokaryotic cells. Recent data suggest that the spatial organization contributes to the temporal regulation of these processes.

  9. Synthetic RNA-protein complex shaped like an equilateral triangle

    NASA Astrophysics Data System (ADS)

    Ohno, Hirohisa; Kobayashi, Tetsuhiro; Kabata, Rinko; Endo, Kei; Iwasa, Takuma; Yoshimura, Shige H.; Takeyasu, Kunio; Inoue, Tan; Saito, Hirohide

    2011-02-01

    Synthetic nanostructures consisting of biomacromolecules such as nucleic acids have been constructed using bottom-up approaches. In particular, Watson-Crick base pairing has been used to construct a variety of two- and three-dimensional DNA nanostructures. Here, we show that RNA and the ribosomal protein L7Ae can form a nanostructure shaped like an equilateral triangle that consists of three proteins bound to an RNA scaffold. The construction of the complex relies on the proteins binding to kink-turn (K-turn) motifs in the RNA, which allows the RNA to bend by ~60° at three positions to form a triangle. Functional RNA-protein complexes constructed with this approach could have applications in nanomedicine and synthetic biology.

  10. Structural and evolutionary versatility in protein complexes with uneven stoichiometry.

    PubMed

    Marsh, Joseph A; Rees, Holly A; Ahnert, Sebastian E; Teichmann, Sarah A

    2015-03-16

    Proteins assemble into complexes with diverse quaternary structures. Although most heteromeric complexes of known structure have even stoichiometry, a significant minority have uneven stoichiometry--that is, differing numbers of each subunit type. To adopt this uneven stoichiometry, sequence-identical subunits must be asymmetric with respect to each other, forming different interactions within the complex. Here we first investigate the occurrence of uneven stoichiometry, demonstrating that it is common in vitro and is likely to be common in vivo. Next, we elucidate the structural determinants of uneven stoichiometry, identifying six different mechanisms by which it can be achieved. Finally, we study the frequency of uneven stoichiometry across evolution, observing a significant enrichment in bacteria compared with eukaryotes. We show that this arises due to a general increased tendency for bacterial proteins to self-assemble and form homomeric interactions, even within the context of a heteromeric complex.

  11. Molecular dynamics simulations of a membrane protein/amphipol complex.

    PubMed

    Perlmutter, Jason D; Popot, Jean-Luc; Sachs, Jonathan N

    2014-10-01

    Amphipathic polymers known as "amphipols" provide a highly stabilizing environment for handling membrane proteins in aqueous solutions. A8-35, an amphipol with a polyacrylate backbone and hydrophobic grafts, has been extensively characterized and widely employed for structural and functional studies of membrane proteins using biochemical and biophysical approaches. Given the sensitivity of membrane proteins to their environment, it is important to examine what effects amphipols may have on the structure and dynamics of the proteins they complex. Here we present the first molecular dynamics study of an amphipol-stabilized membrane protein, using Escherichia coli OmpX as a model. We begin by describing the structure of the complexes formed by supplementing OmpX with increasing amounts of A8-35, in order to determine how the amphipol interacts with the transmembrane and extramembrane surfaces of the protein. We then compare the dynamics of the protein in either A8-35, a detergent, or a lipid bilayer. We find that protein dynamics on all accessible length scales is restrained by A8-35, which provides a basis to understanding some of the stabilizing and functional effects of amphipols that have been experimentally observed.

  12. Biclustering Protein Complex Interactions with a Biclique FindingAlgorithm

    SciTech Connect

    Ding, Chris; Zhang, Anne Ya; Holbrook, Stephen

    2006-12-01

    Biclustering has many applications in text mining, web clickstream mining, and bioinformatics. When data entries are binary, the tightest biclusters become bicliques. We propose a flexible and highly efficient algorithm to compute bicliques. We first generalize the Motzkin-Straus formalism for computing the maximal clique from L{sub 1} constraint to L{sub p} constraint, which enables us to provide a generalized Motzkin-Straus formalism for computing maximal-edge bicliques. By adjusting parameters, the algorithm can favor biclusters with more rows less columns, or vice verse, thus increasing the flexibility of the targeted biclusters. We then propose an algorithm to solve the generalized Motzkin-Straus optimization problem. The algorithm is provably convergent and has a computational complexity of O(|E|) where |E| is the number of edges. It relies on a matrix vector multiplication and runs efficiently on most current computer architectures. Using this algorithm, we bicluster the yeast protein complex interaction network. We find that biclustering protein complexes at the protein level does not clearly reflect the functional linkage among protein complexes in many cases, while biclustering at protein domain level can reveal many underlying linkages. We show several new biologically significant results.

  13. Architecture and function of IFT complex proteins in ciliogenesis.

    PubMed

    Taschner, Michael; Bhogaraju, Sagar; Lorentzen, Esben

    2012-02-01

    Cilia and flagella (interchangeable terms) are evolutionarily conserved organelles found on many different types of eukaryotic cells where they fulfill important functions in motility, sensory reception and signaling. The process of Intraflagellar Transport (IFT) is of central importance for both the assembly and maintenance of cilia, as it delivers building blocks from their site of synthesis in the cell body to the ciliary assembly site at the tip of the cilium. A key player in this process is the multi-subunit IFT-complex, which acts as an adapter between the motor proteins required for movement and the ciliary cargo proteins. Since the discovery of IFT more than 15 years ago, considerable effort has gone into the purification and characterization of the IFT complex proteins. Even though this has led to very interesting findings and has greatly improved our knowledge of the IFT process, we still know very little about the overall architecture of the IFT complex and the specific functions of the various subunits. In this review we will give an update on the knowledge of the structure and function of individual IFT proteins, and the way these proteins interact to form the complex that facilitates IFT.

  14. Single-protein study of photoresistance of pigment-protein complex in lipid bilayer

    NASA Astrophysics Data System (ADS)

    Uchiyama, Daisuke; Hoshino, Hajime; Otomo, Kohei; Kato, Taro; Onda, Ken-ichi; Watanabe, Akira; Oikawa, Hiroyuki; Fujiyoshi, Satoru; Matsushita, Michio; Nango, Mamoru; Watanabe, Natsuko; Sumino, Ayumi; Dewa, Takehisa

    2011-07-01

    Photoresistance of a pigment-binding membrane protein, light-harvesting 2 (LH2) complex from the photosynthetic bacterium, Rhodopseudomonas acidophila, was investigated by fluorescence of single LH2 complexes at a temperature of 296 K. Before irreversibly stopping fluorescence, a single LH2 complex in phospholipid bilayer of dimyristoylphosphatidylcholine (DMPC) emitted on average 4 times more fluorescence photons than a complex in detergent micelle of octylglucopyranoside (OG). Fluorescence-excitation spectrum of single LH2 complexes taken at 5 K showed that the LH2 complex is structurally less perturbed in DMPC bilayer than in OG micelle, suggesting that structural instability reduces photoresistance of LH2.

  15. Heat capacity changes in carbohydrates and protein-carbohydrate complexes.

    PubMed

    Chavelas, Eneas A; García-Hernández, Enrique

    2009-05-13

    Carbohydrates are crucial for living cells, playing myriads of functional roles that range from being structural or energy-storage devices to molecular labels that, through non-covalent interaction with proteins, impart exquisite selectivity in processes such as molecular trafficking and cellular recognition. The molecular bases that govern the recognition between carbohydrates and proteins have not been fully understood yet. In the present study, we have obtained a surface-area-based model for the formation heat capacity of protein-carbohydrate complexes, which includes separate terms for the contributions of the two molecular types. The carbohydrate model, which was calibrated using carbohydrate dissolution data, indicates that the heat capacity contribution of a given group surface depends on its position in the saccharide molecule, a picture that is consistent with previous experimental and theoretical studies showing that the high abundance of hydroxy groups in carbohydrates yields particular solvation properties. This model was used to estimate the carbohydrate's contribution in the formation of a protein-carbohydrate complex, which in turn was used to obtain the heat capacity change associated with the protein's binding site. The model is able to account for protein-carbohydrate complexes that cannot be explained using a previous model that only considered the overall contribution of polar and apolar groups, while allowing a more detailed dissection of the elementary contributions that give rise to the formation heat capacity effects of these adducts.

  16. Electrophoretic separation of proteins via complexation with a polyelectrolyte

    NASA Astrophysics Data System (ADS)

    Baskin, E. M.; Shklovskii, B. I.; Zilberstein, G. V.

    2003-01-01

    We suggest to augment standard isoelectric focusing for separation of proteins in a gradient of pH by a similar focusing in the presence of a strongly charged polyelectrolyte (PE). Proteins which have the same isoelectric point but different “hidden” charges of both signs in this point make complexes with PE, which focus in different pH. This is a result of charge inversion of such proteins by adsorbed PE molecules, which is sensitive to the hidden charge. Hence, the hidden charge is a new separation parameter.

  17. p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. IMPORTANCE Eukaryotic cell adaptor molecules

  18. Unwrapping of DNA-protein complexes under external stretching.

    PubMed

    Sakaue, Takahiro; Löwen, Hartmut

    2004-08-01

    A DNA-protein complex modeled by a semiflexible chain and an attractive spherical core is studied in the situation when an external stretching force is acting on one end monomer of the chain while the other end monomer is kept fixed in space. Without a stretching force, the chain is wrapped around the core. By applying an external stretching force, unwrapping of the complex is induced. We study the statics and dynamics of the unwrapping process by computer simulations and simple phenomenological theory. We find two different scenarios depending on the chain stiffness: For a flexible chain, the extension of the complex scales linearly with the external force applied. The sphere-chain complex is disordered; i.e., there is no clear winding of the chain around the sphere. For a stiff chain, on the other hand, the complex structure is ordered, which is reminiscent of nucleosome. There is a clear winding number, and the unwrapping process under external stretching is discontinuous with jumps of the distance-force curve. This is associated with discrete unwinding processes of the complex. Our predictions are of relevance for experiments, which measure force-extension curves of DNA-protein complexes, such as nucleosome, using optical tweezers.

