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Sample records for adhesion adaptor protein

  1. 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.

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

    PubMed

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

    2014-04-01

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

  3. Anti-adaptors provide multiple modes for regulation of the RssB adaptor protein

    PubMed Central

    Battesti, Aurelia; Hoskins, Joel R.; Tong, Song; Milanesio, Paola; Mann, Jessica M.; Kravats, Andrea; Tsegaye, Yodit M.; Bougdour, Alexandre; Wickner, Sue; Gottesman, Susan

    2013-01-01

    RpoS, an RNA polymerase σ factor, controls the response of Escherichia coli and related bacteria to multiple stress responses. During nonstress conditions, RpoS is rapidly degraded by ClpXP, mediated by the adaptor protein RssB, a member of the response regulator family. In response to stress, RpoS degradation ceases. Small anti-adaptor proteins—IraP, IraM, and IraD, each made under a different stress condition—block RpoS degradation. RssB mutants resistant to either IraP or IraM were isolated and analyzed in vivo and in vitro. Each of the anti-adaptors is unique in its interaction with RssB and sensitivity to RssB mutants. One class of mutants defined an RssB N-terminal region close to the phosphorylation site and critical for interaction with IraP but unnecessary for IraM and IraD function. A second class, in the RssB C-terminal PP2C-like domain, led to activation of RssB function. These mutants allowed the response regulator to act in the absence of phosphorylation but did not abolish interaction with anti-adaptors. This class of mutants is broadly resistant to the anti-adaptors and bears similarity to constitutively activated mutants found in a very different PP2C protein. The mutants provide insight into how the anti-adaptors perturb RssB response regulator function and activation. PMID:24352426

  4. Small-molecule control of protein degradation using split adaptors.

    PubMed

    Davis, Joseph H; Baker, Tania A; Sauer, Robert T

    2011-11-18

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  5. Small-molecule control of protein degradation using split adaptors

    PubMed Central

    Davis, Joseph H.; Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    Targeted intracellular degradation provides a method to study the biological function of proteins and has numerous applications in biotechnology. One promising approach uses adaptor proteins to target substrates with genetically encoded degradation tags for proteolysis. Here, we describe an engineered split-adaptor system, in which adaptor assembly and delivery of substrates to the ClpXP protease depends on a small molecule (rapamycin). This degradation system does not require modification of endogenous proteases, functions robustly over a wide range of adaptor concentrations, and does not require new synthesis of adaptors or proteases to initiate degradation. We demonstrate the efficacy of this system in E. coli by degrading tagged variants of LacI repressor and FtsA, an essential cell-division protein. In the latter case, addition of rapamycin causes pronounced filamentation because daughter cells cannot divide. Strikingly, washing rapamycin away reverses this phenotype. Our system is highly modular, with clearly-defined interfaces for substrate binding, protease binding, and adaptor assembly, providing a clear path to extend this system to other degradation tags, proteases, or induction systems. Together, these new reagents should be useful in controlling protein degradation in bacteria. PMID:21866931

  6. Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity.

    PubMed

    Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M; Shen, Kang

    2016-05-01

    The cardinal feature of neuronal polarization is the establishment and maintenance of axons and dendrites. How axonal and dendritic proteins are sorted and targeted to different compartments is poorly understood. Here, we identified distinct dileucine motifs that are necessary and sufficient to target transmembrane proteins to either the axon or the dendrite through direct interactions with the clathrin-associated adaptor protein complexes (APs) in C. elegans. Axonal targeting requires AP-3, while dendritic targeting is mediated by AP-1. The axonal dileucine motif binds to AP-3 with higher efficiency than to AP-1. Both AP-3 and AP-1 are localized to the Golgi but occupy adjacent domains. We propose that AP-3 and AP-1 directly select transmembrane proteins and target them to axon and dendrite, respectively, by sorting them into distinct vesicle pools. PMID:27151641

  7. Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase*S⃞

    PubMed Central

    Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.; Chen, Kan; Vaynberg, Julia; Mao, Xian; Tu, Yizeng; Zuo, Xiaobing; Wang, Jinbu; Wang, Yun-xing; Wu, Chuanyue; Tjandra, Nico; Qin, Jun

    2009-01-01

    The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK·PINCH complex (28 kDa, KD ∼ 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication. PMID:19117955

  8. Structural Basis of Focal Adhesion Localization of LIM-only Adaptor PINCH by Integrin-linked Kinase

    SciTech Connect

    Yang, Yanwu; Wang, Xiaoxia; Hawkins, Cheryl A.; Chen, Kan; Vaynberg, Julia; Mao, Xian; Tu, Yizeng; Zuo, Xiaobing; Wang, Jinbu; Wang, Yun-xing; Wu, Chuanyue; Tjandra, Nico; Qin, Jun

    2010-11-22

    The LIM-only adaptor PINCH (the particularly interesting cysteine- and histidine-rich protein) plays a pivotal role in the assembly of focal adhesions (FAs), supramolecular complexes that transmit mechanical and biochemical information between extracellular matrix and actin cytoskeleton, regulating diverse cell adhesive processes such as cell migration, cell spreading, and survival. A key step for the PINCH function is its localization to FAs, which depends critically on the tight binding of PINCH to integrin-linked kinase (ILK). Here we report the solution NMR structure of the core ILK {center_dot} PINCH complex (28 kDa, K{sub D} {approx} 68 nm) involving the N-terminal ankyrin repeat domain (ARD) of ILK and the first LIM domain (LIM1) of PINCH. We show that the ILK ARD exhibits five sequentially stacked ankyrin repeat units, which provide a large concave surface to grip the two contiguous zinc fingers of the PINCH LIM1. The highly electrostatic interface is evolutionally conserved but differs drastically from those of known ARD and LIM bound to other types of protein domains. Consistently mutation of a hot spot in LIM1, which is not conserved in other LIM domains, disrupted the PINCH binding to ILK and abolished the PINCH targeting to FAs. These data provide atomic insight into a novel modular recognition and demonstrate how PINCH is specifically recruited by ILK to mediate the FA assembly and cell-extracellular matrix communication.

  9. Protein mediated membrane adhesion

    NASA Astrophysics Data System (ADS)

    Carlson, Andreas; Mahadevan, L.

    2015-05-01

    Adhesion in the context of mechanical attachment, signaling, and movement in cellular dynamics is mediated by the kinetic interactions between membrane-embedded proteins in an aqueous environment. Here, we present a minimal theoretical framework for the dynamics of membrane adhesion that accounts for the kinetics of protein binding, the elastic deformation of the membrane, and the hydrodynamics of squeeze flow in the membrane gap. We analyze the resulting equations using scaling estimates to characterize the spatiotemporal features of the adhesive patterning and corroborate them using numerical simulations. In addition to characterizing aspects of cellular dynamics, our results might also be applicable to a range of phenomena in physical chemistry and materials science where flow, deformation, and kinetics are coupled to each other in slender geometries.

  10. Molecular physiology of the tensin brotherhood of integrin adaptor proteins.

    PubMed

    Haynie, Donald T

    2014-07-01

    Numerous proteins have been identified as constituents of the adhesome, the totality of molecular components in the supramolecular assemblies known as focal adhesions, fibrillar adhesions and other kinds of adhesive contact. The transmembrane receptor proteins called integrins are pivotal adhesome members, providing a physical link between the extracellular matrix (ECM) and the actin cytoskeleton. Tensins are ever more widely investigated intracellular adhesome constituents. Involved in cell attachment and migration, cytoskeleton reorganization, signal transduction and other processes relevant to cancer research, tensins have recently been linked to functional properties of deleted in liver cancer 1 (DLC1) and a mitogen-activated protein kinases (MAPK), to cell migration in breast cancer, and to metastasis suppression in the kidney. Tensins are close relatives of phosphatase homolog/tensin homolog (PTEN), an extensively studied tumor suppressor. Such findings are recasting the earlier vision of tensin (TNS) as an actin-filament (F-actin) capping protein in a different light. This critical review aims to summarize current knowledge on tensins and thus to highlight key points concerning the expression, structure, function, and evolution of the various members of the TNS brotherhood. Insight is sought by comparisons with homologous proteins. Some historical points are added for perspective. PMID:24634006

  11. 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

  12. 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

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

    PubMed

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

    2014-08-25

    Periplasmic adaptor proteins are key components of bacterial tripartite efflux pumps. The 2.85 Å resolution structure of an MFS (major facilitator superfamily) pump adaptor, Aquifex aeolicus EmrA, shows linearly arranged α-helical coiled-coil, lipoyl, and β-barrel domains, but lacks the fourth membrane-proximal domain shown in other pumps to interact with the inner membrane transporter. The adaptor α-hairpin, which binds outer membrane TolC, is exceptionally long at 127 Å, and the β-barrel contains a conserved disordered loop. The structure extends the view of adaptors as flexible, modular components that mediate diverse pump assembly, and suggests that in MFS tripartite pumps a hexamer of adaptors could provide a periplasmic seal. PMID:24996185

  14. Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin receptor substrates (IRSs) are adaptor proteins that link signaling from upstream activators to multiple downstream effectors to modulate normal growth, metabolism, survival, and differentiation. Recent cell culture studies have shown that IRSs can interact with, and are functionally require...

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. Recruitment of the Adaptor Protein Grb2 to EGFR Tetramers

    PubMed Central

    2015-01-01

    Adaptor protein Grb2 binds phosphotyrosines in the epidermal growth factor (EGF) receptor (EGFR) and thereby links receptor activation to intracellular signaling cascades. Here, we investigated how recruitment of Grb2 to EGFR is affected by the spatial organization and quaternary state of activated EGFR. We used the techniques of image correlation spectroscopy (ICS) and lifetime-detected Förster resonance energy transfer (also known as FLIM-based FRET or FLIM–FRET) to measure ligand-induced receptor clustering and Grb2 binding to activated EGFR in BaF/3 cells. BaF/3 cells were stably transfected with fluorescently labeled forms of Grb2 (Grb2–mRFP) and EGFR (EGFR–eGFP). Following stimulation of the cells with EGF, we detected nanometer-scale association of Grb2–mRFP with EGFR–eGFP clusters, which contained, on average, 4 ± 1 copies of EGFR–eGFP per cluster. In contrast, the pool of EGFR–eGFP without Grb2–mRFP had an average cluster size of 1 ± 0.3 EGFR molecules per punctum. In the absence of EGF, there was no association between EGFR–eGFP and Grb2–mRFP. To interpret these data, we extended our recently developed model for EGFR activation, which considers EGFR oligomerization up to tetramers, to include recruitment of Grb2 to phosphorylated EGFR. The extended model, with adjustment of one new parameter (the ratio of the Grb2 and EGFR copy numbers), is consistent with a cluster size distribution where 2% of EGFR monomers, 5% of EGFR dimers, <1% of EGFR trimers, and 94% of EGFR tetramers are associated with Grb2. Together, our experimental and modeling results further implicate tetrameric EGFR as the key signaling unit and call into question the widely held view that dimeric EGFR is the predominant signaling unit. PMID:24697349

  18. The Emerging and Diverse Roles of Src-Like Adaptor Proteins in Health and Disease

    PubMed Central

    Marton, Nikolett; Baricza, Eszter; Érsek, Barbara; Buzás, Edit I.; Nagy, György

    2015-01-01

    Although Src-like adaptor proteins (SLAP-1 and SLAP-2) were mainly studied in lymphocytes, where they act as negative regulators and provide fine control of receptor signaling, recently, several other functions of these proteins were discovered. In addition to the well-characterized immunoregulatory functions, SLAP proteins appear to have an essential role in the pathogenesis of type I hypersensitivity, osteoporosis, and numerous malignant diseases. Both adaptor proteins are expressed in a wide variety of tissues, where they have mostly inhibitory effects on multiple intracellular signaling pathways. In this review, we summarize the diverse effects of SLAP proteins. PMID:26339145

  19. Adaptor Protein-3-Mediated Trafficking of TLR2 Ligands Controls Specificity of Inflammatory Responses but Not Adaptor Complex Assembly.

    PubMed

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

    2015-11-01

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

  20. Distinct adaptor proteins assist exit of Kre2-family proteins from the yeast ER

    PubMed Central

    Noda, Yoichi; Hara, Takehiro; Ishii, Minako; Yoda, Koji

    2014-01-01

    ABSTRACT The Svp26 protein of S. cerevisiae is an ER- and Golgi-localized integral membrane protein with 4 potential membrane-spanning domains. It functions as an adaptor protein that facilitates the ER exit of Ktr3, a mannosyltransferase required for biosynthesis of O-linked oligosaccharides, and the ER exit of Mnn2 and Mnn5, mannosyltransferases, which participate in the biosynthesis of N-linked oligosaccharides. Ktr3 belongs to the Kre2 family, which consists of 9 members of type-II membrane proteins sharing sequence similarities. In this report, we examined all Kre2 family members and found that the Golgi localizations of two others, Kre2 and Ktr1, were dependent on Svp26 by immunofluorescence microscopy and cell fractionations in sucrose density gradients. We show that Svp26 functions in facilitating the ER exit of Kre2 and Ktr1 by an in vitro COPII budding assay. Golgi localization of Ktr4 was not dependent on Svp26. Screening null mutants of the genes encoding abundant COPII membrane proteins for those showing mislocalization of Ktr4 in the ER revealed that Erv41 and Erv46 are required for the correct Golgi localization of Ktr4. We provide biochemical evidence that the Erv41-Erv46 complex functions as an adaptor protein for ER exit of Ktr4. This is the first demonstration of the molecular function of this evolutionally conserved protein complex. The domain switching experiments show that the lumenal domain of Ktr4 is responsible for recognition by the Erv41-Erv46 complex. Thus, ER exit of Kre2-family proteins is dependent on distinct adaptor proteins and our results provide new insights into the traffic of Kre2-family mannosyltransferases. PMID:24585773

  1. Identification of a new transmembrane adaptor protein that constitutively binds Grb2 in B cells

    PubMed Central

    Liu, Yan; Zhang, Weiguo

    2008-01-01

    Transmembrane adaptor proteins couple antigen receptor engagement to downstream signaling cascades in lymphocytes. One example of these proteins is the linker for activation of T cells (LAT), which plays an indispensable role in T cell activation and development. Here, we report identification of a new transmembrane adaptor molecule, namely growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein, transmembrane (GAPT), which is expressed in B cells and myeloid cells. Similar to LAT, GAPT has an extracellular domain, a transmembrane domain, and a cytoplasmic tail with multiple Grb2-binding motifs. In contrast to other transmembrane adaptor proteins, GAPT is not phosphorylated upon BCR ligation but associates with Grb2 constitutively through its proline-rich region. Targeted disruption of the gapt gene in mice affects neither B cell development nor a nitrophenylacetyl-specific antibody response. However, in the absence of GAPT, B cell proliferation after BCR cross-linking is enhanced. In aged GAPT−/− mice, the number of marginal zone (MZ) B cells is increased, and other B cell subsets are normal. The serum concentrations of IgM, IgG2b, and IgG3 are also elevated in these mice. These data indicate that GAPT might play an important role in control of B cell activation and proper maintenance of MZ B cells. PMID:18559951

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-03-01

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

  4. Adhesives from modified soy protein

    DOEpatents

    Sun, Susan; Wang, Donghai; Zhong, Zhikai; Yang, Guang

    2008-08-26

    The, present invention provides useful adhesive compositions having similar adhesive properties to conventional UF and PPF resins. The compositions generally include a protein portion and modifying ingredient portion selected from the group consisting of carboxyl-containing compounds, aldehyde-containing compounds, epoxy group-containing compounds, and mixtures thereof. The composition is preferably prepared at a pH level at or near the isoelectric point of the protein. In other preferred forms, the adhesive composition includes a protein portion and a carboxyl-containing group portion.