  19. Protein corona - from molecular adsorption to physiological complexity.

    PubMed

    Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

    2015-01-01

    In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.

  20. Protein corona – from molecular adsorption to physiological complexity

    PubMed Central

    Docter, Dominic; Maskos, Michael

    2015-01-01

    Summary In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP–protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs. PMID:25977856

  1. Immunoprecipitation and Characterization of Membrane Protein Complexes from Yeast

    ERIC Educational Resources Information Center

    Parra-Belky, Karlett; McCulloch, Kathryn; Wick, Nicole; Shircliff, Rebecca; Croft, Nicolas; Margalef, Katrina; Brown, Jamie; Crabill, Todd; Jankord, Ryan; Waldo, Eric

    2005-01-01

    In this undergraduate biochemistry laboratory experiment, the vacuolar ATPase protein complex is purified from yeast cell extracts by doing immunoprecipitations under nondenaturing conditions. Immunoprecipitations are performed using monoclonal antibodies to facilitate data interpretation, and subunits are separated on the basis of their molecular…

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

    PubMed Central

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

    2016-01-01

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

  3. Elucidation of Novel Structural Scaffold in Rohu TLR2 and Its Binding Site Analysis with Peptidoglycan, Lipoteichoic Acid and Zymosan Ligands, and Downstream MyD88 Adaptor Protein

    PubMed Central

    Sahoo, Bikash Ranjan; Basu, Madhubanti; Swain, Banikalyan; Dikhit, Manas Ranjan; Jayasankar, Pallipuram; Samanta, Mrinal

    2013-01-01

    Toll-like receptors (TLRs) play key roles in sensing wide array of microbial signatures and induction of innate immunity. TLR2 in fish resembles higher eukaryotes by sensing peptidoglycan (PGN) and lipoteichoic acid (LTA) of bacterial cell wall and zymosan of yeasts. However, in fish TLR2, no study yet describes the ligand binding motifs in the leucine rich repeat regions (LRRs) of the extracellular domain (ECD) and important amino acids in TLR2-TIR (toll/interleukin-1 receptor) domain that could be engaged in transmitting downstream signaling. We predicted these in a commercially important freshwater fish species rohu (Labeo rohita) by constructing 3D models of TLR2-ECD, TLR2-TIR, and MyD88-TIR by comparative modeling followed by 40 ns (nanosecond) molecular dynamics simulation (MDS) for TLR2-ECD and 20 ns MDS for TLR2-TIR and MyD88-TIR. Protein (TLR2-ECD)–ligands (PGN, LTA, and zymosan) docking in rohu by AutoDock4.0, FlexX2.1, and GOLD4.1 anticipated LRR16–19, LRR12–14, and LRR20-CT as the most important ligand binding motifs. Protein (TLR2-TIR)—protein (MyD88-TIR) interaction by HADDOCK and ZDOCK predicted BB loop, αB-helix, αC-helix, and CD loop in TLR2-TIR and BB loop, αB-helix, and CD loop in MyD88-TIR as the critical binding domains. This study provides ligands recognition and downstream signaling. PMID:23956969

  4. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

    PubMed Central

    Lockard, Meghan A.; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B.; Terwilliger, Thomas C.; Waldo, Geoffrey S.

    2011-01-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit. PMID:21642284

  5. A high-throughput immobilized bead screen for stable proteins and multi-protein complexes.

    PubMed

    Lockard, Meghan A; Listwan, Pawel; Pedelacq, Jean-Denis; Cabantous, Stéphanie; Nguyen, Hau B; Terwilliger, Thomas C; Waldo, Geoffrey S

    2011-07-01

    We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1-10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon(®) resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length 'breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit.

  6. Comparative Study of Elastic Network Model and Protein Contact Network for Protein Complexes: The Hemoglobin Case

    PubMed Central

    Di Paola, Luisa; Liang, Zhongjie; Giuliani, Alessandro

    2017-01-01

    The overall topology and interfacial interactions play key roles in understanding structural and functional principles of protein complexes. Elastic Network Model (ENM) and Protein Contact Network (PCN) are two widely used methods for high throughput investigation of structures and interactions within protein complexes. In this work, the comparative analysis of ENM and PCN relative to hemoglobin (Hb) was taken as case study. We examine four types of structural and dynamical paradigms, namely, conformational change between different states of Hbs, modular analysis, allosteric mechanisms studies, and interface characterization of an Hb. The comparative study shows that ENM has an advantage in studying dynamical properties and protein-protein interfaces, while PCN is better for describing protein structures quantitatively both from local and from global levels. We suggest that the integration of ENM and PCN would give a potential but powerful tool in structural systems biology. PMID:28243596

  7. Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry.

    PubMed

    Li, Huilin; Sheng, Yuewei; McGee, William; Cammarata, Michael; Holden, Dustin; Loo, Joseph A

    2017-03-07

    Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving noncovalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and intermolecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with preactivation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

  8. Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex.

    PubMed

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-08-20

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase.

  9. Transmembrane Protein 147 (TMEM147) Is a Novel Component of the Nicalin-NOMO Protein Complex*

    PubMed Central

    Dettmer, Ulf; Kuhn, Peer-Hendrik; Abou-Ajram, Claudia; Lichtenthaler, Stefan F.; Krüger, Marcus; Kremmer, Elisabeth; Haass, Christian; Haffner, Christof

    2010-01-01

    Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely γ-secretase and the Nicalin-NOMO (Nodal modulator) complex. The γ-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the β-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200–220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A ∼22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to γ-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with γ-secretase. PMID:20538592

  10. Modeling of Protein Binary Complexes Using Structural Mass Spectrometry Data

    SciTech Connect

    Amisha Kamal,J.; Chance, M.

    2008-01-01

    In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints--positive and/or negative--in the docking step and are also used to decide the type of energy filter--electrostatics or desolvation--in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure.

  11. Solution structure of the focal adhesion adaptor PINCH LIM1 domain and characterization of its interaction with the integrin-linked kinase ankyrin repeat domain.

    PubMed

    Velyvis, A; Yang, Y; Wu, C; Qin, J

    2001-02-16

    PINCH is a recently identified adaptor protein that comprises an array of five LIM domains. PINCH functions through LIM-mediated protein-protein interactions that are involved in cell adhesion, growth, and differentiation. The LIM1 domain of PINCH interacts with integrin-linked kinase (ILK), thereby mediating focal adhesions via a specific integrin/ILK signaling pathway. We have solved the NMR structure of the PINCH LIM1 domain and characterized its binding to ILK. LIM1 contains two contiguous zinc fingers of the CCHC and CCCH types and adopts a global fold similar to that of functionally distinct LIM domains from cysteine-rich protein and cysteine-rich intestinal protein families with CCHC and CCCC zinc finger types. Gel-filtration and NMR experiments demonstrated a 1:1 complex between PINCH LIM1 and the ankyrin repeat domain of ILK. A chemical shift mapping experiment identified regions in PINCH LIM1 that are important for interaction with ILK. Comparison of surface features between PINCH LIM1 and other functionally different LIM domains indicated that the LIM motif might have a highly variable mode in recognizing various target proteins.

  12. Adaptor proteins MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and are specific for mitochondrial fission.

    PubMed

    Palmer, Catherine S; Elgass, Kirstin D; Parton, Robert G; Osellame, Laura D; Stojanovski, Diana; Ryan, Michael T

    2013-09-20

    Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.

  13. Exploration of the Dynamic Properties of Protein Complexes Predicted from Spatially Constrained Protein-Protein Interaction Networks

    PubMed Central

    Yen, Eric A.; Tsay, Aaron; Waldispuhl, Jerome; Vogel, Jackie

    2014-01-01

    Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden

  14. Annexin A2 complexes with S100 proteins: structure, function and pharmacological manipulation

    PubMed Central

    Liu, Yidong; Myrvang, Helene K; Dekker, Lodewijk V

    2015-01-01

    Annexin A2 (AnxA2) was originally identified as a substrate of the pp60v-src oncoprotein in transformed chicken embryonic fibroblasts. It is an abundant protein that associates with biological membranes as well as the actin cytoskeleton, and has been implicated in intracellular vesicle fusion, the organization of membrane domains, lipid rafts and membrane-cytoskeleton contacts. In addition to an intracellular role, AnxA2 has been reported to participate in processes localized to the cell surface including extracellular protease regulation and cell-cell interactions. There are many reports showing that AnxA2 is differentially expressed between normal and malignant tissue and potentially involved in tumour progression. An important aspect of AnxA2 function relates to its interaction with small Ca2+-dependent adaptor proteins called S100 proteins, which is the topic of this review. The interaction between AnxA2 and S100A10 has been very well characterized historically; more recently, other S100 proteins have been shown to interact with AnxA2 as well. The biochemical evidence for the occurrence of these protein interactions will be discussed, as well as their function. Recent studies aiming to generate inhibitors of S100 protein interactions will be described and the potential of these inhibitors to further our understanding of AnxA2 S100 protein interactions will be discussed. PMID:25303710

  15. Molecular Signatures of Membrane Protein Complexes Underlying Muscular Dystrophy*

    PubMed Central

    Turk, Rolf; Hsiao, Jordy J.; Smits, Melinda M.; Ng, Brandon H.; Pospisil, Tyler C.; Jones, Kayla S.; Campbell, Kevin P.; Wright, Michael E.