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-09-01

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

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

    PubMed

    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

  9. 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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Wiley, H Steven; VanHook, Annalisa M

    2016-01-01

    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. PMID:27405978

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

    PubMed

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

    2016-03-25

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

  14. Large hepatitis delta antigen is a novel clathrin adaptor-like protein.

    PubMed

    Huang, Cheng; Chang, Shin C; Yu, I-Chen; Tsay, Yeou-Guang; Chang, Ming-Fu

    2007-06-01

    Clathrin-mediated endocytosis is a common pathway for viral entry, but little is known about the direct association of viral protein with clathrin in the cytoplasm. In this study, a putative clathrin box known to be conserved in clathrin adaptors was identified at the C terminus of the large hepatitis delta antigen (HDAg-L). Similar to clathrin adaptors, HDAg-L directly interacted with the N terminus of the clathrin heavy chain through the clathrin box. HDAg-L is a nucleocytoplasmic shuttle protein important for the assembly of hepatitis delta virus (HDV). Here, we demonstrated that brefeldin A and wortmannin, inhibitors of clathrin-mediated exocytosis and endosomal trafficking, respectively, specifically blocked HDV assembly but had no effect on the assembly of the small surface antigen of hepatitis B virus. In addition, cytoplasm-localized HDAg-L inhibited the clathrin-mediated endocytosis of transferrin and the degradation of epidermal growth factor receptor. These results indicate that HDAg-L is a new clathrin adaptor-like protein, and it may be involved in the maturation and pathogenesis of HDV coinfection or superinfection with hepatitis B virus through interaction with clathrin. PMID:17376909

  15. Differential Regulation of Clathrin and Its Adaptor Proteins during Membrane Recruitment for Endocytosis.

    PubMed

    Wang, Chao; Hu, Tianwei; Yan, Xu; Meng, Tingting; Wang, Yutong; Wang, Qingmei; Zhang, Xiaoyue; Gu, Ying; Sánchez-Rodríguez, Clara; Gadeyne, Astrid; Lin, Jinxing; Persson, Staffan; Van Damme, Daniël; Li, Chuanyou; Bednarek, Sebastian Y; Pan, Jianwei

    2016-05-01

    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

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

    PubMed

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

    2014-06-01

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

  18. 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. PMID:27072891

  19. Surfactant Protein A Enhances Constitutive Immune Functions of Clathrin Heavy Chain and Clathrin Adaptor Protein 2.

    PubMed

    Moulakakis, Christina; Steinhäuser, Christine; Biedziak, Dominika; Freundt, Katja; Reiling, Norbert; Stamme, Cordula

    2016-07-01

    NF-κB transcription factors are key regulators of pulmonary inflammatory disorders and repair. Constitutive lung cell type- and microenvironment-specific NF-κB/inhibitor κBα (IκB-α) regulation, however, is poorly understood. Surfactant protein (SP)-A provides both a critical homeostatic and lung defense control, in part by immune instruction of alveolar macrophages (AMs) via clathrin-mediated endocytosis. The central endocytic proteins, clathrin heavy chain (CHC) and the clathrin adaptor protein (AP) complex AP2, have pivotal alternative roles in cellular homeostasis that are endocytosis independent. Here, we dissect endocytic from alternative functions of CHC, the α-subunit of AP2, and dynamin in basal and SP-A-modified LPS signaling of macrophages. As revealed by pharmacological inhibition and RNA interference in primary AMs and RAW264.7 macrophages, respectively, CHC and α-adaptin, but not dynamin, prevent IκB-α degradation and TNF-α release, independent of their canonical role in membrane trafficking. Kinetics studies employing confocal microscopy, Western analysis, and immunomagnetic sorting revealed that SP-A transiently enhances the basal protein expression of CHC and α-adaptin, depending on early activation of protein kinase CK2 (former casein kinase II) and Akt1 in primary AMs from rats, SP-A(+/+), and SP-A(-/-) mice, as well as in vivo when intratracheally administered to SP-A(+/+) mice. Constitutive immunomodulation by SP-A, but not SP-A-mediated inhibition of LPS-induced NF-κB activity and TNF-α release, requires CHC, α-adaptin, and dynamin. Our data demonstrate that endocytic proteins constitutively restrict NF-κB activity in macrophages and provide evidence that SP-A enhances the immune regulatory capacity of these proteins, revealing a previously unknown pathway of microenvironment-specific NF-κB regulation in the lung. PMID:26771574

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-06-14

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

  2. 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. PMID:16984970

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

    Naudin, Cécile; Chevalier, Clément; Roche, Serge

    2016-01-01

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

  5. The adaptor protein CIN85 assembles intracellular signaling clusters for B cell activation.

    PubMed

    Kühn, Julius; Wong, Leo E; Pirkuliyeva, Sona; Schulz, Kathrin; Schwiegk, Claudia; Fünfgeld, Kevser Gencalp; Keppler, Selina; Batista, Facundo D; Urlaub, Henning; Habeck, Michael; Becker, Stefan; Griesinger, Christian; Wienands, Jürgen

    2016-01-01

    The adaptor molecule Cbl-interacting protein of 85 kD (CIN85) regulates signaling from a number of cell surface receptors, such as growth factor receptors and antigen receptors on lymphocytes. Because of its multidomain structure, CIN85 is thought to act as a classical adaptor protein that connects functionally distinct components of a given signaling pathway through diverse protein domains. However, we found that in B lymphocytes, CIN85 functions to oligomerize SLP-65, which is the central effector protein of the B cell receptor (BCR). Therefore, CIN85 trimerizes through a carboxyl-terminal, coiled-coil domain. The multiple Src homology 3 (SH3) domains of trimeric CIN85 molecules associated with multiple SLP-65 molecules, which recruited further CIN85 trimers, thereby perpetuating the oligomerization process. Formation of this oligomeric signaling complex in resting B cells rendered the cells poised for the efficient initiation of intracellular signaling upon BCR stimulation. Our data suggest that the functionality of signaling cascades does not rely solely on the qualitative linkage of their various components but requires a critical number of effectors to become concentrated in signaling complexes. PMID:27353366

  6. Nephrin Suppresses Hippo Signaling through the Adaptor Proteins Nck and WTIP.

    PubMed

    Keyvani Chahi, Ava; Martin, Claire E; Jones, Nina

    2016-06-10

    Podocytes are key components of the kidney blood filtration barrier, and their ability to withstand hemodynamic strain is proposed to be closely tied to their unique and flexible cytoarchitecture. However, the mechanisms that control such mechanotransduction are poorly understood. We have previously established that tyrosine phosphorylation of the transmembrane protein nephrin promotes recruitment of the Nck1/2 cytoskeletal adaptor proteins and downstream actin remodeling. We now reveal that Nck integrates nephrin with the Hippo kinase cascade through association with the adaptor protein WTIP. Using mutational analysis, we show that Nck sequesters WTIP and its binding partner Lats1 to phosphorylated nephrin, resulting in decreased phospho-activation of Lats1. We further demonstrate that, coincident with nephrin dephosphorylation in a transient model of podocyte injury in mice, Lats1 is rapidly activated, and this precedes significant down-regulation of the transcription regulator Yap. Moreover, we show reduced levels of Yap protein in mice with chronic disruption of nephrin phospho-signaling. Together, these findings support the existence of a dynamic molecular link between nephrin signaling and the canonical Hippo pathway in podocytes, which may facilitate the conversion of mechanical cues to biochemical signals promoting podocyte viability. PMID:27033705

  7. 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.

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

    PubMed

    Horn, Anselm H C; Sticht, Heinrich

    2015-01-01

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

  9. 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

  10. Alterations in cell adhesion proteins and cardiomyopathy

    PubMed Central

    Li, Jifen

    2014-01-01

    Cell adhesive junction is specialized intercellular structure composed of cell adhesion proteins. They are essential to connect adjacent heart muscle cell and make heart contraction effectively and properly. Clinical and genetic studies have revealed close relationship between cell adhesive proteins and the occurrence of various cardiomyopathies. Here we will review recent development on the disease phenotype, potential cellular and molecular mechanism related to cell adhesion molecules, with particular disease pathogenesis learned from genetic manipulated murine models. PMID:24944760

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

    PubMed

    Mavuluri, Jayadev; Beesetti, Swarnalatha; Surabhi, Rohan; Kremerskothen, Joachim; Venkatraman, Ganesh; Rayala, Suresh K

    2016-05-01

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

  12. Valosin-containing protein (VCP)-Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor.

    PubMed

    Xue, Liang; Blythe, Emily E; Freiberger, Elyse C; Mamrosh, Jennifer L; Hebert, Alexander S; Reitsma, Justin M; Hess, Sonja; Coon, Joshua J; Deshaies, Raymond J

    2016-09-01

    Protein quality control (PQC) plays an important role in stemming neurodegenerative diseases and is essential for the growth of some cancers. Valosin-containing protein (VCP)/p97 plays a pivotal role in multiple PQC pathways by interacting with numerous adaptors that link VCP to specific PQC pathways and substrates and influence the post-translational modification state of substrates. However, our poor understanding of the specificity and architecture of the adaptors, and the dynamic properties of their interactions with VCP hinders our understanding of fundamental features of PQC and how modulation of VCP activity can best be exploited therapeutically. In this study we use multiple mass spectrometry-based proteomic approaches combined with biophysical studies to characterize the interaction of adaptors with VCP. Our results reveal that most VCP-adaptor interactions are characterized by rapid dynamics that in some cases are modulated by the VCP inhibitor NMS873. These findings have significant implications for both the regulation of VCP function and the impact of VCP inhibition on different VCP-adaptor complexes. PMID:27406709

  13. The Adaptor Protein Rai/ShcC Promotes Astrocyte-Dependent Inflammation during Experimental Autoimmune Encephalomyelitis.

    PubMed

    Ulivieri, Cristina; Savino, Maria Teresa; Luccarini, Ilaria; Fanigliulo, Emanuela; Aldinucci, Alessandra; Bonechi, Elena; Benagiano, Marisa; Ortensi, Barbara; Pelicci, Giuliana; D'Elios, Mario Milco; Ballerini, Clara; Baldari, Cosima Tatiana

    2016-07-15

    Th17 cells have been casually associated to the pathogenesis of autoimmune disease. We have previously demonstrated that Rai/ShcC, a member of the Shc family of adaptor proteins, negatively regulates Th17 cell differentiation and lupus autoimmunity. In this study, we have investigated the pathogenic outcome of the Th17 bias associated with Rai deficiency on multiple sclerosis development, using the experimental autoimmune encephalomyelitis (EAE) mouse model. We found that, unexpectedly, EAE was less severe in Rai(-/-) mice compared with their wild-type counterparts despite an enhanced generation of myelin-specific Th17 cells that infiltrated into the CNS. Nevertheless, when adoptively transferred into immunodeficient Rai(+/+) mice, these cells promoted a more severe disease compared with wild-type encephalitogenic Th17 cells. This paradoxical phenotype was caused by a dampened inflammatory response of astrocytes, which were found to express Rai, to IL-17. The results provide evidence that Rai plays opposite roles in Th17 cell differentiation and astrocyte activation, with the latter dominant over the former in EAE, highlighting this adaptor as a potential novel target for the therapy of multiple sclerosis. PMID:27288534

  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. PMID:26585312

  15. 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.

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

    PubMed

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

    2015-02-24

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

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

    PubMed Central

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

    2015-01-01

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

  18. 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

  19. Vaccinia virus protein A46R targets multiple Toll-like-interleukin-1 receptor adaptors and contributes to virulence.

    PubMed

    Stack, Julianne; Haga, Ismar R; Schröder, Martina; Bartlett, Nathan W; Maloney, Geraldine; Reading, Patrick C; Fitzgerald, Katherine A; Smith, Geoffrey L; Bowie, Andrew G

    2005-03-21

    Viral immune evasion strategies target key aspects of the host antiviral response. Recently, it has been recognized that Toll-like receptors (TLRs) have a role in innate defense against viruses. Here, we define the function of the vaccinia virus (VV) protein A46R and show it inhibits intracellular signalling by a range of TLRs. TLR signalling is triggered by homotypic interactions between the Toll-like-interleukin-1 resistance (TIR) domains of the receptors and adaptor molecules. A46R contains a TIR domain and is the only viral TIR domain-containing protein identified to date. We demonstrate that A46R targets the host TIR adaptors myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like, TIR domain-containing adaptor inducing IFN-beta (TRIF), and the TRIF-related adaptor molecule and thereby interferes with downstream activation of mitogen-activated protein kinases and nuclear factor kappaB. TRIF mediates activation of interferon (IFN) regulatory factor 3 (IRF3) and induction of IFN-beta by TLR3 and TLR4 and suppresses VV replication in macrophages. Here, A46R disrupted TRIF-induced IRF3 activation and induction of the TRIF-dependent gene regulated on activation, normal T cell expressed and secreted. Furthermore, we show that A46R is functionally distinct from another described VV TLR inhibitor, A52R. Importantly, VV lacking the A46R gene was attenuated in a murine intranasal model, demonstrating the importance of A46R for VV virulence. PMID:15767367

  20. Merkel cell polyomavirus small T antigen targets the NEMO adaptor protein to disrupt inflammatory signaling.

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2015-11-01

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

  2. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis.

    PubMed

    Dixelius, J; Larsson, H; Sasaki, T; Holmqvist, K; Lu, L; Engström, A; Timpl, R; Welsh, M; Claesson-Welsh, L

    2000-06-01

    Endostatin, which corresponds to the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis. Fibroblast growth factor-2 (FGF-2)-induced angiogenesis in the chicken chorioallantoic membrane was inhibited by endostatin, but not by an endostatin mutant R158/270A, lacking heparin-binding ability. Endostatin was internalized by endothelial cells, but not by mouse fibroblasts. Treatment of murine brain endothelial (IBE) cells with endostatin reduced the proportion of cells in S phase, whereas growth-arrested IBE cells in collagen gels treated with endostatin displayed enhanced tubular morphogenesis. IBE cells overexpressing Shb, an adaptor protein implicated in angiostatin-induced apoptosis, displayed elevated apoptosis and decreased tubular morphogenesis in collagen gels in response to endostatin when added together with FGF-2. Induction of apoptosis was dependent on the heparin-binding ability of endostatin and the expression of Shb with a functional Src homology 2 (SH2)-domain. Endostatin treatment for 10 minutes or 24 hours induced tyrosine phosphorylation of Shb and formation of multiprotein complexes. An Shb SH2 domain fusion protein precipitated a 125-kd phosphotyrosyl protein in endostatin-treated cells. The 125-kd component either contained intrinsic tyrosine kinase activity or occurred in complex with a tyrosine kinase. In conclusion, our data show that endostatin induces tyrosine kinase activity and enhanced apoptosis in FGF-treated endothelial cells. PMID:10828022

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  5. 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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

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

    PubMed

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

    2016-06-30

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

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

    PubMed Central

    Symmons, Martyn F.; Marshall, Robert L.

    2015-01-01

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

  10. The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.

    PubMed

    Dasgupta, Preeta; Dorsey, Nicolas J; Li, Jiaqi; Qi, Xiulan; Smith, Elizabeth P; Yamaji-Kegan, Kazuyo; Keegan, Achsah D

    2016-01-01

    Insulin receptor substrate 2 (IRS2) is an adaptor protein that becomes tyrosine-phosphorylated in response to the cytokines interleukin-4 (IL-4) and IL-13, which results in activation of the phosphoinositide 3-kinase (PI3K)-Akt pathway. IL-4 and IL-13 contribute to allergic lung inflammation. To examine the role of IRS2 in allergic disease, we evaluated the responses of IRS2-deficient (IRS2(-/-)) mice. Unexpectedly, loss of IRS2 resulted in a substantial increase in the expression of a subset of genes associated with the generation of alternatively activated macrophages (AAMs) in response to IL-4 or IL-13 in vitro. AAMs secrete factors that enhance allergic responses and promote airway remodeling. Moreover, compared to IRS2(+/+) mice, IRS2(+/-) and IRS2(-/-) mice developed enhanced pulmonary inflammation, accumulated eosinophils and AAMs, and exhibited airway and vascular remodeling upon allergen stimulation, responses that partially depended on macrophage-intrinsic IRS2 signaling. Both in unstimulated and IL-4-stimulated macrophages, lack of IRS2 enhanced phosphorylation of Akt and ribosomal S6 protein. Thus, we identified a critical inhibitory loop downstream of IRS2, demonstrating an unanticipated and previously unrecognized role for IRS2 in suppressing allergic lung inflammation and remodeling. PMID:27330190

  11. 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.

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

    PubMed

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

    2015-01-01

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

  13. Cysteine-based regulation of the CUL3 adaptor protein Keap1

    SciTech Connect

    Sekhar, Konjeti R.; Rachakonda, Girish; Freeman, Michael L.

    2010-04-01

    Nrf2 (NF-E2-related factor 2) is a master transcription factor containing a powerful acidic transcriptional activation domain. Nrf2-dependent gene expression impacts cancer chemoprevention strategies, inflammatory responses, and progression of neurodegenerative diseases. Under basal conditions, association of Nrf2 with the CUL3 adaptor protein Keap1 results in the rapid Nrf2 ubiquitylation and proteasome-dependent degradation. Inhibition of Keap1 function blocks ubiquitylation of Nrf2, allowing newly synthesized Nrf2 to translocate into the nucleus, bind to ARE sites and direct target gene expression. Site-directed mutagenesis experiments coupled with proteomic analysis support a model in which Keap1 contains at least 2 distinct cysteine motifs. The first is located at Cys 151 in the BTB domain. The second is located in the intervening domain and centers around Cys 273 and 288. Adduction or oxidation at Cys151 has been shown to produce a conformational change in Keap1 that results in dissociation of Keap1 from CUL3, thereby inhibiting Nrf2 ubiquitylation. Thus, adduction captures specific chemical information and translates it into biochemical information via changes in structural conformation.

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

    PubMed Central

    Halova, Ivana; Draber, Petr

    2016-01-01

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

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

    PubMed

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

    2015-03-01

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

  16. PLEKHA7: Cytoskeletal adaptor protein at center stage in junctional organization and signaling.

    PubMed

    Shah, Jimit; Guerrera, Diego; Vasileva, Ekaterina; Sluysmans, Sophie; Bertels, Eva; Citi, Sandra

    2016-06-01

    PLEKHA7 is a recently characterized component of the cytoplasmic region of epithelial adherens junctions (AJ). It comprises two WW domains, a pleckstrin-homology domain, and proline-rich and coiled-coil domains. PLEKHA7 interacts with cytoplasmic components of the AJ (p120-catenin, paracingulin, afadin), stabilizes the E-cadherin complex by linking it to the minus ends of noncentrosomal microtubules, and stabilizes junctional nectins through the newly identified interactor PDZD11. Similarly to afadin, and unlike E-cadherin and p120-catenin, the localization of PLEKHA7 at AJ is strictly zonular (in the zonula adhaerens subdomain of AJ), and does not extend along the basolateral contacts. Genome-wide association studies and experiments on animal and cellular models show that although PLEKHA7 is not required for organism viability, it is implicated in cardiovascular physiology, hypertension, primary angle closure glaucoma, susceptibility to staphylococcal α-toxin, and epithelial morphogenesis and growth. Thus, PLEKHA7 is a cytoskeletal adaptor protein important for AJ organization, and at the center of junction-associated signaling pathways which fine-tune important pathophysiological processes. PMID:27072621

  17. Adaptor protein LNK is a negative regulator of brain neural stem cell proliferation after stroke.

    PubMed

    Ahlenius, Henrik; Devaraju, Karthikeyan; Monni, Emanuela; Oki, Koichi; Wattananit, Somsak; Darsalia, Vladimer; Iosif, Robert E; Torper, Olof; Wood, James C; Braun, Sebastian; Jagemann, Lucas; Nuber, Ulrike A; Englund, Elisabet; Jacobsen, Sten-Eirik W; Lindvall, Olle; Kokaia, Zaal

    2012-04-11

    Ischemic stroke causes transient increase of neural stem and progenitor cell (NSPC) proliferation in the subventricular zone (SVZ), and migration of newly formed neuroblasts toward the damaged area where they mature to striatal neurons. The molecular mechanisms regulating this plastic response, probably involved in structural reorganization and functional recovery, are poorly understood. The adaptor protein LNK suppresses hematopoietic stem cell self-renewal, but its presence and role in the brain are poorly understood. Here we demonstrate that LNK is expressed in NSPCs in the adult mouse and human SVZ. Lnk(-/-) mice exhibited increased NSPC proliferation after stroke, but not in intact brain or following status epilepticus. Deletion of Lnk caused increased NSPC proliferation while overexpression decreased mitotic activity of these cells in vitro. We found that Lnk expression after stroke increased in SVZ through the transcription factors STAT1/3. LNK attenuated insulin-like growth factor 1 signaling by inhibition of AKT phosphorylation, resulting in reduced NSPC proliferation. Our findings identify LNK as a stroke-specific, endogenous negative regulator of NSPC proliferation, and suggest that LNK signaling is a novel mechanism influencing plastic responses in postischemic brain. PMID:22496561

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

    PubMed Central

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

    2015-01-01

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

  19. The methyltransferase adaptor protein Trm112 is involved in biogenesis of both ribosomal subunits

    PubMed Central

    Sardana, Richa; Johnson, Arlen W.

    2012-01-01

    We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates. PMID:22956767

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

    PubMed Central

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

    2014-01-01

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

  1. The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis

    PubMed Central

    Li, Rui-Fang; Chen, Gang; Ren, Jian-Gang; Zhang, Wei; Wu, Zhong-Xing; Liu, Bing; Zhao, Yi-Fang

    2014-01-01

    Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases. PMID:25163928

  2. The SH2B1 Adaptor Protein Associates with a Proximal Region of the Erythropoietin Receptor*

    PubMed Central

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

    2012-01-01

    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. PMID:22669948

  3. 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. PMID:22669948

  4. Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins.

    PubMed

    Morvan, Joëlle; de Craene, Johan-Owen; Rinaldi, Bruno; Addis, Vanessa; Misslin, Cédric; Friant, Sylvie

    2015-02-15

    Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3Δ mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2. PMID:25512335

  5. Cell biological characterization of a multidomain adaptor protein, ArgBP2, in epithelial NMuMG cells, and identification of a novel short isoform.

    PubMed

    Murase, Kana; Ito, Hidenori; Kanoh, Hiroyuki; Sudo, Kaori; Iwamoto, Ikuko; Morishita, Rika; Soubeyran, Philippe; Seishima, Mariko; Nagata, Koh-Ichi

    2012-12-01

    ArgBP2 is a member of the SoHo (sorbin-homology) family of adaptor proteins believed to play roles in cell adhesion, cytoskeletal organization, and signaling. We show here a novel splicing isoform of ArgBP2, i.e., ArgBP2™, composed of only three SH3 (src-homology 3) domains and structurally similar to vinexinß. We then characterized the biochemical and cell biological properties of ArgBP2 to compare these with vinexin. Similar to vinexin, ArgBP2 was enriched at focal adhesions in REF52 fibroblast cells and induced anchorage-dependent extracellular signal-regulated kinase activation in NIH3T3 fibroblast cells. In epithelial NMuMG cells, immunofluorescence analyses revealed localization of ArgBP2 at tight junctions (TJs), whereas vinexin was distributed in cytoplasm as well as cell-cell boundaries. During TJ formation, recruitment of ZO-1 to TJs was followed by ArgBP2. Based on mutation analyses, a second SH3 domain was found to be important for ArgBP2 localization to the cell-cell contact sites. These data suggest some role of ArgBP2 in NMuMG cells at TJs that may be distinct from the function of vinexin. PMID:22431180

  6. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.

    PubMed Central

    Chen, Y; Grall, D; Salcini, A E; Pelicci, P G; Pouysségur, J; Van Obberghen-Schilling, E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors. Images PMID:8605873

  7. Molecular mechanics of mussel adhesion proteins

    NASA Astrophysics Data System (ADS)

    Qin, Zhao; Buehler, Markus J.

    2014-01-01

    Mussel foot protein (mfp), a natural glue produced by marine mussel, is an intriguing material because of its superior ability for adhesion in various environments. For example, a very small amount of this material is sufficient to affix a mussel to a substrate in water, providing structural support under extreme forces caused by the dynamic effects of waves. Towards a more complete understanding of its strength and underwater workability, it is necessary to understand the microscropic mechanisms by which the protein structure interacts with various substrates. However, none of the mussel proteins' structure is known, preventing us from directly using atomistic modeling to probe their structural and mechanical properties. Here we use an advanced molecular sampling technique to identify the molecular structures of two mussel foot proteins (mfp-3 and mfp-5) and use those structures to study their mechanics of adhesion, which is then incorporated into a continuum model. We calculate the adhesion energy of the mussel foot protein on a silica substrate, compute the adhesion strength based on results obtained from molecular modeling, and compare with experimental data. Our results show good agreement with experimental measurements, which validates the multiscale model. We find that the molecular structure of the folded mussel foot protein (ultimately defined by its genetic sequence) favors strong adhesion to substrates, where L-3,4-dihydroxyphenylalanine (or DOPA) protein subunits work in a cooperative manner to enhance adhesion. Our experimental data suggests a peak attachment force of 0.4±0.1 N, which compares favorably with the prediction from the multiscale model of Fc=0.21-0.33 N. The principles learnt from those results could guide the fabrication of new interfacial materials (e.g. composites) to integrate organic with inorganic surfaces in an effective manner.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-02-15

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

  10. The 3A Protein from Multiple Picornaviruses Utilizes the Golgi Adaptor Protein ACBD3 To Recruit PI4KIIIβ

    PubMed Central

    Greninger, Alexander L.; Knudsen, Giselle M.; Betegon, Miguel; Burlingame, Alma L.

    2012-01-01

    The activity of phosphatidylinositol 4-kinase class III beta (PI4KIIIβ) has been shown to be required for the replication of multiple picornaviruses; however, it is unclear whether a physical association between PI4KIIIβ and the viral replication machinery exists and, if it does, whether association is necessary. We examined the ability of the 3A protein from 18 different picornaviruses to form a complex with PI4KIIIβ by affinity purification of Strep-Tagged transiently transfected constructs followed by mass spectrometry and Western blotting for putative interacting targets. We found that the 3A proteins of Aichi virus, bovine kobuvirus, poliovirus, coxsackievirus B3, and human rhinovirus 14 all copurify with PI4KIIIβ. Furthermore, we found that multiple picornavirus 3A proteins copurify with the Golgi adaptor protein acyl coenzyme A (acyl-CoA) binding domain protein 3 (ACBD3/GPC60), including those from Aichi virus, bovine kobuvirus, human rhinovirus 14, poliovirus, and coxsackievirus B2, B3, and B5. Affinity purification of ACBD3 confirmed interaction with multiple picornaviral 3A proteins and revealed the ability to bind PI4KIIIβ in the absence of 3A. Mass-spectrometric analysis of transiently expressed Aichi virus, bovine kobuvirus, and human klassevirus 3A proteins demonstrated that the N-terminal glycines of these 3A proteins are myristoylated. Alanine-scanning mutagenesis along the entire length of Aichi virus 3A followed by transient expression and affinity purification revealed that copurification of PI4KIIIβ could be eliminated by mutation of specific residues, with little or no effect on recruitment of ACBD3. One mutation at the N terminus, I5A, significantly reduced copurification of both ACBD3 and PI4KIIIβ. The dependence of Aichi virus replication on the activity of PI4KIIIβ was confirmed by both chemical and genetic inhibition. Knockdown of ACBD3 by small interfering RNA (siRNA) also prevented replication of both Aichi virus and poliovirus

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

    PubMed

    Städeli, Reto; Basler, Konrad

    2005-11-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-05-01

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

  14. Soy and cottonseed protein blends as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an environmentally friendlier alternative to adhesives from petroleum feedstock, soy proteins are currently being formulated as wood adhesives. Cottonseed proteins have also been found to provide good adhesive properties. In at least some cases, cottonseed proteins appear to form greater shear ...

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2016-08-12

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

  17. 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. PMID:17429078

  18. Nck adaptor proteins control the organization of neuronal circuits important for walking.

    PubMed

    Fawcett, James P; Georgiou, John; Ruston, Julie; Bladt, Friedhelm; Sherman, Andrew; Warner, Neil; Saab, Bechara J; Scott, Rizaldy; Roder, John C; Pawson, Tony

    2007-12-26

    The intracellular signaling targets used by mammalian axon guidance receptors to organize the nervous system in vivo are unclear. The Nck1 and Nck2 SH2/SH3 adaptors (collectively Nck) can couple phosphotyrosine (pTyr) signals to reorganization of the actin cytoskeleton and are therefore candidates for linking guidance cues to the regulatory machinery of the cytoskeleton. We find that selective inactivation of Nck in the murine nervous system causes a hopping gait and a defect in the spinal central pattern generator, which is characterized by synchronous firing of bilateral ventral motor neurons. Nck-deficient mice also show abnormal projections of corticospinal tract axons and defective development of the posterior tract of the anterior commissure. These phenotypes are consistent with a role for Nck in signaling initiated by different classes of guidance receptors, including the EphA4 receptor tyrosine kinase. Our data indicate that Nck adaptors couple pTyr guidance signals to cytoskeletal events required for the ipsilateral projections of spinal cord neurons and thus for normal limb movement. PMID:18093944

  19. 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

  20. RNF11 is a GGA protein cargo and acts as a molecular adaptor for GGA3 ubiquitination mediated by Itch.

    PubMed

    Santonico, E; Mattioni, A; Panni, S; Belleudi, F; Mattei, M; Torrisi, M R; Cesareni, G; Castagnoli, L

    2015-06-01

    Ring finger protein 11 (RNF11) is a RING (really interesting new gene)-H2 E3 ligase that is overexpressed in several human tumor tissues. The mature protein, which is anchored to membranes via a double acylation, localizes to early endosome and recycling compartments. Apart from its subcellular localization, additional lines of evidence implicate RNF11 in the mechanisms underlying vesicle traffic. Here we identify two acidic-cluster dileucine (Ac-LL) motifs, which are recognized by the VHS domains of Golgi-localized, gamma adaptin era-containing, ADP-ribosylation factor-binding protein (GGA) adaptors, as the molecular determinants governing RNF11 sorting at the trans-Golgi network and its internalization from the plasma membrane. We also show that RNF11 recruits itch to drive the ubiquitination of GGA3. This function is experimentally detectable only in cells overexpressing an RNF11 variant that is inactivated in the RING domain, indicating that RNF11 recruits GGA3 and controls its ubiquitination by regulating itch activity. Accordingly, our data demonstrate the involvement of itch in regulating GGA3 stability. Indeed, we observe that the endogenous levels of GGA3 are increased in cells knocked down for itch and endogenous GGA3 is hyperubiquitinated in an itch-dependent manner in a cell line expressing catalytically inactive RNF11. Our data are consistent with a model whereby the RING E3 ligase RNF11 is a novel GGA cargo actively participating in regulating the ubiquitination of the GGA protein family. The results that we are presenting put RNF11 at the center of a finally regulated system where it acts both as an adaptor and a modulator of itch-mediated control of ubiquitination events underlying membrane traffic. PMID:25195858

  1. Toll-Interleukin 1 Receptor domain-containing adaptor protein positively regulates BV2 cell M1 polarization.

    PubMed

    Gong, Leilei; Wang, Hanxiang; Sun, Xiaolei; Liu, Chun; Duan, Chengwei; Cai, Rixin; Gu, Xingxing; Zhu, Shunxing