    2016-01-01

    Mutations in genes encoding components of the sarcolemmal dystrophin-glycoprotein complex (DGC) are responsible for a large number of muscular dystrophies. As such, molecular dissection of the DGC is expected to both reveal pathological mechanisms, and provides a biological framework for validating new DGC components. Establishment of the molecular composition of plasma-membrane protein complexes has been hampered by a lack of suitable biochemical approaches. Here we present an analytical workflow based upon the principles of protein correlation profiling that has enabled us to model the molecular composition of the DGC in mouse skeletal muscle. We also report our analysis of protein complexes in mice harboring mutations in DGC components. Bioinformatic analyses suggested that cell-adhesion pathways were under the transcriptional control of NFκB in DGC mutant mice, which is a finding that is supported by previous studies that showed NFκB-regulated pathways underlie the pathophysiology of DGC-related muscular dystrophies. Moreover, the bioinformatic analyses suggested that inflammatory and compensatory mechanisms were activated in skeletal muscle of DGC mutant mice. Additionally, this proteomic study provides a molecular framework to refine our understanding of the DGC, identification of protein biomarkers of neuromuscular disease, and pharmacological interrogation of the DGC in adult skeletal muscle https://www.mda.org/disease/congenital-muscular-dystrophy/research. PMID:27099343

  16. Heterodimeric Drosophila gap gene protein complexes acting as transcriptional repressors.

    PubMed Central

    Sauer, F; Jäckle, H

    1995-01-01

    The Drosophila gap gene Krüppel (Kr) encodes a transcriptional regulator. It acts both as an integral part of the Drosophila segmentation gene in the early blastoderm and in a variety of tissues and organs at later stages of embryogenesis. In transfected tissue culture cells, the Kr protein (Kr) was shown to both activate and repress gene expression in a concentration-dependent manner when acting from a single binding site close to the promoter. Here we show that KR can associate with the transcription factors encoded by the gap genes knirps (kni) and hunchback (hb) which affect KR-dependent gene expression in Drosophila tissue culture cells. The association of DNA-bound hb protein or free kni protein with distinct but different regions of KR results in the formation of DNA-bound transcriptional repressor complexes. Our results suggest that individual transcription factors can associate to form protein complexes which act as direct repressors of transcription. The interactions shown here add an unexpected level of complexity to the control of gene expression. Images PMID:7588607

  17. Characterization of the human GARP (Golgi associated retrograde protein) complex

    SciTech Connect

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank . E-mail: stenlie@t-online.de

    2005-05-15

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

  18. Changes in protein structure at the interface accompanying complex formation.

    PubMed

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H; Chakrabarti, Pinak

    2015-11-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein-Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial.

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

    SciTech Connect

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

    2011-12-31

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

  20. The RCP-Rab11 complex regulates endocytic protein sorting.

    PubMed

    Peden, Andrew A; Schonteich, Eric; Chun, John; Junutula, Jagath R; Scheller, Richard H; Prekeris, Rytis

    2004-08-01

    Rab 11 GTPase is an important regulator of endocytic membrane traffic. Recently, we and others have identified a novel family of Rab11 binding proteins, known as Rab11-family interacting proteins (FIPs). One of the family members, Rab coupling protein (RCP), was identified as a protein binding to both Rab4 and Rab11 GTPases. RCP was therefore suggested to serve a dual function as Rab4 and Rab11 binding protein. In this study, we characterized the cellular functions of RCP and mapped its interactions with Rab4 and Rab11. Our data show that RCP interacts only weakly with Rab4 in vitro and does not play the role of coupling Rab11 and Rab4 in vivo. Furthermore, our data indicate that the RCP-Rab11 complex regulates the sorting of transferrin receptors from the degradative to the recycling pathway. We therefore propose that RCP functions primarily as a Rab11 binding protein that regulates protein sorting in tubular endosomes.

  1. 2D depiction of nonbonding interactions for protein complexes.

    PubMed

    Zhou, Peng; Tian, Feifei; Shang, Zhicai

    2009-04-30

    A program called the 2D-GraLab is described for automatically generating schematic representation of nonbonding interactions across the protein binding interfaces. The input file of this program takes the standard PDB format, and the outputs are two-dimensional PostScript diagrams giving intuitive and informative description of the protein-protein interactions and their energetics properties, including hydrogen bond, salt bridge, van der Waals interaction, hydrophobic contact, pi-pi stacking, disulfide bond, desolvation effect, and loss of conformational entropy. To ensure these interaction information are determined accurately and reliably, methods and standalone programs employed in the 2D-GraLab are all widely used in the chemistry and biology community. The generated diagrams allow intuitive visualization of the interaction mode and binding specificity between two subunits in protein complexes, and by providing information on nonbonding energetics and geometric characteristics, the program offers the possibility of comparing different protein binding profiles in a detailed, objective, and quantitative manner. We expect that this 2D molecular graphics tool could be useful for the experimentalists and theoreticians interested in protein structure and protein engineering.

  2. Homology modelling of protein-protein complexes: a simple method and its possibilities and limitations

    PubMed Central

    Launay, Guillaume; Simonson, Thomas

    2008-01-01

    Background Structure-based computational methods are needed to help identify and characterize protein-protein complexes and their function. For individual proteins, the most successful technique is homology modelling. We investigate a simple extension of this technique to protein-protein complexes. We consider a large set of complexes of known structures, involving pairs of single-domain proteins. The complexes are compared with each other to establish their sequence and structural similarities and the relation between the two. Compared to earlier studies, a simpler dataset, a simpler structural alignment procedure, and an additional energy criterion are used. Next, we compare the Xray structures to models obtained by threading the native sequence onto other, homologous complexes. An elementary requirement for a successful energy function is to rank the native structure above any threaded structure. We use the DFIREβ energy function, whose quality and complexity are typical of the models used today. Finally, we compare near-native models to distinctly non-native models. Results If weakly stable complexes are excluded (defined by a binding energy cutoff), as well as a few unusual complexes, a simple homology principle holds: complexes that share more than 35% sequence identity share similar structures and interaction modes; this principle was less clearcut in earlier studies. The energy function was then tested for its ability to identify experimental structures among sets of decoys, produced by a simple threading procedure. On average, the experimental structure is ranked above 92% of the alternate structures. Thus, discrimination of the native structure is good but not perfect. The discrimination of near-native structures is fair. Typically, a single, alternate, non-native binding mode exists that has a native-like energy. Some of the associated failures may correspond to genuine, alternate binding modes and/or native complexes that are artefacts of the crystal

  3. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    PubMed

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  4. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  5. A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

    PubMed Central

    Yeakley, J M; Morfin, J P; Rosenfeld, M G; Fu, X D

    1996-01-01

    A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8755518

  6. A liquid crystal of ascorbyl palmitate, used as vaccine platform, provides sustained release of antigen and has intrinsic pro-inflammatory and adjuvant activities which are dependent on MyD88 adaptor protein.

    PubMed

    Sánchez Vallecillo, María F; Minguito de la Escalera, María M; Aguirre, María V; Ullio Gamboa, Gabriela V; Palma, Santiago D; González-Cintado, Leticia; Chiodetti, Ana L; Soldano, Germán; Morón, Gabriel; Allemandi, Daniel A; Ardavín, Carlos; Pistoresi-Palencia, María C; Maletto, Belkys A

    2015-09-28

    Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1β, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.

  7. Changes in protein structure at the interface accompanying complex formation

    PubMed Central

    Chakravarty, Devlina; Janin, Joël; Robert, Charles H.; Chakrabarti, Pinak

    2015-01-01

    Protein interactions are essential in all biological processes. The changes brought about in the structure when a free component forms a complex with another molecule need to be characterized for a proper understanding of molecular recognition as well as for the successful implementation of docking algorithms. Here, unbound (U) and bound (B) forms of protein structures from the Protein–Protein Interaction Affinity Database are compared in order to enumerate the changes that occur at the interface atoms/residues in terms of the solvent-accessible surface area (ASA), secondary structure, temperature factors (B factors) and disorder-to-order transitions. It is found that the interface atoms optimize contacts with the atoms in the partner protein, which leads to an increase in their ASA in the bound interface in the majority (69%) of the proteins when compared with the unbound interface, and this is independent of the root-mean-square deviation between the U and B forms. Changes in secondary structure during the transition indicate a likely extension of helices and strands at the expense of turns and coils. A reduction in flexibility during complex formation is reflected in the decrease in B factors of the interface residues on going from the U form to the B form. There is, however, no distinction in flexibility between the interface and the surface in the monomeric structure, thereby highlighting the potential problem of using B factors for the prediction of binding sites in the unbound form for docking another protein. 16% of the proteins have missing (disordered) residues in the U form which are observed (ordered) in the B form, mostly with an irregular conformation; the data set also shows differences in the composition of interface and non-interface residues in the disordered polypeptide segments as well as differences in their surface burial. PMID:26594372

  8. Protein Complex Affinity Capture from Cryomilled Mammalian Cells.

    PubMed

    LaCava, John; Jiang, Hua; Rout, Michael P

    2016-12-09

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

  9. Protein Complex Affinity Capture from Cryomilled Mammalian Cells

    PubMed Central

    LaCava, John; Jiang, Hua; Rout, Michael P.

    2016-01-01

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets. PMID:28060343

  10. Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.

    PubMed

    Knowles, Timothy J; Scott-Tucker, Anthony; Overduin, Michael; Henderson, Ian R

    2009-03-01

    The folding of transmembrane proteins into the outer membrane presents formidable challenges to Gram-negative bacteria. These proteins must migrate from the cytoplasm, through the inner membrane and into the periplasm, before being recognized by the beta-barrel assembly machinery, which mediates efficient insertion of folded beta-barrels into the outer membrane. Recent discoveries of component structures and accessory interactions of this complex are yielding insights into how cells fold membrane proteins. Here, we discuss how these structures illuminate the mechanisms responsible for the biogenesis of outer membrane proteins.

  11. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    NASA Astrophysics Data System (ADS)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  12. A profile of protein-protein interaction: Crystal structure of a lectin-lectin complex.

    PubMed

    Surya, Sukumaran; Abhilash, Joseph; Geethanandan, Krishnan; Sadasivan, Chittalakkottu; Haridas, Madhathilkovilakathu

    2016-06-01

    Proteins may utilize complex networks of interactions to create/proceed signaling pathways of highly adaptive responses such as programmed cell death. Direct binary interactions study of proteins may help propose models for protein-protein interaction. Towards this goal we applied a combination of thermodynamic kinetics and crystal structure analyses to elucidate the complexity and diversity in such interactions. By determining the heat change on the association of two galactose-specific legume lectins from Butea monosperma (BML) and Spatholobus parviflorus (SPL) belonging to Fabaceae family helped to compute the binding equilibrium. It was extended further by X-ray structural analysis of BML-SPL binary complex. In order to chart the proteins interacting mainly through their interfaces, identification of the nature of forces which stabilized the association of the lectin-lectin complex was examined. Comprehensive analysis of the BMLSPL complex by isothermal titration calorimetry and X-ray crystal structure threw new light on the lectin-lectin interactions suggesting of their use in diverse areas of glycobiology.

  13. Characterization of known protein complexes using k-connectivity and other topological measures

    PubMed Central

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  14. DMS Footprinting of Structured RNAs and RNA-Protein Complexes

    PubMed Central

    Tijerina, Pilar; Mohr, Sabine; Russell, Rick

    2008-01-01

    We describe a protocol in which dimethyl sulfate (DMS) modification of the base-pairing faces of unpaired adenosine and cytidine nucleotides is used for structural analysis of RNAs and RNA-protein complexes (RNPs). The protocol is optimized for RNAs of small to moderate size (≤500 nucleotides). The RNA or RNP is first exposed to DMS under conditions that promote formation of the folded structure or complex, as well as ‘control’ conditions that do not allow folding or complex formation. The positions and extents of modification are then determined by primer extension, polyacrylamide gel electrophoresis (PAGE), and quantitative analysis. From changes in the extent of modification upon folding or protein binding (appearance of a ‘footprint’), it is possible to detect local changes in RNA secondary and tertiary structure, as well as the formation of RNA-protein contacts. This protocol takes 1.5–3 days to complete, depending on the type of analysis used. PMID:17948004

  15. Protein fragment bimolecular fluorescence complementation analyses for the in vivo study of protein-protein interactions and cellular protein complex localizations

    PubMed Central

    Waadt, Rainer; Schlücking, Kathrin; Schroeder, Julian I.; Kudla, Jörg

    2014-01-01

    Summary The analyses of protein-protein interactions is crucial for understanding cellular processes including signal transduction, protein trafficking and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo. Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms, and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further we discuss different BiFC applications and provide examples for proper BiFC analyses in planta. PMID:24057390

  16. Radioprotection by polyethylene glycol-protein complexes in mice

    SciTech Connect

    Gray, B.H.; Stull, R.W.

    1983-03-01

    Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before /sup 60/Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following /sup 60/Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors.

  17. Small-Angle X-Ray Scattering From RNA, Proteins, And Protein Complexes

    SciTech Connect

    Lipfert, Jan; Doniach, Sebastian; /Stanford U., Phys. Dept. /Stanford U., Appl. Phys. Dept. /SLAC, SSRL

    2007-09-18

    Small-angle X-ray scattering (SAXS) is increasingly used to characterize the structure and interactions of biological macromolecules and their complexes in solution. Although still a low-resolution technique, the advent of high-flux synchrotron sources and the development of algorithms for the reconstruction of 3-D electron density maps from 1-D scattering profiles have made possible the generation of useful low-resolution molecular models from SAXS data. Furthermore, SAXS is well suited for the study of unfolded or partially folded conformational ensembles as a function of time or solution conditions. Here, we review recently developed algorithms for 3-D structure modeling and applications to protein complexes. Furthermore, we discuss the emerging use of SAXS as a tool to study membrane protein-detergent complexes. SAXS is proving useful to study the folding of functional RNA molecules, and finally we discuss uses of SAXS to study ensembles of denatured proteins.

  18. Integrating computational methods and experimental data for understanding the recognition mechanism and binding affinity of protein-protein complexes.

    PubMed

    Gromiha, M Michael; Yugandhar, K

    2017-01-07

    Protein-protein interactions perform several functions inside the cell. Understanding the recognition mechanism and binding affinity of protein-protein complexes is a challenging problem in experimental and computational biology. In this review, we focus on two aspects (i) understanding the recognition mechanism and (ii) predicting the binding affinity. The first part deals with computational techniques for identifying the binding site residues and the contribution of important interactions for understanding the recognition mechanism of protein-protein complexes in comparison with experimental observations. The second part is devoted to the methods developed for discriminating high and low affinity complexes, and predicting the binding affinity of protein-protein complexes using three-dimensional structural information and just from the amino acid sequence. The overall view enhances our understanding of the integration of experimental data and computational methods, recognition mechanism of protein-protein complexes and the binding affinity.

  19. 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.

  20. Optimization and dynamics of protein-protein complexes using B-splines.

    PubMed

    Gillilan, Richard E; Lilien, Ryan H

    2004-10-01

    A moving-grid approach for optimization and dynamics of protein-protein complexes is introduced, which utilizes cubic B-spline interpolation for rapid energy and force evaluation. The method allows for the efficient use of full electrostatic potentials joined smoothly to multipoles at long distance so that multiprotein simulation is possible. Using a recently published benchmark of 58 protein complexes, we examine the performance and quality of the grid approximation, refining cocrystallized complexes to within 0.68 A RMSD of interface atoms, close to the optimum 0.63 A produced by the underlying MMFF94 force field. We quantify the theoretical statistical advantage of using minimization in a stochastic search in the case of two rigid bodies, and contrast it with the underlying cost of conjugate gradient minimization using B-splines. The volumes of conjugate gradient minimization basins of attraction in cocrystallized systems are generally orders of magnitude larger than well volumes based on energy thresholds needed to discriminate native from nonnative states; nonetheless, computational cost is significant. Molecular dynamics using B-splines is doubly efficient due to the combined advantages of rapid force evaluation and large simulation step sizes. Large basins localized around the native state and other possible binding sites are identifiable during simulations of protein-protein motion. In addition to providing increased modeling detail, B-splines offer new algorithmic possibilities that should be valuable in refining docking candidates and studying global complex behavior.

  1. Distinct Roles for TGN/Endosome Epsin-like Adaptors Ent3p and Ent5p

    PubMed Central

    Costaguta, Giancarlo; Duncan, Mara C.; Fernández, G. Esteban; Huang, Grace H.

    2006-01-01

    Clathrin adaptors are key factors in clathrin-coated vesicle formation, coupling clathrin to cargo and/or the lipid bilayer. A physically interacting network of three classes of adaptors participate in clathrin-mediated traffic between the trans-Golgi network (TGN) and endosomes: AP-1, Gga proteins, and epsin-like proteins. Here we investigate functional relationships within this network through transport assays and protein localization analysis in living yeast cells. We observed that epsin-like protein Ent3p preferentially localized with Gga2p, whereas Ent5p distributed equally between AP-1 and Gga2p. Ent3p was mislocalized in Gga-deficient but not in AP-1–deficient cells. In contrast, Ent5p retained localization in cells lacking either or both AP-1 and Gga proteins. The Ent proteins were dispensable for AP-1 or Gga localization. Synthetic genetic growth and α-factor maturation defects were observed when ent5Δ but not ent3Δ was introduced together with deletions of the GGA genes. In AP-1–deficient cells, ent3Δ and to a lesser extent ent5Δ caused minor α-factor maturation defects, but together resulted in a near-lethal phenotype. Deletions of ENT3 and ENT5 also displayed synthetic defects similar to, but less severe than, synthetic effects of AP-1 and Gga inactivation. These results differentiate Ent3p and Ent5p function in vivo, suggesting that Ent3p acts primarily with Gga proteins, whereas Ent5p acts with both AP-1 and Gga proteins but is more critical for AP-1–mediated transport. The data also support a model in which the Ent adaptors provide important accessory functions to AP-1 and Gga proteins in TGN/endosome traffic. PMID:16790491

  2. Promoter chromatin remodeling of immediate-early genes is mediated through H3 phosphorylation at either serine 28 or 10 by the MSK1 multi-protein complex

    PubMed Central

    Drobic, Bojan; Pérez-Cadahía, Beatriz; Yu, Jenny; Kung, Sam Kam-Pun; Davie, James R.