    2016-06-01

    Microglial activation, including classical (M1) and alternative (M2) activation, plays important roles in the development of several central nervous system disorders and promotes tissue reconstruction. Toll-like receptor (TLR)4 is important for microglial polarization. TIR domain-containing adaptor protein (TIRAP) is an intracellular adaptor protein, which is responsible for the early phase of TLR4 activation. The role of TIRAP in BV2 cell M1 polarization is still unknown. In this study, we showed that TIRAP expression is greatly elevated in lipopolysaccharide (LPS)/interferon (IFN)-γ-treated microglia. TIRAP overexpression promoted BV2 microglial M1 polarization by increasing M1-related marker production (inducible nitric oxide synthase, CD86, interleukin-6, interleukin-1β and tumour necrosis factor-α). In contrast, TIRAP knockdown prevented M1-related marker production. Mechanistically, TIRAP could interact with TNF Receptor-Associated Factor 6 (TRAF6) to increase M1-related marker production in TIRAP overexpressed and LPS/IFN-γ-treated BV2 cells. In addition, silencing of TIRAP effectively inhibited the activation of the Transforming Growth Factor-Beta-Activated Kinase 1/I-Kappa-B Kinase /Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-Cells (TAK1/IKK/NF-κB) signalling pathway and the phosphorylation of Akt and mitogen-activated protein kinases, which were activated by LPS/IFN-γ stimulation. Thus, our results suggest that TIRAP positively regulated BV2 microglial M1 polarization through TLR4-mediated TAK1/IKK/NF-κB, mitogen-activated protein kinases and Akt signalling pathways. PMID:27061018

  2. Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

    PubMed

    Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

    2016-08-26

    Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. PMID:27311859

  3. How to develop globular proteins into adhesives.

    PubMed

    van der Leeden, M C; Rutten, A A; Frens, G

    2000-05-26

    To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2

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

    PubMed

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

    2011-06-10

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

  5. Proteins recruited by SH3 domains of Ruk/CIN85 adaptor identified by LC-MS/MS

    PubMed Central

    Havrylov, Serhiy; Rzhepetskyy, Yuriy; Malinowska, Agata; Drobot, Lyudmyla; Redowicz, Maria Jolanta

    2009-01-01

    Background Ruk/CIN85 is a mammalian adaptor molecule with three SH3 domains. Using its SH3 domains Ruk/CIN85 can cluster multiple proteins and protein complexes, and, consequently, facilitates organisation of elaborate protein interaction networks with diverse regulatory roles. Previous research linked Ruk/CIN85 with the regulation of vesicle-mediated transport and cancer cell invasiveness. Despite the recent findings, precise molecular functions of Ruk/CIN85 in these processes remain largely elusive and further research is hampered by a lack of complete lists of its partner proteins. Results In the present study we employed a LC-MS/MS-based experimental pipeline to identify a considerable number (over 100) of proteins recruited by the SH3 domains of Ruk/CIN85 in vitro. Most of these identifications are novel Ruk/CIN85 interaction candidates. The identified proteins have diverse molecular architectures and can interact with other proteins, as well as with lipids and nucleic acids. Some of the identified proteins possess enzymatic activities. Functional profiling analyses and literature mining demonstrate that many of the proteins recruited by the SH3 domains of Ruk/CIN85 identified in this work were involved in the regulation of membranes and cytoskeletal structures necessary for vesicle-mediated transport and cancer cell invasiveness. Several groups of the proteins were also associated with few other cellular processes not previously related to Ruk/CIN85, most prominently with cell division. Conclusion Obtained data support the notion that Ruk/CIN85 regulates vesicle-mediated transport and cancer cell invasiveness through the assembly of multimeric protein complexes governing coordinated remodelling of membranes and underlying cytoskeletal structures, and imply its important roles in formation of coated vesicles and biogenesis of invadopodia. In addition, this study points to potential involvement of Ruk/CIN85 in other cellular processes, chiefly in cell division

  6. Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

    PubMed

    Kimpler, Lisa A; Glosson, Nicole L; Downs, Deanna; Gonyo, Patrick; May, Nathan A; Hudson, Amy W

    2014-01-01

    The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes. PMID:24901711

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

    PubMed

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

    2016-07-29

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

  8. The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

    PubMed Central

    Hollopeter, Gunther; Lange, Jeffrey J; Zhang, Ying; Vu, Thien N; Gu, Mingyu; Ailion, Michael; Lambie, Eric J; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Jorgensen, Erik M

    2014-01-01

    The AP2 clathrin adaptor complex links protein cargo to the endocytic machinery but it is unclear how AP2 is activated on the plasma membrane. Here we demonstrate that the membrane-associated proteins FCHo and SGIP1 convert AP2 into an open, active conformation. We screened for Caenorhabditis elegans mutants that phenocopy the loss of AP2 subunits and found that AP2 remains inactive in fcho-1 mutants. A subsequent screen for bypass suppressors of fcho-1 nulls identified 71 compensatory mutations in all four AP2 subunits. Using a protease-sensitivity assay we show that these mutations restore the open conformation in vivo. The domain of FCHo that induces this rearrangement is not the F-BAR domain or the µ-homology domain, but rather is an uncharacterized 90 amino acid motif, found in both FCHo and SGIP proteins, that directly binds AP2. Thus, these proteins stabilize nascent endocytic pits by exposing membrane and cargo binding sites on AP2. DOI: http://dx.doi.org/10.7554/eLife.03648.001 PMID:25303366

  9. Structural basis of HIV-1 Vpu-mediated BST2 antagonism via hijacking of the clathrin adaptor protein complex 1

    PubMed Central

    Jia, Xiaofei; Weber, Erin; Tokarev, Andrey; Lewinski, Mary; Rizk, Maryan; Suarez, Marissa; Guatelli, John; Xiong, Yong

    2014-01-01

    BST2/tetherin, an antiviral restriction factor, inhibits the release of enveloped viruses from the cell surface. Human immunodeficiency virus-1 (HIV-1) antagonizes BST2 through viral protein u (Vpu), which downregulates BST2 from the cell surface. We report the crystal structure of a protein complex containing Vpu and BST2 cytoplasmic domains and the core of the clathrin adaptor protein complex 1 (AP1). This, together with our biochemical and functional validations, reveals how Vpu hijacks the AP1-dependent membrane trafficking pathways to mistraffick BST2. Vpu mimics a canonical acidic dileucine-sorting motif to bind AP1 in the cytosol, while simultaneously interacting with BST2 in the membrane. These interactions enable Vpu to build on an intrinsic interaction between BST2 and AP1, presumably causing the observed retention of BST2 in juxtanuclear endosomes and stimulating its degradation in lysosomes. The ability of Vpu to hijack AP-dependent trafficking pathways suggests a potential common theme for Vpu-mediated downregulation of host proteins. DOI: http://dx.doi.org/10.7554/eLife.02362.001 PMID:24843023

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

    PubMed Central

    Korpos, Éva; Deák, Ferenc; Kiss, Ibolya

    2015-01-01

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

  11. Asc1p, a WD40-domain containing adaptor protein, is required for the interaction of the RNA-binding protein Scp160p with polysomes.

    PubMed Central

    Baum, Sonja; Bittins, Margarethe; Frey, Steffen; Seedorf, Matthias

    2004-01-01

    Scp160p interacts in an mRNA-dependent manner with translating ribosomes via multiple RNA-binding heterogeneous nuclear ribonucleoprotein K-homology (KH) domains. In the present study, we show by protein-protein cross-linking that Scp160p is in close proximity to translation elongation factor 1A and the WD40 (Trp-Asp 40)-repeat containing protein Asc1p at ribosomes. Analysis of a truncation mutant revealed that the C-terminus of Scp160p is essential for ribosome binding and that Cys(1067) at the C-terminus of Scp160p is required to obtain these cross-links. The interaction of Scp160p with ribosomes depends on Asc1p. In fast-growing yeast cells, nearly all Asc1p is tightly bound to ribosomes, but it can also be present in a ribosome-free form depending on growth conditions. The functional homologue of Asc1p, mammalian RACK1 (receptor of activated C kinase), was previously characterized as an adaptor protein bridging activated signalling molecules with their substrates. Our results suggest that Scp160p connects specific mRNAs, ribosomes and a translation factor with an adaptor for signalling molecules. These interactions might regulate the translation activity of ribosomes programmed with specific mRNAs. PMID:15012629

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

    PubMed

    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

  13. 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

  14. SorLA/LR11 regulates processing of amyloid precursor protein via interaction with adaptors GGA and PACS-1.

    PubMed

    Schmidt, Vanessa; Sporbert, Anje; Rohe, Michael; Reimer, Tatjana; Rehm, Armin; Andersen, Olav M; Willnow, Thomas E

    2007-11-01

    SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation. PMID:17855360

  15. An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

    PubMed Central

    Wilmington, Shameika R.; Matouschek, Andreas

    2016-01-01

    A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels. PMID:27043013

  16. Distinct Involvement of the Gab1 and Grb2 Adaptor Proteins in Signal Transduction by the Related Receptor Tyrosine Kinases RON and MET

    PubMed Central

    Chaudhuri, Amitabha; Xie, Ming-Hong; Yang, Becky; Mahapatra, Kaushiki; Liu, Jinfeng; Marsters, Scot; Bodepudi, Sweta; Ashkenazi, Avi

    2011-01-01

    Although the signal transduction mechanisms of the receptor tyrosine kinase MET are well defined, less is known about its close relative RON. MET initiates intracellular signaling by autophosphorylation on specific cytoplasmic tyrosines that form docking sites for the adaptor proteins Grb2 and Gab1. Grb2 binds directly and is essential for all of the biological activities of MET. Gab1 docks either directly or indirectly via Grb2 and controls only a subset of MET functions. Because MET and RON possess similar adaptor binding sites, it was anticipated that their adaptor interactions would be conserved. Here we show that in contrast to MET, RON relies primarily on Gab1 for signal transmission. Surprisingly, disruption of the Grb2 docking site of RON or Grb2 depletion augments activity, whereas enhancement of Grb2 binding attenuates Gab1 recruitment and signaling. Hence, RON and MET differ in their adaptor interactions; furthermore, Grb2 performs a novel antagonistic role in the context of RON signaling. PMID:21784853

  17. The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development

    PubMed Central

    Lin, Wai W.; Yi, Zuoan; Stunz, Laura L.; Maine, Christian J.; Sherman, Linda A.; Bishop, Gail A.

    2016-01-01

    Tumor necrosis factor receptor–associated factor 3 (TRAF3) is an adaptor protein that inhibits signaling by CD40 and by the receptor for B cell–activating factor (BAFF) and negatively regulates homeostatic B cell survival. Loss-of-function mutations in TRAF3 are associated with human B cell malignancies, in particular multiple myeloma. The cytokine interleukin-6 (IL-6) supports the differentiation and survival of normal and neoplastic plasma cells. We found that mice with a deficiency in TRAF3 specifically in B cells (B-Traf3−/− mice) had about twice as many plasma cells as did their littermate controls. TRAF3-deficient B cells had enhanced responsiveness to IL-6, and genetic loss of IL-6 in B-Traf3−/− mice restored their plasma cell numbers to normal. TRAF3 inhibited IL-6 receptor (IL-6R)–mediated signaling by facilitating the association of PTPN22 (a nonreceptor protein tyrosine phosphatase) with the kinase Janus-activated kinase 1 (Jak1), which in turn blocked phosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3). Consistent with these results, the number of plasma cells in the PTPN22-deficient mice was increased compared to that in the wild-type mice. Our findings identify TRAF3 and PTPN22 as inhibitors of IL-6R signaling in B cells and reveal a previously uncharacterized role for TRAF3 in the regulation of plasma cell differentiation. PMID:26329582

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2015-12-25

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

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

    PubMed

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

    2016-01-01

    The expressions and activities of hepatic drug-metabolizing enzymes and transporters (DMETs) are altered during infection and inflammation. Inflammatory responses in the liver are mediated primarily by Toll-like receptor (TLR)-signaling, which involves recruitment of Toll/interleukin (IL)-1 receptor (TIR) domain containing adaptor protein (TIRAP) and TIR domain containing adaptor inducing interferon (IFN)-β (TRIF) that eventually leads to induction of proinflammatory cytokines and mitogen-activated protein kinases (MAPKs). Lipopolysaccharide (LPS) activates the Gram-negative bacterial receptor TLR4 and polyinosinic:polycytidylic acid (polyI:C) activates the viral receptor TLR3. TLR4 signaling involves TIRAP and TRIF, whereas TRIF is the only adaptor protein involved in the TLR3 pathway. We have shown previously that LPS-mediated downregulation of DMETs is independent of TIRAP. To determine the role of TRIF, we treated TRIF(+/+) and TRIF(-/-) mice with LPS or polyI:C. LPS downregulated (∼40%-60%) Cyp3a11, Cyp2a4, Ugt1a1, Mrp2 mRNA levels, whereas polyI:C downregulated (∼30%-60%) Cyp3a11, Cyp2a4, Cyp1a2, Cyp2b10, Ugt1a1, Mrp2, and Mrp3 mRNA levels in TRIF(+/+) mice. This downregulation was not attenuated in TRIF(-/-) mice. Induction of cytokines by LPS was observed in both TRIF(+/+) and TRIF(-/-) mice. Cytokine induction was delayed in polyI:C-treated TRIF(-/-) mice, indicating that multiple mechanisms mediating polyI:C signaling exist. To assess the role of MAPKs, primary hepatocytes were pretreated with specific inhibitors before treatment with LPS/polyI:C. We found that only the c-jun-N-terminal kinase (JNK) inhibitor attenuated the down-regulation of DMETs. These results show that TRIF-independent pathways can be involved in the downregulation of DMETs through TLR4 and 3. JNK-dependent mechanisms likely mediate this downregulation. PMID:26470915

  2. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    PubMed Central

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  3. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins.

    PubMed

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N; Patil, Navinkumar J; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-01-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives. PMID:26508080

  4. Mussel adhesion is dictated by time-regulated secretion and molecular conformation of mussel adhesive proteins

    NASA Astrophysics Data System (ADS)

    Petrone, Luigi; Kumar, Akshita; Sutanto, Clarinda N.; Patil, Navinkumar J.; Kannan, Srinivasaraghavan; Palaniappan, Alagappan; Amini, Shahrouz; Zappone, Bruno; Verma, Chandra; Miserez, Ali

    2015-10-01

    Interfacial water constitutes a formidable barrier to strong surface bonding, hampering the development of water-resistant synthetic adhesives. Notwithstanding this obstacle, the Asian green mussel Perna viridis attaches firmly to underwater surfaces via a proteinaceous secretion (byssus). Extending beyond the currently known design principles of mussel adhesion, here we elucidate the precise time-regulated secretion of P. viridis mussel adhesive proteins. The vanguard 3,4-dihydroxy-L-phenylalanine (Dopa)-rich protein Pvfp-5 acts as an adhesive primer, overcoming repulsive hydration forces by displacing surface-bound water and generating strong surface adhesion. Using homology modelling and molecular dynamics simulations, we find that all mussel adhesive proteins are largely unordered, with Pvfp-5 adopting a disordered structure and elongated conformation whereby all Dopa residues reside on the protein surface. Time-regulated secretion and structural disorder of mussel adhesive proteins appear essential for optimizing extended nonspecific surface interactions and byssus' assembly. Our findings reveal molecular-scale principles to help the development of wet-resistant adhesives.

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

    PubMed

    Jay, Kyle A; Smith, Dana L; Blackburn, Elizabeth H

    2016-07-15

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

  6. 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

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

    PubMed Central

    Jay, Kyle A.; Smith, Dana L.

    2016-01-01

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

  8. Investigation of modified cottonseed protein adhesives for wood composites

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several modified cottonseed protein isolates were studied and compared to corresponding soy protein isolates for their adhesive properties when bonded to wood composites. Modifications included treatments with alkali, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and urea. Wood composites...

  9. The interaction of Kinesin-1 with its adaptor protein JIP1 can be regulated via proteins binding to the JIP1-PTB domain

    PubMed Central

    2013-01-01

    Background The regulatory mechanisms of motor protein-dependent intracellular transport are still not fully understood. The kinesin-1-binding protein, JIP1, can function as an adaptor protein that links kinesin-1 and other JIP1-binding “cargo” proteins. However, it is unknown whether these “cargo” proteins influence the JIP1–kinesin-1 binding. Results We show here that JIP1–kinesin-1 binding in Neuro2a cells was dependent on conserved amino acid residues in the JIP1-phosphotyrosine binding (PTB) domain, including F687. In addition, mutation of F687 severely affected the neurite tip localization of JIP1. Proteomic analysis revealed another kinesin-1 binding protein, JIP3, as a major JIP1 binding protein. The association between JIP1 and JIP3 was dependent on the F687 residue in JIP1, and this association induced the formation of a stable ternary complex with kinesin-1. On the other hand, the binding of JIP1 and JIP3 was independent of kinesin-1 binding. We also show that other PTB binding proteins can interrupt the formation of the ternary complex. Conclusions The formation of the JIP1–kinesin-1 complex depends on the protein binding-status of the JIP1 PTB domain. This may imply a regulatory mechanism of kinesin-1-dependent intracellular transport. PMID:23496950

  10. Structure of a putative ClpS N-end rule adaptor protein from the malaria pathogen Plasmodium falciparum.

    PubMed

    AhYoung, Andrew P; Koehl, Antoine; Vizcarra, Christina L; Cascio, Duilio; Egea, Pascal F

    2016-03-01

    The N-end rule pathway uses an evolutionarily conserved mechanism in bacteria and eukaryotes that marks proteins for degradation by ATP-dependent chaperones and proteases such as the Clp chaperones and proteases. Specific N-terminal amino acids (N-degrons) are sufficient to target substrates for degradation. In bacteria, the ClpS adaptor binds and delivers N-end rule substrates for their degradation upon association with the ClpA/P chaperone/protease. Here, we report the first crystal structure, solved at 2.7 Å resolution, of a eukaryotic homolog of bacterial ClpS from the malaria apicomplexan parasite Plasmodium falciparum (Pfal). Despite limited sequence identity, Plasmodium ClpS is very similar to bacterial ClpS. Akin to its bacterial orthologs, plasmodial ClpS harbors a preformed hydrophobic pocket whose geometry and chemical properties are compatible with the binding of N-degrons. However, while the N-degron binding pocket in bacterial ClpS structures is open and accessible, the corresponding pocket in Plasmodium ClpS is occluded by a conserved surface loop that acts as a latch. Despite the closed conformation observed in the crystal, we show that, in solution, Pfal-ClpS binds and discriminates peptides mimicking bona fide N-end rule substrates. The presence of an apicoplast targeting peptide suggests that Pfal-ClpS localizes to this plastid-like organelle characteristic of all Apicomplexa and hosting most of its Clp machinery. By analogy with the related ClpS1 from plant chloroplasts and cyanobacteria, Plasmodium ClpS likely functions in association with ClpC in the apicoplast. Our findings open new venues for the design of novel anti-malarial drugs aimed at disrupting parasite-specific protein quality control pathways. PMID:26701219

  11. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation.

    PubMed

    Chen, Bill B; Coon, Tiffany A; Glasser, Jennifer R; McVerry, Bryan J; Zhao, Jing; Zhao, Yutong; Zou, Chunbin; Ellis, Bryon; Sciurba, Frank C; Zhang, Yingze; Mallampalli, Rama K

    2013-05-01

    Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance. PMID:23542741

  12. Adhesion of mussel foot proteins to different substrate surfaces

    PubMed Central

    Lu, Qingye; Danner, Eric; Waite, J. Herbert; Israelachvili, Jacob N.; Zeng, Hongbo; Hwang, Dong Soo

    2013-01-01

    Mussel foot proteins (mfps) have been investigated as a source of inspiration for the design of underwater coatings and adhesives. Recent analysis of various mfps by a surface forces apparatus (SFA) revealed that mfp-1 functions as a coating, whereas mfp-3 and mfp-5 resemble adhesive primers on mica surfaces. To further refine and elaborate the surface properties of mfps, the force–distance profiles of the interactions between thin mfp (i.e. mfp-1, mfp-3 or mfp-5) films and four different surface chemistries, namely mica, silicon dioxide, polymethylmethacrylate and polystyrene, were measured by an SFA. The results indicate that the adhesion was exquisitely dependent on the mfp tested, the substrate surface chemistry and the contact time. Such studies are essential for understanding the adhesive versatility of mfps and related/similar adhesion proteins, and for translating this versatility into a new generation of coatings and (including in vivo) adhesive materials. PMID:23173195

  13. 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

  14. The adaptor protein alpha-syntrophin regulates adipocyte lipid droplet growth.

    PubMed

    Eisinger, Kristina; Rein-Fischboeck, Lisa; Pohl, Rebekka; Meier, Elisabeth M; Krautbauer, Sabrina; Buechler, Christa

    2016-07-01

    The scaffold protein alpha-syntrophin (SNTA) regulates lipolysis indicating a role in lipid homeostasis. Adipocytes are the main lipid storage cells in the body, and here, the function of SNTA has been analyzed in 3T3-L1 cells. SNTA is expressed in preadipocytes and is induced early during adipogenesis. Knock-down of SNTA in preadipocytes increases their proliferation. Proteins which are induced during adipogenesis like adiponectin and caveolin-1, and the inflammatory cytokine IL-6 are at normal levels in the mature cells differentiated from preadipocytes with low SNTA. This suggests that SNTA does neither affect differentiation nor inflammation. Expression of proteins with a role in cholesterol and triglyceride homeostasis is unchanged. Consequently, basal and epinephrine induced lipolysis as well as insulin stimulated phosphorylation of Akt and ERK1/2 are normal. Importantly, adipocytes with low SNTA form smaller lipid droplets and store less triglycerides. Stearoyl-CoA reductase and MnSOD are reduced upon SNTA knock-down but do not contribute to lower lipid levels. Oleate uptake is even increased in cells with SNTA knock-down. In summary, current data show that SNTA is involved in the expansion of lipid droplets independent of adipogenesis. Enhanced preadipocyte proliferation and capacity to store surplus fatty acids may protect adipocytes with low SNTA from lipotoxicity in obesity. PMID:27242274

  15. A dual role for the adaptor protein DRK in Drosophila olfactory learning and memory

    PubMed Central

    Moressis, Anastasios; Friedrich, Anke R.; Pavlopoulos, Elias; Davis, Ronald L.; Skoulakis, Efthimios M. C.

    2009-01-01

    Participation of RAS, RAF and MAPK in learning and memory has been demonstrated in a number of studies, but the molecular events requisite for cascade activation and regulation have not been explored. We demonstrate that the adapter protein DRK which is essential for signaling to RAS in developmental contexts, is preferentially distributed in the adult mushroom bodies, centers for olfactory learning and memory. We demonstrate that drk mutant heterozygotes exhibit deficits in olfactory learning and memory, apparent under limited training conditions, but are not impaired in sensory responses requisite for the association of the stimuli, or brain neuroanatomy. Furthermore we demonstrate that the protein is required acutely within mushroom body neurons to mediate efficient learning, a process that requires RAF activation. Importantly, 90-minute memory remained impaired, even after differential training yielding equivalent learning in animals with compromised DRK levels and controls, and did not require RAF. Sustained MAPK activation is compromised in drk mutants and surprisingly is negatively regulated by constitutive RAF activity. The data establish a role for DRK in Drosophila behavioral neuroplasticity and suggest a dual role for the protein, first in RAF activation-dependent learning and additionally in RAF-inhibition dependent sustained MAPK activation essential for memory formation or stability. PMID:19244537

  16. Soy protein isolate molecular level contributions to bulk adhesive properties

    NASA Astrophysics Data System (ADS)

    Shera, Jeanne Norton

    Increasing environmental awareness and the recognized health hazards of formaldehyde-based resins has prompted a strong demand for environmentally-responsible adhesives for wood composites. Soy protein-based adhesives have been shown to be commercially viable with 90-day shelf stability and composite physical properties comparable to those of commercial formaldehyde-based particleboards. The main research focus is to isolate and characterize the molecular level features in soy protein isolate responsible for providing mechanical properties, storage stability, and water resistance during adhesive formulation, processing, and wood composite fabrication. Commercial composite board will be reviewed to enhance our understanding of the individual components and processes required for particleboard production. The levels of protein structure will be defined and an overview of current bio-based technology will be presented. In the process, the logic for utilizing soy protein as a sole binder in the adhesive will be reinforced. Variables such as adhesive components, pH, divalent ions, blend aging, protein molecular weight, formulation solids content, and soy protein functionalization will relate the bulk properties of soy protein adhesives to the molecular configuration of the soybean protein. This work has demonstrated that when intermolecular beta-sheet interactions and protein long-range order is disrupted, viscosity and mechanical properties decrease. Storage stability can be maintained through the stabilization of intermolecular beta-sheet interactions. When molecular weight is reduced through enzymatic digestion, long-range order is disrupted and viscosity and mechanical properties decrease accordingly. Processibility and physical properties must be balanced to increase solids while maintaining low viscosity, desirable mechanical properties, and adequate storage stability. The structure of the soybean protein must be related to the particleboard bulk mechanical

  17. 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

  18. 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

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

    PubMed

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

    2014-01-01

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

  20. Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus

    PubMed Central

    Boutry, Céline; Wahl, Astrid; Delplace, Brigitte; Clippe, André; Fontaine, Laetitia

    2012-01-01

    In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermophilus genome revealed the presence of a homolog of mecA (mecASt), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecASt represses late competence genes without affecting the early competence stage under conditions that are not permissive for competence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains deleted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA interacts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus. PMID:22287513

  1. Detachment-Based Equilibrium of Anoikic Cell Death and Autophagic Cell Survival Through Adaptor Protein p66(Shc).

    PubMed

    Cai, Zeyuan; Zhao, Dan; Sun, Yanan; Gao, Dan; Li, Xia; Yang, Jie; Ma, Zhenyi

    2016-03-01

    Anoikis (detachment-induced cell death) confers a tumor-suppressive function in metastatic cancer cells. Autophagy, a conserved self-degradative process, enhances the anoikis resistance of detached cancer cells by maintaining cellular homeostasis. However, the mechanism of regulating cell fate-decision by balancing anoikis and autophagy has been poorly understood. Our previous studies have shown that the adaptor protein p66(Shc) mediates anoikis through RhoA activation and inhibits tumor metastasis in vivo. We also found that p66(Shc) depletion mitigates nutrient-deprivation-induced autophagy. These findings suggest p66(Shc) may coordinately regulate these two processes. To verify this hypothesis, we investigated the effect of p66(Shc) on the cell death of detached lung cancer cells, and measured autophagy markers and autophagic flux. Results showed that p66(Shc) depletion significantly inhibited anoikis, and reduced the formation of LC3B-II and the degradation of Sequestosome 1 (SQSTM1, p62) in detachment-induced cells. Using monodansylcadaverine (MDC)-LysoTracker double staining and monomeric Cherry (mCherry)-GFP-LC3 assay, we found that the autophagic flux was also mitigated by p66(Shc) depletion. In addition, p66(Shc) knockdown increased the formation of full-length X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1), which enhances anoikis sensitivity. In conclusion, p66(Shc) plays an essential role in detachment-based equilibrium of anoikic cell death and autophagic cell survival. Anat Rec, 299:325-333, 2016. © 2015 Wiley Periodicals, Inc. PMID:26643258

  2. The Wnt Adaptor Protein ATP6AP2 Regulates Multiple Stages of Adult Hippocampal Neurogenesis

    PubMed Central

    Han, Jinju; Pena, Monique; von Bohlen und Halbach, Oliver; Peters, Jörg; Gage, Fred H.

    2015-01-01

    In the mammalian hippocampus, canonical Wnt signals provided by the microenvironment regulate the differentiation of adult neural stem cells (NSCs) toward the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways and the role of Wnt/Planar cell polarity (PCP) signaling in adult neurogenesis remains unknown. Using in vitro assays on differentiating adult NSCs, we identified a transition of Wnt signaling responsiveness from Wnt/β-catenin to Wnt/PCP signaling. In mice, retroviral knockdown strategies against ATP6AP2, a recently discovered core protein involved in both signaling pathways, revealed that its dual role is critical for granule cell fate and morphogenesis. We were able to confirm its dual role in neurogenic Wnt signaling in vitro for both canonical Wnt signaling in proliferating adult NSCs and non-canonical Wnt signaling in differentiating neuroblasts. Although LRP6 appeared to be critical for granule cell fate determination, in vivo knockdown of PCP core proteins FZD3 and CELSR1-3 revealed severe maturational defects without changing the identity of newborn granule cells. Furthermore, we found that CELSR1-3 control distinctive aspects of PCP-mediated granule cell morphogenesis with CELSR1 regulating the direction of dendrite initiation sites and CELSR2/3 controlling radial migration and dendritic patterning. The data presented here characterize distinctive roles for Wnt/β-catenin signaling in granule cell fate determination and for Wnt/PCP signaling in controlling the morphological maturation of differentiating neuroblasts. PMID:25810528

  3. The Wnt adaptor protein ATP6AP2 regulates multiple stages of adult hippocampal neurogenesis.

    PubMed

    Schafer, Simon T; Han, Jinju; Pena, Monique; von Bohlen Und Halbach, Oliver; Peters, Jörg; Gage, Fred H

    2015-03-25

    In the mammalian hippocampus, canonical Wnt signals provided by the microenvironment regulate the differentiation of adult neural stem cells (NSCs) toward the neuronal lineage. Wnts are part of a complex and diverse set of signaling pathways and the role of Wnt/Planar cell polarity (PCP) signaling in adult neurogenesis remains unknown. Using in vitro assays on differentiating adult NSCs, we identified a transition of Wnt signaling responsiveness from Wnt/β-catenin to Wnt/PCP signaling. In mice, retroviral knockdown strategies against ATP6AP2, a recently discovered core protein involved in both signaling pathways, revealed that its dual role is critical for granule cell fate and morphogenesis. We were able to confirm its dual role in neurogenic Wnt signaling in vitro for both canonical Wnt signaling in proliferating adult NSCs and non-canonical Wnt signaling in differentiating neuroblasts. Although LRP6 appeared to be critical for granule cell fate determination, in vivo knockdown of PCP core proteins FZD3 and CELSR1-3 revealed severe maturational defects without changing the identity of newborn granule cells. Furthermore, we found that CELSR1-3 control distinctive aspects of PCP-mediated granule cell morphogenesis with CELSR1 regulating the direction of dendrite initiation sites and CELSR2/3 controlling radial migration and dendritic patterning. The data presented here characterize distinctive roles for Wnt/β-catenin signaling in granule cell fate determination and for Wnt/PCP signaling in controlling the morphological maturation of differentiating neuroblasts. PMID:25810528

  4. Invertebrate and Vertebrate Class III Myosins Interact with MORN Repeat-Containing Adaptor Proteins

    PubMed Central

    Mecklenburg, Kirk L.; Freed, Stephanie A.; Raval, Manmeet; Quintero, Omar A.; Yengo, Christopher M.; O'Tousa, Joseph. E.

    2015-01-01

    In Drosophila photoreceptors, the NINAC-encoded myosin III is found in a complex with a small, MORN-repeat containing, protein Retinophilin (RTP). Expression of these two proteins in other cell types showed NINAC myosin III behavior is altered by RTP. NINAC deletion constructs were used to map the RTP binding site within the proximal tail domain of NINAC. In vertebrates, the RTP ortholog is MORN4. Co-precipitation experiments demonstrated that human MORN4 binds to human myosin IIIA (MYO3A). In COS7 cells, MORN4 and MYO3A, but not MORN4 and MYO3B, co-localize to actin rich filopodia extensions. Deletion analysis mapped the MORN4 binding to the proximal region of the MYO3A tail domain. MYO3A dependent MORN4 tip localization suggests that MYO3A functions as a motor that transports MORN4 to the filopodia tips and MORN4 may enhance MYO3A tip localization by tethering it to the plasma membrane at the protrusion tips. These results establish conserved features of the RTP/MORN4 family: they bind within the tail domain of myosin IIIs to control their behavior. PMID:25822849

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  7. Spatial distribution of proteins in the quagga mussel adhesive apparatus.