    2010-01-01

    Upon activation of the ERK and p38 MAPK pathways, the MSK1/2-mediated nucleosomal response, including H3 phosphorylation at serine 28 or 10, is coupled with the induction of immediate-early (IE) gene transcription. The outcome of this response, varying with the stimuli and cellular contexts, ranges from neoplastic transformation to neuronal synaptic plasticity. Here, we used sequential co-immunoprecipitation assays and sequential chromatin immunoprecipitation (ChIP) assays on mouse fibroblast 10T1/2 and MSK1 knockdown 10T1/2 cells to show that H3 serine 28 and 10 phosphorylation leads to promoter remodeling. MSK1, in complexes with phospho-serine adaptor 14-3-3 proteins and BRG1 the ATPase subunit of the SWI/SNF remodeler, is recruited to the promoter of target genes by transcription factors such as Elk-1 or NF-κB. Following MSK1-mediated H3 phosphorylation, BRG1 associates with the promoter of target genes via 14-3-3 proteins, which act as scaffolds. The recruited SWI/SNF remodels nucleosomes at the promoter of IE genes enabling the binding of transcription factors like JUN and the onset of transcription. PMID:20129940

  3. Biodegradation of the chitin-protein complex in crustacean cuticle

    USGS Publications Warehouse

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

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

    PubMed Central

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

    2009-01-01

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

  5. Protein Complexes are Central in the Yeast Genetic Landscape

    PubMed Central

    Michaut, Magali; Baryshnikova, Anastasia; Costanzo, Michael; Myers, Chad L.; Andrews, Brenda J.; Boone, Charles; Bader, Gary D.

    2011-01-01

    If perturbing two genes together has a stronger or weaker effect than expected, they are said to genetically interact. Genetic interactions are important because they help map gene function, and functionally related genes have similar genetic interaction patterns. Mapping quantitative (positive and negative) genetic interactions on a global scale has recently become possible. This data clearly shows groups of genes connected by predominantly positive or negative interactions, termed monochromatic groups. These groups often correspond to functional modules, like biological processes or complexes, or connections between modules. However it is not yet known how these patterns globally relate to known functional modules. Here we systematically study the monochromatic nature of known biological processes using the largest quantitative genetic interaction data set available, which includes fitness measurements for ∼5.4 million gene pairs in the yeast Saccharomyces cerevisiae. We find that only 10% of biological processes, as defined by Gene Ontology annotations, and less than 1% of inter-process connections are monochromatic. Further, we show that protein complexes are responsible for a surprisingly large fraction of these patterns. This suggests that complexes play a central role in shaping the monochromatic landscape of biological processes. Altogether this work shows that both positive and negative monochromatic patterns are found in known biological processes and in their connections and that protein complexes play an important role in these patterns. The monochromatic processes, complexes and connections we find chart a hierarchical and modular map of sensitive and redundant biological systems in the yeast cell that will be useful for gene function prediction and comparison across phenotypes and organisms. Furthermore the analysis methods we develop are applicable to other species for which genetic interactions will progressively become more available. PMID

  6. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    SciTech Connect

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S. E-mail: mkukrishna@rediffmail.com

    2005-08-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V{sub M}) of 3.3 Å{sup 3} Da{sup −1}, corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin.

  7. Spectroscopic analysis of protein Fe-NO complexes.

    PubMed

    Bellota-Antón, César; Munnoch, John; Robb, Kirsty; Adamczyk, Katrin; Candelaresi, Marco; Parker, Anthony W; Dixon, Ray; Hutchings, Matthew I; Hunt, Neil T; Tucker, Nicholas P

    2011-10-01

    The toxic free radical NO (nitric oxide) has diverse biological roles in eukaryotes and bacteria, being involved in signalling, vasodilation, blood clotting and immunity, and as an intermediate in microbial denitrification. The predominant biological mechanism of detecting NO is through the formation of iron nitrosyl complexes, although this is a deleterious process for other iron-containing enzymes. We have previously applied techniques such as UV-visible and EPR spectroscopy to the analysis of protein Fe-NO complex formation in order to study how NO controls the activity of the bacterial transcriptional regulators NorR and NsrR. These studies have analysed NO-dependent biological activity both in vitro and in vivo using diverse biochemical, molecular and spectroscopic methods. Recently, we have applied ultrafast 2D-IR (two-dimensional IR) spectroscopy to the analysis of NO-protein interactions using Mb (myoglobin) and Cc (cytochrome c) as model haem proteins. The ultrafast fluctuations of Cc and Mb show marked differences, indicating altered flexibility of the haem pockets. We have extended this analysis to bacterial catalase enzymes that are known to play a role in the nitrosative stress response by detoxifying peroxynitrite. The first 2D-IR analysis of haem nitrosylation and perspectives for the future are discussed.

  8. Inferring drug-disease associations based on known protein complexes.

    PubMed

    Yu, Liang; Huang, Jianbin; Ma, Zhixin; Zhang, Jing; Zou, Yapeng; Gao, Lin

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html.

  9. Inferring drug-disease associations based on known protein complexes

    PubMed Central

    2015-01-01

    Inferring drug-disease associations is critical in unveiling disease mechanisms, as well as discovering novel functions of available drugs, or drug repositioning. Previous work is primarily based on drug-gene-disease relationship, which throws away many important information since genes execute their functions through interacting others. To overcome this issue, we propose a novel methodology that discover the drug-disease association based on protein complexes. Firstly, the integrated heterogeneous network consisting of drugs, protein complexes, and disease are constructed, where we assign weights to the drug-disease association by using probability. Then, from the tripartite network, we get the indirect weighted relationships between drugs and diseases. The larger the weight, the higher the reliability of the correlation. We apply our method to mental disorders and hypertension, and validate the result by using comparative toxicogenomics database. Our ranked results can be directly reinforced by existing biomedical literature, suggesting that our proposed method obtains higher specificity and sensitivity. The proposed method offers new insight into drug-disease discovery. Our method is publicly available at http://1.complexdrug.sinaapp.com/Drug_Complex_Disease/Data_Download.html. PMID:26044949

  10. Anticancer osmium complex inhibitors of the HIF-1α and p300 protein-protein interaction

    PubMed Central

    Yang, Chao; Wang, Wanhe; Li, Guo-Dong; Zhong, Hai-Jing; Dong, Zhen-Zhen; Wong, Chun-Yuen; Kwong, Daniel W. J.; Ma, Dik-Lung; Leung, Chung-Hang

    2017-01-01

    The hypoxia inducible factor (HIF) pathway has been considered to be an attractive anti-cancer target. One strategy to inhibit HIF activity is through the disruption of the HIF-1α–p300 protein-protein interaction. We report herein the identification of an osmium(II) complex as the first metal-based inhibitor of the HIF-1α–p300 interaction. We evaluated the effect of complex 1 on HIF-1α signaling pathway in vitro and in cellulo by using the dual luciferase reporter assay, co-immunoprecipitation assay, and immunoblot assay. Complex 1 exhibited a dose-dependent inhibition of HRE-driven luciferase activity, with an IC50 value of 1.22 μM. Complex 1 interfered with the HIF-1α–p300 interaction as revealed by a dose-dependent reduction of p300 co-precipitated with HIF-1α as the concentration of complex 1 was increased. Complex 1 repressed the phosphorylation of SRC, AKT and STAT3, and had no discernible effect on the activity of NF-κB. We anticipate that complex 1 could be utilized as a promising scaffold for the further development of more potent HIF-1α inhibitors for anti-cancer treatment. PMID:28225008

  11. Analysis of the interface variability in NMR structure ensembles of protein-protein complexes.

    PubMed

    Calvanese, Luisa; D'Auria, Gabriella; Vangone, Anna; Falcigno, Lucia; Oliva, Romina

    2016-06-01

    NMR structures consist in ensembles of conformers, all satisfying the experimental restraints, which exhibit a certain degree of structural variability. We analyzed here the interface in NMR ensembles of protein-protein heterodimeric complexes and found it to span a wide range of different conservations. The different exhibited conservations do not simply correlate with the size of the systems/interfaces, and are most probably the result of an interplay between different factors, including the quality of experimental data and the intrinsic complex flexibility. In any case, this information is not to be missed when NMR structures of protein-protein complexes are analyzed; especially considering that, as we also show here, the first NMR conformer is usually not the one which best reflects the overall interface. To quantify the interface conservation and to analyze it, we used an approach originally conceived for the analysis and ranking of ensembles of docking models, which has now been extended to directly deal with NMR ensembles. We propose this approach, based on the conservation of the inter-residue contacts at the interface, both for the analysis of the interface in whole ensembles of NMR complexes and for the possible selection of a single conformer as the best representative of the overall interface. In order to make the analyses automatic and fast, we made the protocol available as a web tool at: https://www.molnac.unisa.it/BioTools/consrank/consrank-nmr.html.

  12. Identification of CMS as a cytosolic adaptor of the human pTalpha chain involved in pre-TCR function.

    PubMed

    Navarro, María N; Nusspaumer, Gretel; Fuentes, Patricia; González-García, Sara; Alcain, Juan; Toribio, María L

    2007-12-15

    The T-cell receptor beta (TCRbeta)/pre-TCRalpha (pTalpha) pre-TCR complex (pre-TCR) signals the expansion and differentiation of de-veloping thymocytes. Functional pro-perties of the pre-TCR rely on its unique pTalpha chain, which suggests the participation of specific intracellular adaptors. However, pTalpha-interacting molecules remain unknown. Here, we identified a polyproline-arginine sequence in the human pTalpha cytoplasmic tail that interacted in vitro with SH3 domains of the CIN85/CMS family of adaptors, and mediated the recruitment of multiprotein complexes involving all (CMS, CIN85, and CD2BP3) members. Supporting the physiologic relevance of this interaction, we found that 1 such adaptor, CMS, interacted in vivo with human pTalpha, and its expression was selectively up-regulated during human thymopoiesis in pre-TCR-activated thymocytes. Upon activation, pre-TCR clustering was induced, and CMS and polymerized actin were simultaneously recruited to the pre-TCR activation site. CMS also associated via its C-terminal region to the actin cytoskeleton in the endocytic compartment, where it colocalized with internalized pTalpha in traffic to lysosomal degradation. Notably, deletion of the pTalpha CIN85/CMS-binding motif impaired pre-TCR-mediated Ca(2+) mobilization and NFAT transcriptional activity, and precluded activation induced by overexpression of a CMS-SH3 N-terminal mutant. These results provide the first molecular evidence for a pTalpha intracellular adaptor involved in pre-TCR function.

  13. Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

    PubMed

    Huser, Sonja; Suri, Gregor; Crottet, Pascal; Spiess, Martin

    2013-02-15

    The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

  14. Force-induced remodelling of proteins and their complexes

    PubMed Central

    Chen, Yun; Radford, Sheena E; Brockwell, David J

    2015-01-01

    Force can drive conformational changes in proteins, as well as modulate their stability and the affinity of their complexes, allowing a mechanical input to be converted into a biochemical output. These properties have been utilised by nature and force is now recognised to be widely used at the cellular level. The effects of force on the biophysical properties of biological systems can be large and varied. As these effects are only apparent in the presence of force, studies on the same proteins using traditional ensemble biophysical methods can yield apparently conflicting results. Where appropriate, therefore, force measurements should be integrated with other experimental approaches to understand the physiological context of the system under study. PMID:25710390

  15. Evidence for a protein-protein complex during iron loading into ferritin by ceruloplasmin.

    PubMed

    Reilly, C A; Sorlie, M; Aust, S D

    1998-06-01

    The formation of a protein-protein complex for the loading of iron into ferritin by ceruloplasmin was investigated. Ferritin stimulated the ferroxidase activity of ceruloplasmin unless the ferritin was fully loaded, in which case it inhibited the ferroxidase activity of ceruloplasmin. The apparent association constant for the interaction of ferritin and ceruloplasmin was 24 nM. Isothermal titration calorimetry indicated that the interaction of ceruloplasmin and ferritin was endothermic, driven by positive changes in entropy. The association constants for complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7 x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized forms of ceruloplasmin, respectively. The oxidized form of ceruloplasmin was retained on an affinity column with ferritin immobilized as the ligand and remained bound to the column with mobile phases of increased hydrophobicity, but was eluted with increased ionic strength. The ability of ceruloplasmin to remain bound to the affinity resin was affected by the species from which ceruloplasmin was isolated. Gradient ultracentrifugation also provided evidence that the two proteins were associated, since ferritin promoted migration of ceruloplasmin through the gradient. Including ferrous iron in the gradient resulted in reduction of ceruloplasmin and increased the mobility of ceruloplasmin with ferritin. These data provide evidence that ferritin and ceruloplasmin form a protein-protein complex during iron loading into ferritin, which may limit redox cycling of iron in vivo.

  16. Rediscovery of halogen bonds in protein-ligand complexes.

    PubMed

    Zhou, P; Tian, F; Zou, J; Shang, Z

    2010-04-01

    Although the halogen bond has attracted much interest in chemistry and material science communities, its implications for drug design are just now coming to light. The protein-ligand interactions through short halogen-oxygen/nitrogen/sulfur contacts have been observed in crystal structures for a long time, but only in recent years, with the experimental and theoretical progress in weak biological interactions, especially the pioneering works contributed by Ho and co-workers (Auffinger, P.; Hays, F. A.; Westhof, E.; Ho, P. S. Proc. Natl. Acad. Sci. USA 2004, 101, 16789-16794), these short contacts involving halogens in biomolecules were rediscovered and re-recognized as halogen bonds to stress their shared similarities with hydrogen bonds in strength and directionality. Crystal structure determinations of protein complexes with halogenated ligands preliminarily unveiled the functionality of halogen bonds in protein-ligand recogni-tion. Database surveys further revealed a considerable number of short halogen-oxygen contacts between proteins and halogenated ligands. Theoretical calculations on model and real systems eventually gave a quantitative pronouncement for the substantial contribution of halogen bonds to ligand binding. All of these works forebode that the halogen bond can be exploited as a new and versatile tool for rational drug design and bio-crystal engineering.

  17. Adaptor for Measuring Principal Strains with Tuckerman Strain Gage

    NASA Technical Reports Server (NTRS)

    Mcpherson, A E

    1943-01-01

    An adapter is described which uses three Tuckerman optical strain gages to measure the displacement of the three vortices of an equilateral triangle along lines 120 degrees apart. These displacements are substituted in well-known equations in order to compute the magnitude and direction of the principal strains. Tests of the adaptor indicate that principal strains over a gage length of 1.42 inch may be measured with a systematic error not exceeding 4 percent and a mean observational error of the order of + or minus 0.000006. The maximum observed error in strain was of the order of 0.00006. The directions of principal strains for unidirectional stress were measured with the adaptor with an average error of the order of 1 degree.

  18. The 14-3-3 protein forms a molecular complex with heat shock protein Hsp60 and cellular prion protein.

    PubMed

    Satoh, Jun-ichi; Onoue, Hiroyuki; Arima, Kunimasa; Yamamura, Takashi

    2005-10-01

    The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3zeta and gamma isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.

  19. Structure and fractal dimension of protein-detergent complexes

    NASA Astrophysics Data System (ADS)

    Chen, Sow-Hsin; Teixeira, José

    1986-11-01

    Small-angle neutron-scattering experiments were made on bovine serum albumin (BSA)-lithium dodecyl sulfate (LDS) complexes in buffer solutions. As increasing amounts of LDS are added, the scattering data indicate that BSA molecules are successively transformed into random coil conformations with LDS forming globular micelles randomly decorating the polypeptide backbones. A cross-section formula is developed which successfully fits small-angle neutron-scattering spectra over the entire Q range. The fractal dimension, the micellar size, and the extent of the denatured protein are simultaneously extracted.

  20. Structure and function analysis of protein-nucleic acid complexes

    NASA Astrophysics Data System (ADS)

    Kuznetsova, S. A.; Oretskaya, T. S.

    2016-05-01

    The review summarizes published data on the results and achievements in the field of structure and function analysis of protein-nucleic acid complexes by means of main physical and biochemical methods, including X-ray diffraction, nuclear magnetic resonance spectroscopy, electron and atomic force microscopy, small-angle X-ray and neutron scattering, footprinting and cross-linking. Special attention is given to combined approaches. The advantages and limitations of each method are considered, and the prospects of their application for wide-scale structural studies in vivo are discussed. The bibliography includes 145 references.

  1. Modulation of TCR responsiveness by the Grb2-family adaptor, Gads.

    PubMed

    Lugassy, Jennie; Corso, Jasmin; Beach, Dvora; Petrik, Thomas; Oellerich, Thomas; Urlaub, Henning; Yablonski, Deborah

    2015-01-01

    T cell antigen receptor (TCR) signaling depends on three interacting adaptor proteins: SLP-76, Gads, and LAT. Their mechanisms of signaling have been extensively explored, with the aid of fortuitously isolated LAT- and SLP-76-deficient T cell lines, but no such tools were available for Gads, a Grb2-family adaptor that bridges the TCR-inducible interaction between SLP-76 and LAT. TALEN-directed genome editing was applied to disrupt the first coding exon of human Gads in the Jurkat T cell line. Gads was dispensable for TCR-induced phosphorylation of SLP-76, but was a dose-dependent amplifier of TCR-induced CD69 expression. Gads conferred responsiveness to weak TCR stimuli, leading to PLC-γ1 phosphorylation and calcium flux. TALEN-derived, Gads-deficient T cell lines provide a uniquely tractable genetic platform for exploring its regulatory features, such as Gads phosphorylation at T262, which we observed by mass spectrometry. Upon mutation of this site, TCR responsiveness and sensitivity to weak TCR stimuli were increased. This study demonstrates the feasibility of TALEN-based reverse genetics in Jurkat T cells, while enriching our understanding of Gads as a regulated modulator of TCR sensitivity.

  2. SH2B1beta adaptor is a key enhancer of RET tyrosine kinase signaling.

    PubMed

    Donatello, S; Fiorino, A; Degl'Innocenti, D; Alberti, L; Miranda, C; Gorla, L; Bongarzone, I; Rizzetti, M G; Pierotti, M A; Borrello, M G

    2007-10-04

    The RET gene encodes two main isoforms of a receptor tyrosine kinase (RTK) implicated in various human diseases. Activating germ-line point mutations are responsible for multiple endocrine neoplasia type 2-associated medullary thyroid carcinomas, inactivating germ-line mutations for Hirschsprung's disease, while somatic rearrangements (RET/PTCs) are specific to papillary thyroid carcinomas. SH2B1beta, a member of the SH2B adaptors family, and binding partner for several RTKs, has been recently described to interact with proto-RET. Here, we show that both RET isoforms and its oncogenic derivatives bind to SH2B1beta through the SRC homology 2 (SH2) domain and a kinase activity-dependent mechanism. As a result, RET phosphorylates SH2B1beta, which in turn enhances its autophosphorylation, kinase activity, and downstream signaling. RET tyrosine residues 905 and 981 are important determinants for functional binding of the adaptor, as removal of both autophosphorylation sites displaces its recruitment. Binding of SH2B1beta appears to protect RET from dephosphorylation by protein tyrosine phosphatases, and might represent a likely mechanism contributing to its upregulation. Thus, overexpression of SH2B1beta, by enhancing phosphorylation/activation of RET transducers, potentiates the cellular differentiation and the neoplastic transformation thereby induced, and counteracts the action of RET inhibitors. Overall, our results identify SH2B1beta as a key enhancer of RET physiologic and pathologic activities.

  3. Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo

    PubMed Central

    Liu, Jiangshu; Lee, Donghoon M.; Yu, Cao Guo; Angers, Stephane; Harris, Tony J. C.

    2015-01-01

    Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo. PMID:25540427

  4. Detection of Protein-Protein Interaction Within an RNA-Protein Complex Via Unnatural-Amino-Acid-Mediated Photochemical Crosslinking.