    PubMed

    Rees, David J; Hanifi, Arash; Manion, Joseph; Gantayet, Arpita; Sone, Eli D

    2016-01-01

    The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion. PMID:26825294

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

    PubMed Central

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

    2015-01-01

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

  9. Intrinsic Surface-Drying Properties of Bio-adhesive Proteins

    PubMed Central

    Akdogan, Yasar; Wei, Wei; Huang, Kuo-Ying; Kageyama, Yoshiyuki; Danner, Eric W.; Miller, Dusty R.; Martinez Rodriguez, Nadine R.; Herbert Waite, J.

    2014-01-01

    Sessile marine mussels must “dry” underwater surfaces before adhering to them. Synthetic adhesives have yet to overcome this fundamental challenge. Previous studies of bio-inspired adhesion have largely been performed under applied compressive forces but these are poor predictors of an adhesive’s ability to spontaneously penetrate surface hydration layers. In a force-free approach to measuring molecular-level interaction via the surface water diffusivity, different mussel foot proteins were found to have differential abilities to evict hydration layers from the surfaces—a necessary step for adsorption and adhesion. It was anticipated that Dopa would mediate dehydration given its efficacy forbio-inspired wet adhesion. Instead, hydrophobic side-chains are found to be a critical component in bringing about protein-surface intimacy. This is the first direct measurement of interfacial water dynamics during force-free adsorptive interactions at solid surfaces, and offers guidance for engineering wet adhesives and coatings. PMID:25168789

  10. 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

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

    PubMed

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

    2016-01-01

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

  12. 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

  13. 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. PMID:21039524

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

    PubMed Central

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

    2015-01-01

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

  15. Mussel-mimetic protein-based adhesive hydrogel.

    PubMed

    Kim, Bum Jin; Oh, Dongyeop X; Kim, Sangsik; Seo, Jeong Hyun; Hwang, Dong Soo; Masic, Admir; Han, Dong Keun; Cha, Hyung Joon

    2014-05-12

    Hydrogel systems based on cross-linked polymeric materials which could provide both adhesion and cohesion in wet environment have been considered as a promising formulation of tissue adhesives. Inspired by marine mussel adhesion, many researchers have tried to exploit the 3,4-dihydroxyphenylalanine (DOPA) molecule as a cross-linking mediator of synthetic polymer-based hydrogels which is known to be able to achieve cohesive hardening as well as adhesive bonding with diverse surfaces. Beside DOPA residue, composition of other amino acid residues and structure of mussel adhesive proteins (MAPs) have also been considered important elements for mussel adhesion. Herein, we represent a novel protein-based hydrogel system using DOPA-containing recombinant MAP. Gelation can be achieved using both oxdiation-induced DOPA quinone-mediated covalent and Fe(3+)-mediated coordinative noncovalent cross-linking. Fe(3+)-mediated hydrogels show deformable and self-healing viscoelastic behavior in rheological analysis, which is also well-reflected in bulk adhesion strength measurement. Quinone-mediated hydrogel has higher cohesive strength and can provide sufficient gelation time for easier handling. Collectively, our newly developed MAP hydrogel can potentially be used as tissue adhesive and sealant for future applications. PMID:24650082

  16. Polymer adhesion at surfaces: biological adhesive proteins and their synthetic mimics

    NASA Astrophysics Data System (ADS)

    Messersmith, Phillip

    2008-03-01

    Mussels are famous for their ability to permanently adhere to a wide variety of wet surfaces, such as rocks, metal and polymer ship hulls, and wood structures. They accomplish this through specialized proteins collectively referred to as mussel adhesive proteins (MAPs). The biophysical aspects of MAP adhesion is being revealed through the use of single molecule force measurements. The results provide insight into the adhesive roles of key amino acids found in these proteins, including the magnitude of adhesive forces, cooperative effects, and their self-healing properties. This molecular-level information is being incorporated into designs of biomimetic polymer coatings for a variety of applications. Our biomimetic approach to polymer design will be illustrated by a few examples where adhesive constituents found in MAPs are exploited to make wet-adhesive polymer coatings. In addition, small molecule analogs of MAPs can be used to apply thin functional films onto virtually any material surface using a facile approach. These coatings have a variety of potential uses in microelectronics, water treatment, prevention of environmental biofouling, and for control of biointerfacial phenomena at the surfaces of medical/diagnostic devices.

  17. Novel protein-repellent dental adhesive containing 2-methacryloyloxyethyl phosphorylcholine

    PubMed Central

    Zhang, Ning; Melo, Mary Anne S.; Bai, Yuxing; Xu, Hockin H. K.

    2015-01-01

    Objectives Biofilms at tooth-restoration margins can produce acids and cause secondary caries. A protein-repellent adhesive resin can potentially inhibition bacteria attachment and biofilm growth. However, there has been no report on protein-repellent dental resins. The objectives of this study were to develop a protein-repellent bonding agent incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC), and to investigate its resistance to protein adsorption and biofilm growth for the first time. Methods MPC was incorporated into Scotchbond Multi-Purpose (SBMP) at 0%, 3.75%, 7.5%, 11.25%, and 15% by mass. Extracted human teeth were used to measure dentin shear bond strengths. Protein adsorption onto resins was determined by a micro bicinchoninic acid (BCA) method. A dental plaque microcosm biofilm model with human saliva as inoculum was used to measure biofilm metabolic activity and colony-forming unit (CFU) counts. Results Adding 7.5% MPC into primer and adhesive did not decrease the dentin bond strength, compared to control (p > 0.1). Incorporation of 7.5% of MPC achieved the lowest protein adsorption, which was 20-fold less than that of control. Incorporation of 7.5% of MPC greatly reduced bacterial adhesion, yielding biofilm total microorganism, total streptococci, and mutans streptococci CFU that were an order of magnitude less than control. Conclusions A protein-repellent dental adhesive resin was developed for the first time. Incorporation of MPC into primer and adhesive at 7.5% by mass greatly reduced the protein adsorption and bacterial adhesion, without compromising the dentin bond strength. The novel protein-repellent primer and adhesive are promising to inhibit biofilm formation and acid production, to protect the tooth-restoration margins and prevent secondary caries. PMID:25234652

  18. Mutations in the gene encoding the Sigma 2 subunit of the adaptor protein 1 complex, AP1S2, cause X-linked mental retardation.

    PubMed

    Tarpey, Patrick S; Stevens, Claire; Teague, Jon; Edkins, Sarah; O'Meara, Sarah; Avis, Tim; Barthorpe, Syd; Buck, Gemma; Butler, Adam; Cole, Jennifer; Dicks, Ed; Gray, Kristian; Halliday, Kelly; Harrison, Rachel; Hills, Katy; Hinton, Jonathon; Jones, David; Menzies, Andrew; Mironenko, Tatiana; Perry, Janet; Raine, Keiran; Richardson, David; Shepherd, Rebecca; Small, Alexandra; Tofts, Calli; Varian, Jennifer; West, Sofie; Widaa, Sara; Yates, Andy; Catford, Rachael; Butler, Julia; Mallya, Uma; Moon, Jenny; Luo, Ying; Dorkins, Huw; Thompson, Deborah; Easton, Douglas F; Wooster, Richard; Bobrow, Martin; Carpenter, Nancy; Simensen, Richard J; Schwartz, Charles E; Stevenson, Roger E; Turner, Gillian; Partington, Michael; Gecz, Jozef; Stratton, Michael R; Futreal, P Andrew; Raymond, F Lucy

    2006-12-01

    In a systematic sequencing screen of the coding exons of the X chromosome in 250 families with X-linked mental retardation (XLMR), we identified two nonsense mutations and one consensus splice-site mutation in the AP1S2 gene on Xp22 in three families. Affected individuals in these families showed mild-to-profound mental retardation. Other features included hypotonia early in life and delay in walking. AP1S2 encodes an adaptin protein that constitutes part of the adaptor protein complex found at the cytoplasmic face of coated vesicles located at the Golgi complex. The complex mediates the recruitment of clathrin to the vesicle membrane. Aberrant endocytic processing through disruption of adaptor protein complexes is likely to result from the AP1S2 mutations identified in the three XLMR-affected families, and such defects may plausibly cause abnormal synaptic development and function. AP1S2 is the first reported XLMR gene that encodes a protein directly involved in the assembly of endocytic vesicles. PMID:17186471

  19. 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.

  20. 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.

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

    PubMed

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

    2016-04-15

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

  2. 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

  3. The focal adhesion protein PINCH-1 associates with EPLIN at integrin adhesion sites

    PubMed Central

    Karaköse, Esra; Geiger, Tamar; Flynn, Kevin; Lorenz-Baath, Katrin; Zent, Roy; Mann, Matthias; Fässler, Reinhard

    2015-01-01

    ABSTRACT PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Lims1) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. PMID:25609703

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

    PubMed Central

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

    2012-01-01

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

  5. A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network.

    PubMed

    Guo, Yusong; Zanetti, Giulia; Schekman, Randy

    2013-01-01

    Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.DOI:http://dx.doi.org/10.7554/eLife.00160.001. PMID:23326640

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2016-05-01

    Endocytosis has been suggested to be important in the cellular processes of plant immune responses. However, our understanding of its role during effector-triggered immunity (ETI) is still limited. We have previously shown that plant endocytosis, especially clathrin-coated vesicle formation at the plasma membrane, is mediated by the adaptor protein-2 (AP-2) complex and that loss of the μ subunit of AP-2 (AP2M) affects plant growth and floral organ development. Here, we report that AP2M is required for full-strength ETI mediated by the disease resistance (R) genes RPM1 and RPS2 in Arabidopsis. Reduced ETI was observed in an ap2m mutant plant, measured by growth of Pseudomonas syringae pv. tomato DC3000 strains carrying the corresponding effector genes avrRpm1 or avrRpt2 and by hypersensitive cell death response and defense gene expression triggered by these strains. In contrast, RPS4-mediated ETI and its associated immune responses were not affected by the ap2m mutation. While RPM1 and RPS2 are localized to the plasma membrane, RPS4 is localized to the cytoplasm and nucleus. Our results suggest that AP2M is involved in ETI mediated by plasma membrane-localized R proteins, possibly by mediating endocytosis of the immune receptor complex components from the plasma membrane. PMID:26828402

  8. Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

    PubMed

    Urbina, Patricia; Bersch, Beate; De Angelis, Fabien; Derfoufi, Kheiro-Mouna; Prévost, Martine; Goormaghtigh, Erik; Vandenbussche, Guy

    2016-05-24

    Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site. PMID:27145046

  9. Recombinant production of functional full-length and truncated human TRAM/TICAM-2 adaptor protein involved in Toll-like receptor and interferon signaling.

    PubMed

    Ullah, M Obayed; Valkov, Eugene; Ve, Thomas; Williams, Simon; Mas, Caroline; Mansell, Ashley; Kobe, Bostjan

    2015-02-01

    TRAM/TICAM-2 is used by Toll-like receptor 4 (TLR4) as a bridging adaptor during the mammalian innate immune response. It recruits TRIF, another TIR domain-containing adaptor protein, to TLR4 via TIR domain interactions, which leads to the activation of transcription factors responsible for the production of type-1 interferon and cytokines. The molecular mechanisms of these dual interactions mediated by the TRAM TIR domain are not clear. To understand the molecular basis of TIR:TIR domain interactions, structural and biochemical studies of TRAM TIR domain are necessary, and require a functional soluble protein. In this paper, we report a successful purification and characterization of full-length TRAM. Because full-length TRAM likely contains unstructured regions that may be disadvantageous for structural studies, we also carried out a systematic construct design to determine the boundaries of the TRAM TIR domain. The truncated TRAM constructs were designed based on secondary structure predictions and screened by small-scale expression. Selected constructs were subjected to biophysical analyses. We show that the expressed TRAM TIR domain is functional using in vitro GST pull-down assays that demonstrate a physical interaction with the TLR4 TIR domain. We further show, by site-directed mutagenesis, that the "BB loop" regions of both the TRAM TIR domain and the TLR4 TIR domain are crucial for this physical interaction. PMID:25306876

  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. Protein Modifications Regulate the Role of 14-3-3γ Adaptor Protein in cAMP-induced Steroidogenesis in MA-10 Leydig Cells*

    PubMed Central

    Aghazadeh, Yasaman; Ye, Xiaoying; Blonder, Josip; Papadopoulos, Vassilios

    2014-01-01

    The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser58 phosphorylation and 14-3-3γ Lys49 acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser58 or Lys49. Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser58 phosphorylation and Lys49 acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser58 phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys49 acetylation is important for the cAMP-dependent induction of these interactions. PMID:25086053

  12. Interaction of the protein tyrosine phosphatase PTPL1 with the PtdIns(3,4)P2-binding adaptor protein TAPP1.

    PubMed Central

    Kimber, Wendy A; Deak, Maria; Prescott, Alan R; Alessi, Dario R

    2003-01-01

    It has been postulated that PtdIns(3,4) P (2), one of the immediate breakdown products of PtdIns(3,4,5) P (3), functions as a signalling molecule in insulin- and growth-factor-stimulated pathways. To date, the t andem- P H-domain-containing p rotein- 1 (TAPP1) and related TAPP2 are still the only known PH-domain-containing proteins that interact strongly and specifically with PtdIns(3,4) P (2). In this study we demonstrate that endogenously expressed TAPP1, is constitutively associated with the protein-tyrosine-phosphatase-like protein-1 (PTPL1 also known as FAP-1). We show that PTPL1 binds to TAPP1 and TAPP2, principally though its first PDZ domain [where PDZ is postsynaptic density protein ( P SD-95)/ Drosophila disc large tumour suppressor ( d lg)/tight junction protein ( Z O1)] and show that this renders PTPL1 capable of associating with PtdIns(3,4) P (2) in vitro. Our data suggest that the binding of TAPP1 to PTPL1 does not influence PTPL1 phosphatase activity, but instead functions to maintain PTPL1 in the cytoplasm. Following stimulation of cells with hydrogen peroxide to induce PtdIns(3,4) P (2) production, PTPL1, complexed to TAPP1, translocates to the plasma membrane. This study provides the first evidence that TAPP1 and PtdIns(3,4) P (2) could function to regulate the membrane localization of PTPL1. We speculate that if PTPL1 was recruited to the plasma membrane by increasing levels of PtdIns(3,4) P (2), it could trigger a negative feedback loop in which phosphoinositide-3-kinase-dependent or other signalling pathways could be switched off by the phosphatase-catalysed dephosphorylation of receptor tyrosine kinases or tyrosine phosphorylated adaptor proteins such as IRS1 or IRS2. Consistent with this notion we observed RNA-interference-mediated knock-down of TAPP1 in HEK-293 cells, enhanced activation and phosphorylation of PKB following IGF1 stimulation. PMID:14516276

  13. Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays

    PubMed Central

    Torres, Alexis J.; Vasudevan, Lavanya; Holowka, David; Baird, Barbara A.

    2008-01-01

    Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-FcεRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-FcεRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-FcεRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-FcεRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (α5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-FcεRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-FcεRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the FcεRI β subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-FcεRI has a net positive effect on FcεRI signaling. PMID:19004813

  14. Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein

    PubMed Central

    Sundaramoorthy, Ramasubramanian; Fyfe, Paul K.; Hunter, William N.

    2012-01-01

    Staphylococcus aureus pathogenesis depends on a specialized protein secretion system, ESX-1, that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB; small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacteria tuberculosis suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and / or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins. PMID:18773907

  15. Dancing to Another Tune—Adhesive Moonlighting Proteins in Bacteria

    PubMed Central

    Kainulainen, Veera; Korhonen, Timo K.

    2014-01-01

    Biological moonlighting refers to proteins which express more than one function. Moonlighting proteins occur in pathogenic and commensal as well as in Gram-positive and Gram-negative bacteria. The canonical functions of moonlighting proteins are in essential cellular processes, i.e., glycolysis, protein synthesis, chaperone activity, and nucleic acid stability, and their moonlighting functions include binding to host epithelial and phagocytic cells, subepithelia, cytoskeleton as well as to mucins and circulating proteins of the immune and hemostatic systems. Sequences of the moonlighting proteins do not contain known motifs for surface export or anchoring, and it has remained open whether bacterial moonlighting proteins are actively secreted to the cell wall or whether they are released from traumatized cells and then rebind onto the bacteria. In lactobacilli, ionic interactions with lipoteichoic acids and with cell division sites are important for surface localization of the proteins. Moonlighting proteins represent an abundant class of bacterial adhesins that are part of bacterial interactions with the environment and in responses to environmental changes. Multifunctionality in bacterial surface proteins appears common: the canonical adhesion proteins fimbriae express also nonadhesive functions, whereas the mobility organelles flagella as well as surface proteases express adhesive functions. PMID:24833341

  16. Fibroblast adhesion to recombinant tropoelastin expressed as a protein A-fusion protein.

    PubMed Central

    Grosso, L E; Parks, W C; Wu, L J; Mecham, R P

    1991-01-01

    A bovine tropoelastin cDNA encoding exons 15-36 that includes the elastin-receptor binding site was expressed in Escherichia coli as a fusion protein with Protein A from Staphylococcus aureus. After isolation of the fusion protein by affinity chromatography on Ig-Sepharose, the tropoelastin domain was separated from plasmid-pR1T2T-encoded Protein A (Protein A') by CNBr cleavage. Cell-adhesion assays demonstrated specific adhesion to the recombinant tropoelastin. Furthermore, the data indicate that interactions involving the bovine elastin receptor mediate nuchalligament fibroblast adhesion to the recombinant protein. In agreement with earlier studies of fibroblast chemotaxis to bovine tropoelastin, nuchal-ligament fibroblast adhesion demonstrated developmental regulation of the elastin receptor. Images Fig. 2. Fig. 3. PMID:1996952

  17. Glycosylated Hydroxytryptophan in a Mussel Adhesive Protein from Perna viridis*

    PubMed Central

    Zhao, Hua; Sagert, Jason; Hwang, Dong Soo; Waite, J. Herbert

    2009-01-01

    The 3,4-dihydroxyphenyl-l-alanine (Dopa)-containing proteins of mussel byssus play a critical role in wet adhesion and have inspired versatile new synthetic strategies for adhesives and coatings. Apparently, however, not all mussel adhesive proteins are beholden to Dopa chemistry. The cDNA-deduced sequence of Pvfp-1, a highly aromatic and redox active byssal coating protein in the green mussel Perna viridis, suggests that Dopa may be replaced by a post-translational modification of tryptophan. The N-terminal tryptophan-rich domain of Pvfp-1 contains 42 decapeptide repeats with the consensus sequences ATPKPW1TAW2K and APPPAW1TAW2K. A small collagen domain (18 Gly-X-Y repeats) is also present. Tandem mass spectrometry of isolated tryptic decapeptides has detected both C2-hexosylated tryptophan (W1) and C2-hexosylated hydroxytryptophan (W2), the latter of which is redox active. The UV absorbance spectrum of W2 is consistent with 7-hydroxytryptophan, which represents an intriguing new theme for bioinspired opportunistic wet adhesion. PMID:19584055

  18. The Src homology 2 protein Shb promotes cell cycle progression in murine hematopoietic stem cells by regulation of focal adhesion kinase activity

    SciTech Connect

    Gustafsson, Karin; Heffner, Garrett; Wenzel, Pamela L.; Curran, Matthew; Grawé, Jan; McKinney-Freeman, Shannon L.; Daley, George Q.; Welsh, Michael

    2013-07-15

    The widely expressed adaptor protein Shb has previously been reported to contribute to T cell function due to its association with the T cell receptor and furthermore, several of Shb's known interaction partners are established regulators of blood cell development and function. In addition, Shb deficient embryonic stem cells displayed reduced blood cell colony formation upon differentiation in vitro. The aim of the current study was therefore to explore hematopoietic stem and progenitor cell function in the Shb knockout mouse. Shb deficient bone marrow contained reduced relative numbers of long-term hematopoietic stem cells (LT-HSCs) that exhibited lower proliferation rates. Despite this, Shb knockout LT-HSCs responded promptly by entering the cell cycle in response to genotoxic stress by 5-fluorouracil treatment. In competitive LT-HSC transplantations, Shb null cells initially engrafted as well as the wild-type cells but provided less myeloid expansion over time. Moreover, Shb knockout bone marrow cells exhibited elevated basal activities of focal adhesion kinase/Rac1/p21-activated kinase signaling and reduced responsiveness to Stem Cell Factor stimulation. Consequently, treatment with a focal adhesion kinase inhibitor increased Shb knockout LT-HSC proliferation. The altered signaling characteristics thus provide a plausible mechanistic explanation for the changes in LT-HSC proliferation since these signaling intermediates have all been shown to participate in LT-HSC cell cycle control. In summary, the loss of Shb dependent signaling in bone marrow cells, resulting in elevated focal adhesion kinase activity and reduced proliferative responses in LT-HSCs under steady state hematopoiesis, confers a disadvantage to the maintenance of LT-HSCs over time. -- Highlights: • Shb is an adaptor protein operating downstream of tyrosine kinase receptors. • Shb deficiency reduces hematopoietic stem cell proliferation. • The proliferative effect of Shb occurs via increased

  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. PMID:26546666

  20. Adhesion

    MedlinePlus

    ... adhesions Ovarian cyst References Munireddy S, Kavalukas SL, Barbul A. Intra-abdominal healing: gastrointestinal tract and adhesions. Surg Clin N Am Kulaylat MN, Dayton, MT. Surgical complications. In: Townsend CM Jr, Beauchamp RD, Evers BM, Mattox KL, ...

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

    PubMed

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

    2016-08-15

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

  2. 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

  3. 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

  4. Silk Fibroin Aqueous-Based Adhesives Inspired by Mussel Adhesive Proteins.

    PubMed

    Burke, Kelly A; Roberts, Dane C; Kaplan, David L

    2016-01-11

    Silk fibroin from the domesticated silkworm Bombyx mori is a naturally occurring biopolymer with charged hydrophilic terminal regions that end-cap a hydrophobic core consisting of repeating sequences of glycine, alanine, and serine residues. Taking inspiration from mussels that produce proteins rich in L-3,4-dihydroxyphenylalanine (DOPA) to adhere to a variety of organic and inorganic surfaces, the silk fibroin was functionalized with catechol groups. Silk fibroin was selected for its high molecular weight, tunable mechanical and degradation properties, aqueous processability, and wide availability. The synthesis of catechol-functionalized silk fibroin polymers containing varying amounts of hydrophilic polyethylene glycol (PEG, 5000 g/mol) side chains was carried out to balance silk hydrophobicity with PEG hydrophilicity. The efficiency of the catechol functionalization reaction did not vary with PEG conjugation over the range studied, although tuning the amount of PEG conjugated was essential for aqueous solubility. Adhesive bonding and cell compatibility of the resulting materials were investigated, where it was found that incorporating as little as 6 wt % PEG prior to catechol functionalization resulted in complete aqueous solubility of the catechol conjugates and increased adhesive strength compared with silk lacking catechol functionalization. Furthermore, PEG-silk fibroin conjugates maintained their ability to form β-sheet secondary structures, which can be exploited to reduce swelling. Human mesenchymal stem cells (hMSCs) proliferated on the silks, regardless of PEG and catechol conjugation. These materials represent a protein-based approach to catechol-based adhesives, which we envision may find applicability as biodegradable adhesives and sealants. PMID:26674175

  5. M protein mediates streptococcal adhesion to HEp-2 cells.

    PubMed

    Wang, J R; Stinson, M W

    1994-02-01

    Streptococcus pyogenes adheres to human epithelial cells in vitro and in vivo. To identify adhesins, cell wall components were extracted from S. pyogenes M6 with alkali or by treatment with mutanolysin and lysozyme. HEp-2 cells were incubated with extracts of S. pyogenes M6 and then analyzed by Western blot (immunoblot) assays, using antibodies to S. pyogenes. Only one streptococcal component (62 kDa) was bound to HEp-2 cells and was identified serologically as M6 protein. Experiments with pepsin-cleaved fragments of M protein indicated that the binding site was located at the N-terminal half of the molecule. M protein was bound selectively to two trypsin-sensitive surface components, 97 and 205 kDa, of HEp-2 cells on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels. Tritium-labeled lipoteichoic acid bound to different HEp-2 cell components, 34 and 35 kDa, in a parallel experiment, indicating that lipoteichoic acid was not complexed with M protein and does not mediate M-protein binding. The four HEp-2 components were unrelated to fibronectin since they did not react with specific antibodies. An M-protein-deficient (M-) strain of streptococcus (JRS75), grown in chemically defined medium, showed 73% less adhesion activity to HEp-2 monolayers than an M+ strain (JRS4). Streptococcal adhesion was insensitive to competitive inhibition by selected monosaccharides. These results indicate that M protein binds directly to certain HEp-2 cell membrane components and mediates streptococcal adhesion. PMID:8300205

  6. LINKIN, a new transmembrane protein necessary for cell adhesion

    PubMed Central

    Kato, Mihoko; Chou, Tsui-Fen; Yu, Collin Z; DeModena, John; Sternberg, Paul W

    2014-01-01

    In epithelial collective migration, leader and follower cells migrate while maintaining cell–cell adhesion and tissue polarity. We have identified a conserved protein and interactors required for maintaining cell adhesion during a simple collective migration in the developing C. elegans male gonad. LINKIN is a previously uncharacterized, transmembrane protein conserved throughout Metazoa. We identified seven atypical FG–GAP domains in the extracellular domain, which potentially folds into a β-propeller structure resembling the α-integrin ligand-binding domain. C. elegans LNKN-1 localizes to the plasma membrane of all gonadal cells, with apical and lateral bias. We identified the LINKIN interactors RUVBL1, RUVBL2, and α-tubulin by using SILAC mass spectrometry on human HEK 293T cells and testing candidates for lnkn-1-like function in C. elegans male gonad. We propose that LINKIN promotes adhesion between neighboring cells through its extracellular domain and regulates microtubule dynamics through RUVBL proteins at its intracellular domain. DOI: http://dx.doi.org/10.7554/eLife.04449.001 PMID:25437307

  7. The Src homology and collagen A (ShcA) adaptor protein is required for the spatial organization of the costamere/Z-disk network during heart development.

    PubMed

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

    2015-01-23

    Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere. PMID:25488665

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

    PubMed Central

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

    2015-01-01

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

  9. Evaluation of photodynamic therapy in adhesion protein expression

    PubMed Central

    PACHECO-SOARES, CRISTINA; MAFTOU-COSTA, MAIRA; DA CUNHA MENEZES COSTA, CAROLINA GENÚNCIO; DE SIQUEIRA SILVA, ANDREZA CRISTINA; MORAES, KAREN C.M.

    2014-01-01

    Photodynamic therapy (PDT) is a treatment modality that has clinical applications in both non-neoplastic and neoplastic diseases. PDT involves a light-sensitive compound (photosensitizer), light and molecular oxygen. This procedure may lead to several different cellular responses, including cell death. Alterations in the attachment of cancer cells to the substratum and to each other are important consequences of photodynamic treatment. PDT may lead to changes in the expression of cellular adhesion structure and cytoskeleton integrity, which are key factors in decreasing tumor metastatic potential. HEp-2 cells were photosensitized with aluminum phthalocyanine tetrasulfonate and zinc phthalocyanine, and the proteins β1-integrin and focal adhesion kinase (FAK) were assayed using fluorescence microscopy. The verification of expression changes in the genes for FAK and β1 integrin were performed by reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that HEp-2 cells do not express β-integrin or FAK 12 h following PDT. It was concluded that the PDT reduces the adhesive ability of HEp-2 cells, inhibiting their metastatic potential. The present study aimed to analyze the changes in the expression and organization of cellular adhesion elements and the subsequent metastatic potential of HEp-2 cells following PDT treatment. PMID:25013490

  10. 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

  11. 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

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

    PubMed

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

    2014-07-01

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

  13. Structure, Function and On-Off Switching of a Core Unit Contact between CheA Kinase and CheW Adaptor Protein in the Bacterial Chemosensory Array: A Disulfide Mapping and TAM-IDS Study

    PubMed Central

    Natale, Andrew M.; Duplantis, Jane L.; Piasta, Kene N.; Falke, Joseph J.

    2014-01-01

    The ultrasensitive, ultrastable bacterial chemosensory array of Escherichia coli and Salmonella typhimurium is representative of the large, conserved family of sensory arrays that control the cellular chemotaxis of motile bacteria and Archaea. The core framework of the membrane-bound array is a lattice assembled from three components: a transmembrane receptor, a cytoplasmic His kinase (CheA), and a cytoplasmic adaptor protein (CheW). Structural studies in the field have revealed the global architecture of the array and complexes between specific components, but much remains to be learned about the essential protein-protein interfaces that define array structure and transmit signals between components. This study has focused on the structure, function and on-off switching of a key contact between the kinase and adaptor proteins in the working, membrane-bound array. Specifically, the study addressed interface 1 in the putative kinase-adaptor ring where subdomain 1 of the kinase regulatory domain contacts subdomain 2 of the adaptor protein. Two independent approaches – disulfide mapping and site-directed Trp and Ala mutagenesis – were employed to (i) test the structural model of interface 1 and (ii) investigate its functional roles in both stable kinase incorporation and receptor-regulated kinase on-off switching. Studies were carried out in functional, membrane-bound arrays or in live cells. The findings reveal that crystal structures of binary and ternary complexes accurately depict the native interface in its kinase-activating on state. Furthermore, the findings indicate that at least part of the interface becomes less closely packed in its kinase-inhibiting off state. Together, the evidence shows the interface has a dual structural and signaling function that is crucial for stable kinase incorporation into the array, for kinase activation in the array on state, and likely for attractant-triggered kinase on-off switching. A model is presented that describes the

  14. MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src

    PubMed Central

    Sun, Weiyong; Wei, Xudong; Kesavan, Kamala; Garrington, Timothy P.; Fan, Ruihua; Mei, Junjie; Anderson, Steven M.; Gelfand, Erwin W.; Johnson, Gary L.