    PubMed

    Yeh, Fu-Lung; Tung, Luh; Chang, Tien-Hsien

    2016-01-01

    Although DExD/H-box proteins are known to unwind RNA duplexes and modulate RNA structures in vitro, it is highly plausible that, in vivo, some may function to remodel RNA-protein complexes. Precisely how the latter is achieved remains a mystery. We investigated this critical issue by using yeast Prp28p, an evolutionarily conserved DExD/H-box splicing factor, as a model system. To probe how Prp28p interacts with spliceosome, we strategically placed p-benzoyl-phenylalanine (BPA), a photoactivatable unnatural amino acid, along the body of Prp28p in vivo. Extracts prepared from these engineered strains were then used to assemble in vitro splicing reactions for BPA-mediated protein-protein crosslinkings. This enabled us, for the first time, to "capture" Prp28p in action. This approach may be applicable to studying the roles of other DExD/H-box proteins functioning in diverse RNA-related pathways, as well as to investigating protein-protein contacts within an RNA-protein complex.

  5. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  6. New reagents for increasing ESI multiple charging of proteins and protein complexes.

    PubMed

    Lomeli, Shirley H; Peng, Ivory X; Yin, Sheng; Loo, Rachel R Ogorzalek; Loo, Joseph A

    2010-01-01

    The addition of m-nitrobenzyl alcohol (m-NBA) was shown previously (Lomeli et al., J. Am. Soc. Mass Spectrom. 2009, 20, 593-596) to enhance multiple charging of native proteins and noncovalent protein complexes in electrospray ionization (ESI) mass spectra. Additional new reagents have been found to "supercharge" proteins from nondenaturing solutions; several of these reagents are shown to be more effective than m-NBA for increasing positive charging. Using the myoglobin protein-protoporphyrin IX (heme) complex, the following reagents were shown to increase ESI charging: benzyl alcohol, m-nitroacetophenone, m-nitrobenzonitrile, o-NBA, m-NBA, p-NBA, m-nitrophenyl ethanol, sulfolane (tetramethylene sulfone), and m-(trifluoromethyl)-benzyl alcohol. Based on average charge state, sulfolane displayed a greater charge increase (61%) than m-NBA (21%) for myoglobin in aqueous solutions. The reagents that promote higher ESI charging appear to have low solution-phase basicities and relatively low gas-phase basicities, and are less volatile than water. Another feature of mass spectra from some of the active reagents is that adducts are present on higher charge states, suggesting that a mechanism by which proteins acquire additional charge involves direct interaction with the reagent, in addition to other factors such as surface tension and protein denaturation.

  7. The hypoxic regulator of sterol synthesis Nro1 is a nuclear import adaptor

    PubMed Central

    Yeh, Tzu-Lan; Lee, Chih-Yung S.; Amzel, L. Mario; Espenshade, Peter J.; Bianchet, Mario A.

    2011-01-01

    SUMMARY Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 Å resolution shows an all-α-helical fold that can be divided into two domains: a small N-terminal domain and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response. PMID:21481773

  8. The Hypoxic Regulator of Sterol Synthesis Nro1 Is a Nuclear Import Adaptor

    SciTech Connect

    T Yeh; C Lee; L Amzel; P Espenshade; M Bianchet

    2011-12-31

    Fission yeast protein Sre1, the homolog of the mammalian sterol regulatory element-binding protein (SREBP), is a hypoxic transcription factor required for sterol homeostasis and low-oxygen growth. Nro1 regulates the stability of the N-terminal transcription factor domain of Sre1 (Sre1N) by inhibiting the action of the prolyl 4-hydroxylase-like Ofd1 in an oxygen-dependent manner. The crystal structure of Nro1 determined at 2.2 {angstrom} resolution shows an all-{alpha}-helical fold that can be divided into two domains: a small N-terminal domain, and a larger C-terminal HEAT-repeat domain. Follow-up studies showed that Nro1 defines a new class of nuclear import adaptor that functions both in Ofd1 nuclear localization and in the oxygen-dependent inhibition of Ofd1 to control the hypoxic response.

  9. 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

  10. Single-Molecule FRET Reveals Hidden Complexity in a Protein Energy Landscape

    PubMed Central

    Tsytlonok, Maksym; Ibrahim, Shehu M.; Rowling, Pamela J.E.; Xu, Wenshu; Ruedas-Rama, Maria J.; Orte, Angel; Klenerman, David; Itzhaki, Laura S.

    2015-01-01

    Summary Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured “safety pin” or “staple” from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated. PMID:25565106

  11. Single-molecule FRET reveals hidden complexity in a protein energy landscape.

    PubMed

    Tsytlonok, Maksym; Ibrahim, Shehu M; Rowling, Pamela J E; Xu, Wenshu; Ruedas-Rama, Maria J; Orte, Angel; Klenerman, David; Itzhaki, Laura S

    2015-01-06

    Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

  12. Absence of the inflammasome adaptor ASC reduces hypoxia-induced pulmonary hypertension in mice.

    PubMed

    Cero, Fadila Telarevic; Hillestad, Vigdis; Sjaastad, Ivar; Yndestad, Arne; Aukrust, Pål; Ranheim, Trine; Lunde, Ida Gjervold; Olsen, Maria Belland; Lien, Egil; Zhang, Lili; Haugstad, Solveig Bjærum; Løberg, Else Marit; Christensen, Geir; Larsen, Karl-Otto; Skjønsberg, Ole Henning

    2015-08-15

    Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1β, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC(-/-), and NLRP3(-/-) mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC(-/-) mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P < 0.001), indicating a substantially reduced pulmonary hypertension in mice lacking ASC. Magnetic resonance imaging further supported these findings by demonstrating reduced right ventricular remodeling. RVSP of NLRP3(-/-) mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1β in WT and NLRP3(-/-) mice, this response was absent in ASC(-/-) mice. Moreover, ASC(-/-) mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension.

  13. Spatio-temporal modeling of signaling protein recruitment to EGFR

    PubMed Central

    2010-01-01

    Background A stochastic simulator was implemented to study EGFR signal initiation in 3D with single molecule detail. The model considers previously unexplored contributions to receptor-adaptor coupling, such as receptor clustering and diffusive properties of both receptors and binding partners. The agent-based and rule-based approach permits consideration of combinatorial complexity, a problem associated with multiple phosphorylation sites and the potential for simultaneous binding of adaptors. Results The model was used to simulate recruitment of four different signaling molecules (Grb2, PLCγ1, Stat5, Shc) to the phosphorylated EGFR tail, with rules based on coarse-grained prediction of spatial constraints. Parameters were derived in part from quantitative immunoblotting, immunoprecipitation and electron microscopy data. Results demonstrate that receptor clustering increases the efficiency of individual adaptor retainment on activated EGFR, an effect that is overridden if crowding is imposed by receptor overexpression. Simultaneous docking of multiple proteins is highly dependent on receptor-adaptor stability and independent of clustering. Conclusions Overall, we propose that receptor density, reaction kinetics and membrane spatial organization all contribute to signaling efficiency and influence the carcinogenesis process. PMID:20459599

  14. Small Cofactors May Assist Protein Emergence from RNA World: Clues from RNA-Protein Complexes

    PubMed Central

    Shen, Liang; Ji, Hong-Fang

    2011-01-01

    It is now widely accepted that at an early stage in the evolution of life an RNA world arose, in which RNAs both served as the genetic material and catalyzed diverse biochemical reactions. Then, proteins have gradually replaced RNAs because of their superior catalytic properties in catalysis over time. Therefore, it is important to investigate how primitive functional proteins emerged from RNA world, which can shed light on the evolutionary pathway of life from RNA world to the modern world. In this work, we proposed that the emergence of most primitive functional proteins are assisted by the early primitive nucleotide cofactors, while only a minority are induced directly by RNAs based on the analysis of RNA-protein complexes. Furthermore, the present findings have significant implication for exploring the composition of primitive RNA, i.e., adenine base as principal building blocks. PMID:21789260

  15. Yeast mitochondrial protein-protein interactions reveal diverse complexes and disease-relevant functional relationships.

    PubMed

    Jin, Ke; Musso, Gabriel; Vlasblom, James; Jessulat, Matthew; Deineko, Viktor; Negroni, Jacopo; Mosca, Roberto; Malty, Ramy; Nguyen-Tran, Diem-Hang; Aoki, Hiroyuki; Minic, Zoran; Freywald, Tanya; Phanse, Sadhna; Xiang, Qian; Freywald, Andrew; Aloy, Patrick; Zhang, Zhaolei; Babu, Mohan

    2015-02-06

    Although detailed, focused, and mechanistic analyses of associations among mitochondrial proteins (MPs) have identified their importance in varied biological processes, a systematic understanding of how MPs function in concert both with one another and with extra-mitochondrial proteins remains incomplete. Consequently, many questions regarding the role of mitochondrial dysfunction in the development of human disease remain unanswered. To address this, we compiled all existing mitochondrial physical interaction data for over 1200 experimentally defined yeast MPs and, through bioinformatic analysis, identified hundreds of heteromeric MP complexes having extensive associations both within and outside the mitochondria. We provide support for these complexes through structure prediction analysis, morphological comparisons of deletion strains, and protein co-immunoprecipitation. The integration of these MP complexes with reported genetic interaction data reveals substantial crosstalk between MPs and non-MPs and identifies novel factors in endoplasmic reticulum-mitochondrial organization, membrane structure, and mitochondrial lipid homeostasis. More than one-third of these MP complexes are conserved in humans, with many containing members linked to clinical pathologies, enabling us to identify genes with putative disease function through guilt-by-association. Although still remaining incomplete, existing mitochondrial interaction data suggests that the relevant molecular machinery is modular, yet highly integrated with non-mitochondrial processes.

  16. Protein-Protein Interaction Investigated by Steered Molecular Dynamics: The TCR-pMHC Complex

    PubMed Central

    Cuendet, Michel A.; Michielin, Olivier

    2008-01-01

    We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism. PMID:18621828

  17. Evolution of protein complexity: the blue copper-containing oxidases and related proteins.

    PubMed

    Rydén, L G; Hunt, L T

    1993-01-01

    The blue copper proteins and their relatives have been compared by sequence alignments, by comparison of three-dimensional structures, and by construction of phylogenetic trees. The group contains proteins varying in size from 100 residues to over 2,300 residues in a single chain, containing from zero to nine copper atoms, and with a broad variation in function ranging from electron carrier proteins and oxidases to the blood coagulation factors V and VIII. Difference matrices show the sequence difference to be over 90% for many pairs in the group, yet alignment scores and other evidence suggest that they all evolved from a common ancestor. We have attempted to delineate how this evolution took place and in particular to define the mechanisms by which these proteins acquired an ever-increasing complexity in structure and function. We find evidence for six such mechanisms in this group of proteins: domain enlargement, in which a single domain increases in size from about 100 residues up to 210; domain duplication, which allows for a size increase from about 170 to about 1,000 residues; segment elongation, in which a small segment undergoes multiple successive duplications that can increase the chain size 50-fold; domain recruitment, in which a domain coded elsewhere in the genome is added on to the peptide chain; subunit formation, to form multisubunit proteins; and glycosylation, which in some cases doubles the size of the protein molecule. Size increase allows for the evolution of new catalytic properties, in particular the oxidase function, and for the formation of coagulation factors with multiple interaction sites and regulatory properties. The blood coagulation system is examined as an example in which a system of interacting proteins evolved by successive duplications of larger parts of the genome. The evolution of size, functionality, and diversity is compared with the general question of increase in size and complexity in biology.

  18. CCT complex restricts neuropathogenic protein aggregation via autophagy

    PubMed Central

    Pavel, Mariana; Imarisio, Sara; Menzies, Fiona M.; Jimenez-Sanchez, Maria; Siddiqi, Farah H.; Wu, Xiaoting; Renna, Maurizio; O'Kane, Cahir J.; Crowther, Damian C.; Rubinsztein, David C.

    2016-01-01

    Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity. PMID:27929117

  19. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    PubMed

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

  20. HIV-1 Nucleocapsid Mimics the Membrane Adaptor Syntenin PDZ to Gain Access to ESCRTs and Promote Virus Budding.

    PubMed

    Sette, Paola; O'Connor, Sarah K; Yerramilli, V Siddartha; Dussupt, Vincent; Nagashima, Kunio; Chutiraka, Kasana; Lingappa, Jaisri; Scarlata, Suzanne; Bouamr, Fadila

    2016-03-09

    HIV-1 recruits cellular endosomal sorting complexes required for transport (ESCRTs) to bud virions from the membrane. Disruption of the viral nucleocapsid (NC) domain integrity affects HIV-1 budding. However, the molecular mechanisms of NC's involvement in HIV budding remain unclear. We find that NC mimics the PDZ domains of syntenin, a membrane-binding adaptor involved in cell-to-cell contact/communication, to capture the Bro1 domain of ALIX, which is an ESCRTs recruiting cellular adaptor. NC binds membranes via basic residues in either the distal or proximal zinc fingers, and NC-membrane binding is essential for Bro1 capture and HIV-1 budding. Removal of RNA enhances NC membrane binding, suggesting a dynamic competition between membrane lipids and RNA for the same binding sites in NC. Remarkably, syntenin PDZ can substitute for NC function in HIV-1 budding. Thus, NC mimics syntenin PDZs to function as a membrane-binding adaptor critical for HIV-1 budding at specific microdomains of the membrane.

  1. HIV-1 Nucleocapsid mimics the membrane adaptor Syntenin to gain access to ESCRTs and promote virus budding

    PubMed Central

    Sette, Paola; O’Connor, Sarah K.; Yerramilli, V. Siddartha; Dussupt, Vincent; Nagashima, Kunio; Chutiraka, Kasana; Lingappa, Jaisri; Scarlata, Suzanne; Bouamr, Fadila

    2016-01-01

    Summary HIV-1 recruits cellular Endosomal Sorting Complexes Required for Transport (ESCRTs) to bud virions from the membrane. Disruption of the viral nucleocapsid (NC) domain integrity affects HIV-1 budding. However, the molecular mechanisms of NC’s involvement in HIV budding remain unclear. We find that NC mimics the PDZ domains of syntenin, a membrane-binding adaptor involved in cell-to-cell contact/communication, to capture the Bro1 domain of ALIX, which is an ESCRTs recruiting cellular adaptor. NC binds membranes via basic residues in either the distal or proximal zinc fingers and NC-membrane binding is essential for Bro1 capture and HIV-1 budding. Removal of RNA enhances NC membrane binding suggesting a dynamic competition between membrane lipids and RNA for same binding sites in NC. Remarkably, syntenin PDZ can substitute for NC function in HIV-1 budding. Thus, NC mimics syntenin PDZs to function as a membrane-binding adaptor critical for HIV-1 budding at microdomains of the membrane. PMID:26962944

  2. Protein-Protein Interactions of the Baculovirus Per Os Infectivity Factors in the PIF Complex.

    PubMed

    Zheng, Qin; Shen, Yunwang; Kon, Xiangshuo; Zhang, Jianjia; Feng, Min; Wu, Xiaofeng

    2017-01-28

    After ingestion of occlusion bodies, the occlusion-derived viruses (ODVs) of baculoviruses establish the first round of infection within the larval host midgut cells. Several ODV envelope proteins, called per os infectivity factors (PIFs), have been shown to be essential for oral infection. Eight PIFs have been identified to date, including P74, PIFs1-6, and Ac110. At least six PIFs: P74, PIFs1-4, PIF6, together with three other ODV-specific proteins: Ac5, P95 (Ac83), and Ac108, have been reported to form a complex on the ODV surface. In this study, in order to understand the interactions of these PIFs, the direct protein-protein interactions of the nine components of the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) PIF complex were investigated using yeast two-hybrid (Y2H) combined with bimolecular fluorescence complementation (BiFC) assay. Six direct interactions comprising PIF1-PIF2, PIF1-PIF3, PIF1-PIF4, PIF1-P95, PIF2-PIF3, and PIF3-PIF4, were identified in Y2H analysis, and these results were further verified by BiFC. For P74, PIF6, Ac5 and Ac108, no direct interaction was identified. P95 (Ac83) was identified to interact with PIF1 and further Y2H analysis of the truncations and deletion mutants showed that the predicted P95 chitin-binding domain and PIF1 100-200aa were responsible for P95 interaction with PIF1. Furthermore, a summary of the protein-protein interactions of PIFs reported so far, comprising 10 reciprocal interactions and 2 self-interactions, is presented, which will facilitate our understanding of the characteristic of PIF complex.

  3. CircRNA-protein complexes: IMP3 protein component defines subfamily of circRNPs

    PubMed Central

    Schneider, Tim; Hung, Lee-Hsueh; Schreiner, Silke; Starke, Stefan; Eckhof , Heinrich; Rossbach, Oliver; Reich, Stefan; Medenbach, Jan; Bindereif , Albrecht

    2016-01-01

    Circular RNAs (circRNAs) constitute a new class of noncoding RNAs in higher eukaryotes generated from pre-mRNAs by alternative splicing. Here we investigated in mammalian cells the association of circRNAs with proteins. Using glycerol gradient centrifugation, we characterized in cell lysates circRNA-protein complexes (circRNPs) of distinct sizes. By polysome-gradient fractionation we found no evidence for efficient translation of a set of abundant circRNAs in HeLa cells. To identify circRNPs with a specific protein component, we focused on IMP3 (IGF2BP3, insulin-like growth factor 2 binding protein 3), a known tumor marker and RNA-binding protein. Combining RNA-seq analysis of IMP3-co-immunoprecipitated RNA and filtering for circular-junction reads identified a set of IMP3-associated circRNAs, which were validated and characterized. In sum, our data suggest that specific circRNP families exist defined by a common protein component. In addition, this provides a general approach to identify circRNPs with a given protein component. PMID:27510448

  4. Hierarchical structures made of proteins. The complex architecture of spider webs and their constituent silk proteins.

    PubMed

    Heim, Markus; Römer, Lin; Scheibel, Thomas

    2010-01-01

    Biopolymers fulfil a variety of different functions in nature. They conduct various processes inside and outside cells and organisms, with a functionality ranging from storage of information to stabilization, protection, shaping, transport, cellular division, or movement of whole organisms. Within the plethora of biopolymers, the most sophisticated group is of proteinaceous origin: the cytoskeleton of a cell is made of protein filaments that aid in pivotal processes like intracellular transport, movement, and cell division; geckos use a distinct arrangement of keratin-like filaments on their toes which enable them to walk up smooth surfaces, such as walls, and even upside down across ceilings; and spiders spin silks that are extra-corporally used for protection of offspring and construction of complex prey traps. The following tutorial review describes the hierarchical organization of protein fibers, using spider dragline silk as an example. The properties of a dragline silk thread originate from the strictly controlled assembly of the underlying protein chains. The assembly procedure leads to protein fibers showing a complex hierarchical organization comprising three different structural phases. This structural organization is responsible for the outstanding mechanical properties of individual fibers, which out-compete even those of high-performance artificial fibers like Kevlar. Web-weaving spiders produce, in addition to dragline silk, other silks with distinct properties, based on slightly variant constituent proteins--a feature that allows construction of highly sophisticated spider webs with well designed architect