    2003-01-01

    Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex. PMID:12640115

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

    PubMed

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

    2005-04-01

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

  16. 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 Central

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

    2012-01-01

    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. PMID:22291017

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

    PubMed Central

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

    2016-01-01

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

  18. Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2013-12-01

    The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3-SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3-SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3-SH2 connector, and SH2 domain of SLAP2 (SLAP2-32). While both domains adopt typical folds, the short SH3-SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding. PMID:24018043

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

    PubMed Central

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

    2000-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2010-10-01

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

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

    PubMed

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

    2010-04-22

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

  3. 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

    2015-11-01

    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. PMID:26446278

  4. Adjuvanticity of the oil-in-water emulsion MF59 is independent of Nlrp3 inflammasome but requires the adaptor protein MyD88

    PubMed Central

    Seubert, Anja; Calabro, Samuele; Santini, Laura; Galli, Barbara; Genovese, Alessia; Valentini, Sara; Aprea, Susanna; Colaprico, Annalisa; D'Oro, Ugo; Giuliani, Marzia M.; Pallaoro, Michele; Pizza, Mariagrazia; O'Hagan, Derek T.; Wack, Andreas; Rappuoli, Rino; De Gregorio, Ennio

    2011-01-01

    Oil-in-water emulsions have been successfully used to increase the efficacy, immunogenicity, and cross-protection of human vaccines; however, their mechanism of action is still largely unknown. Nlrp3 inflammasome has been previously associated to the activity of alum, another adjuvant broadly used in human vaccines, and MyD88 adaptor protein is required for the adjuvanticity of most Toll-like receptor agonists. We compared the contribution of Nlrp3 and MyD88 to the adjuvanticity of alum, the oil-in-water emulsion MF59, and complete Freund's adjuvant in mice using a three-component vaccine against serogroup B Neisseria meningitidis (rMenB). Although the basal antibody responses to the nonadjuvanted rMenB vaccine were largely dependent on Nlrp3, the high-level antibody responses induced by alum, MF59, or complete Freund's adjuvant did not require Nlrp3. Surprisingly, we found that MF59 requires MyD88 to enhance bactericidal antibody responses to the rMenB vaccine. Because MF59 did not activate any of the Toll-like receptors in vitro, we propose that MF59 requires MyD88 for a Toll-like receptor-independent signaling pathway. PMID:21690334

  5. Adhesion and structure properties of protein nanomaterials containing hydrophobic and charged amino acids.

    PubMed

    Shen, Xinchun; Mo, Xiaoqun; Moore, Robyn; Frazier, Shawnalea J; Iwamoto, Takeo; Tomich, John M; Sun, Xiuzhi Susan

    2006-03-01

    Protein polymers are being used or considered for biobased adhesives and coating materials. Most adhesives derived from macro protein molecules work through receptors or cross-links to bring about adhesion. The adhesion mechanism of protein polymers would lead to better understanding of adhesives and the discovery of new practical properties of protein polymers at both nano- and macro-scales. The objective of this research work was to study adhesion properties of protein polymers at nanoscale (a peptide adhesive with nanometer-scale units that range in size of several nanometers, defined as protein nanomaterial). Seven protein nanomaterial samples with different degrees of adhesive strength were designed and synthesized using solid phase chemistries. All protein nanomaterials contain a common hydrophobic core flanked by charged amino acid sequences. The adhesion properties of the protein nanomaterials were investigated at different pH values and curing temperatures. The protein nanomaterials self aggregate and interact with the wood surface. The protein nanomaterial KKK-FLIVIGSII-KKK identified in this study had high adhesive strength toward wood. It had the highest shear strength at pH 12, with an amino acid sequence that was very hydrophobic and uncharged. This protein nanomaterial underwent structural analyses using circular dichroism, laser-Fourier transform infrared, and laser desorption mass spectrometry. At pH 12 this peptide adopted a pH-induced beta-like conformation. Adhesive strength reflects contributions of both hydrogen bonding and van der Waals interactions. Ionic and covalent bonds do not appear to be significant factors for adhesion in this study. PMID:16573147

  6. Use of additives to enhance the properties of cottonseed protein as wood adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soy protein is currently being used commercially as a “green” wood adhesive. Previous work in this laboratory has shown that cottonseed protein isolate, tested on maple wood veneer, produced higher adhesive strength and hot water resistance relative to soy protein. In the present study, cottonseed...

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

    PubMed

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

    2016-08-26

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

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

    PubMed

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

    2016-01-01

    Baculovirus has been exploited for use as a novel vaccine vector. To investigate the feasibility and efficacy of recombinant baculoviruses (rBVs) expressing respiratory syncytial virus (RSV) fusion (F) proteins, four constructs (Bac-tF/64, Bac-CF, Bac-CF/tF64 and Bac-CF/tF64-VISA) were generated. Bac-tF64 displays the F ectodomain (tF) on the envelope of rBVs, whereas Bac-CF expresses full-length F protein in transduced mammalian cells. Bac-CF/tF64 not only displays tF on the envelope but also expresses F in cells. Bac-CF/tF64-VISA comprises Bac-CF/tF64 harboring the virus-induced signaling adaptor (VISA) gene. After administration to BALB/c mice, all four vectors elicited RSV neutralizing antibody (Ab), systemic Ab (IgG, IgG1, and IgG2a), and cytokine responses. Compared with Bac-tF64, mice inoculated with Bac-CF and Bac-CF/tF64 exhibited an increased mixed Th1/Th2 cytokine response, increased ratios of IgG2a/IgG1 antibody responses, and reduced immunopathology upon RSV challenge. Intriguingly, co-expression of VISA reduced Th2 cytokine (IL-4, IL-5, and IL-10) production induced by Bac-CF/tF64, thus relieving lung pathology upon a subsequent RSV challenge. Our results indicated that the Bac-CF/tF64 vector incorporated with the VISA molecule may provide an effective vaccine strategy for protection against RSV. PMID:26643933

  9. 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

  10. Focal adhesion protein abnormalities in myelodysplastic mesenchymal stromal cells

    SciTech Connect

    Aanei, Carmen Mariana; Eloae, Florin Zugun; Flandrin-Gresta, Pascale; Tavernier, Emmanuelle; Carasevici, Eugen; Guyotat, Denis; Campos, Lydia

    2011-11-01

    Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y{sup 397}], and HSP90{alpha}/{beta} and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90{alpha}/{beta} client protein, these results suggest the utility of HSP90{alpha}/{beta} inhibition as a target for adjuvant therapy for myelodysplasia.

  11. ScaC, an Adaptor Protein Carrying a Novel Cohesin That Expands the Dockerin-Binding Repertoire of the Ruminococcus flavefaciens 17 Cellulosome

    PubMed Central

    Rincón, Marco T.; Martin, Jennifer C.; Aurilia, Vincenzo; McCrae, Sheila I.; Rucklidge, Garry J.; Reid, Martin D.; Bayer, Edward A.; Lamed, Raphael; Flint, Harry J.

    2004-01-01

    A new gene, designated scaC and encoding a protein carrying a single cohesin, was identified in the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 as part of a gene cluster that also codes for the cellulosome structural components ScaA and ScaB. Phylogenetic analysis showed that the sequence of the ScaC cohesin is distinct from the sequences of other cohesins, including the sequences of R. flavefaciens ScaA and ScaB. The scaC gene product also includes at its C terminus a dockerin module that closely resembles those found in R. flavefaciens enzymes that bind to the cohesins of the primary ScaA scaffoldin. The putative cohesin domain and the C-terminal dockerin module were cloned and overexpressed in Escherichia coli as His6-tagged products (ScaC-Coh and ScaC-Doc, respectively). Affinity probing of protein extracts of R. flavefaciens 17 separated in one-dimensional and two-dimensional gels with recombinant cohesins from ScaC and ScaA revealed that two distinct subsets of native proteins interact with ScaC-Coh and ScaA-Coh. Furthermore, ScaC-Coh failed to interact with the recombinant dockerin module from the enzyme EndB that is recognized by ScaA cohesins. On the other hand, ScaC-Doc was shown to interact specifically with the recombinant cohesin domain from ScaA, and the ScaA-Coh probe was shown to interact with a native 29-kDa protein spot identified as ScaC by matrix-assisted laser desorption ionization—time of flight mass spectrometry. These results suggest that ScaC plays the role of an adaptor scaffoldin that is bound to ScaA via the ScaC dockerin module, which, via the distinctive ScaC cohesin, expands the range of proteins that can bind to the ScaA-based enzyme complex. PMID:15090497

  12. Expression of epithelial adhesion proteins and integrins in chronic inflammation.

    PubMed Central

    Haapasalmi, K.; Mäkelä, M.; Oksala, O.; Heino, J.; Yamada, K. M.; Uitto, V. J.; Larjava, H.

    1995-01-01

    Epithelial cell behavior in chronic inflammation is poorly characterized. During inflammation of tooth-supporting structures (periodontal disease), increased proliferation of epithelial cells into the inflamed connective tissue stroma is commonly seen. In some areas ulceration and degeneration take place. We studied alterations in the expression of adhesion molecules and integrins during chronic periodontal inflammation. In inflamed tissue, laminin-1 and type IV collagen were still present in the basement membrane and surrounding blood vessels, but they were also found extravascularly in inflamed connective tissue stroma. Type VII collagen and laminin-5 (also known as kalinin, epiligrin, or nicein) were poorly preserved in the basement membrane zone, but both were found in unusual streak-like distributions in the subepithelial connective tissue stroma in inflamed tissue. Both fibronectin and tenascin were substantially decreased in chronically inflamed connective tissue, showing only punctate staining at the basement membrane zone. Integrins of the beta 1 family showed two distinct staining patterns in epithelial cells during chronic inflammation; focal losses of beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1) were found in most areas, while in other areas the entire pocket epithelium was found to be strongly positive for beta 1 integrins. No members of the alpha v integrin family were found in any epithelia studied. Expression of the alpha 6 beta 4 integrin was high in basal cells of healthy tissue, but weak in epithelium associated with chronic inflammation. Chronic inflammation therefore involves alterations in both adhesion proteins and integrins expressed by epithelial cells. Basement membrane components found at abnormal sites in stroma in chronic inflammation might serve as new adhesive ligands for various cell types in inflamed stroma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:7541610

  13. Adaptor protein Ste50p links the Ste11p MEKK to the HOG pathway through plasma membrane association

    PubMed Central

    Wu, Cunle; Jansen, Gregor; Zhang, Jianchun; Thomas, David Y.; Whiteway, Malcolm

    2006-01-01

    In a variety of yeast cellular pathways, the Ste50p protein regulates the kinase function of the mitogen extracellular signal-regulated kinase kinase (MEKK) Ste11p. Both Ste11p and Ste50p contain sterile α motif (SAM) domains; these are interchangeable, and can be replaced by other protein-interacting modules. Furthermore, the function of the Ras association (RA)-like domain of Ste50p can be mimicked by a plasma membrane recruiting signal, and direct plasma membrane targeting of Ste11p bypasses the requirement of Ste50p for Ste11p function. Thus the regulatory role of Ste50p requires both the N-terminal SAM domain to bind Ste11p and the C-terminal RA-like domain to direct kinase localization. We have identified Opy2p, an integral membrane protein that can interact with Ste50p, as a new component in the Sho1p–Ste11p/Ste50p signaling branch of the high-osmolarity glycerol (HOG) pathway. We propose that Opy2p can serve as a membrane anchor for the Ste50p/Ste11p module in the activation of the HOG pathway. PMID:16543225

  14. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of β1 integrin

    PubMed Central

    Chen, Lixiang; Hughes, Richard A.; Baines, Anthony J.; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-01-01

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R−/− mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R−/− keratinocytes, and 4.1R−/− mice exhibited defective epidermal wound healing. Cultured 4.1R−/− keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  15. Protein 4.1R regulates cell adhesion, spreading, migration and motility of mouse keratinocytes by modulating surface expression of beta1 integrin.

    PubMed

    Chen, Lixiang; Hughes, Richard A; Baines, Anthony J; Conboy, John; Mohandas, Narla; An, Xiuli

    2011-07-15

    Protein 4.1R is a membrane-cytoskeleton adaptor protein that has diverse roles in controlling the cell surface expression and/or function of transmembrane proteins, and in organizing F-actin. 4.1R is expressed in keratinocytes, but its role in these cells has not been explored. Here, we have investigated the role of 4.1R in skin using 4.1R(-/-) mice. Cell adhesion, spreading, migration and motility were significantly impaired in 4.1R(-/-) keratinocytes, and 4.1R(-/-) mice exhibited defective epidermal wound healing. Cultured 4.1R(-/-) keratinocytes on fibronectin failed to form actin stress fibres and focal adhesions. Furthermore, in the absence of 4.1R, the surface expression, and consequently the activity of β1 integrin were reduced. These data enabled the identification of a functional role for protein 4.1R in keratinocytes by modulating the surface expression of β1 integrin, possibly through a direct association between 4.1R and β1 integrin. PMID:21693581

  16. Boronate Complex Formation with Dopa Containing Mussel Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica

    PubMed Central

    Israelachvili, Jacob N.; Chen, Yunfei; Waite, J. Herbert

    2014-01-01

    The biochemistry of mussel adhesion has inspired the design of surface primers, adhesives, coatings and gels for technological applications. These mussel-inspired systems often focus on incorporating the amino acid 3,4-dihydroxyphenyl-L-alanine (Dopa) or a catecholic analog into a polymer. Unfortunately, effective use of Dopa is compromised by its susceptibility to auto-oxidation at neutral pH. Oxidation can lead to loss of adhesive function and undesired covalent cross-linking. Mussel foot protein 5 (Mfp-5), which contains ∼30 mole % Dopa, is a superb adhesive under reducing conditions but becomes nonadhesive after pH-induced oxidation. Here we report that the bidentate complexation of borate by Dopa to form a catecholato-boronate can be exploited to retard oxidation. Although exposure of Mfp-5 to neutral pH typically oxidizes Dopa, resulting in a>95% decrease in adhesion, inclusion of borate retards oxidation at the same pH. Remarkably, this Dopa-boronate complex dissociates upon contact with mica to allow for a reversible Dopa-mediated adhesion. The borate protection strategy allows for Dopa redox stability and maintained adhesive function in an otherwise oxidizing environment. PMID:25303409

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

    PubMed

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

    2015-08-01

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

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

    PubMed 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

  19. Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells is Revealed by the ShcD/RaLP Adaptor Protein

    PubMed Central

    Turco, Margherita Y; Furia, Laura; Dietze, Anja; Fernandez Diaz, Luis; Ronzoni, Simona; Sciullo, Anna; Simeone, Antonio; Constam, Daniel; Faretta, Mario; Lanfrancone, Luisa

    2012-01-01

    The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. Stem Cells2012;30:2423–2436 PMID:22948967

  20. Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity.

    PubMed

    Saether, Per C; Hoelsbrekken, Sigurd E; Fossum, Sigbjørn; Dissen, Erik

    2011-12-15

    Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family. PMID:22084441

  1. 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

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

    PubMed Central

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

    2011-01-01

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

  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. PMID:25691625

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

    PubMed

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

    2011-06-01

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

  5. 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. PMID:25196215

  6. Interaction of the enteropathogenic Escherichia coli protein, translocated intimin receptor (Tir), with focal adhesion proteins.

    PubMed

    Freeman, N L; Zurawski, D V; Chowrashi, P; Ayoob, J C; Huang, L; Mittal, B; Sanger, J M; Sanger, J W

    2000-12-01

    When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning regions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody staining of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit a number of focal adhesion proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli. PMID:11093251

  7. Enhancement of cell surface expression and receptor functions of membrane progestin receptor α (mPRα) by progesterone receptor membrane component 1 (PGRMC1): evidence for a role of PGRMC1 as an adaptor protein for steroid receptors.

    PubMed

    Thomas, Peter; Pang, Yefei; Dong, Jing

    2014-03-01

    A variety of functions have been proposed for progesterone receptor membrane component 1 (PGRMC1), including acting as a component of a membrane progestin receptor and as an adaptor protein. Here we show that stable overexpression of human PGRMC1 in nuclear progesterone receptor (PR)-negative breast cancer cell lines causes increased expression of PGRMC1 and membrane progesterone receptor α (mPRα) on cell membranes that is associated with increased specific [(3)H]progesterone binding. The membrane progestin binding affinity and specificity were characteristic of mPRα, with a Kd of 4.7 nM and high affinity for the mPR-specific agonist, Org OD 02-0, and low affinity for corticosteroids. Progestin treatment caused activation of G proteins, further evidence for increased expression of functional mPRs on PGRMC1-transfected cell membranes. Immunocytochemical and coimmunoprecipitation studies showed a close association of PGRMC1 with mPRα in cell membranes. Transfection of PGRMC1 into spontaneously immortalized rat granulosa cells was associated with membrane expression of PGRMC1 and mPRα as well as antiapoptotic effects of progestins that were abolished after cotransfection with small interfering RNA for mPRα. These data demonstrate that PGRMC1 can act as an adaptor protein, transporting mPRα to the cell surface, and that the progestin binding and apoptotic functions previously ascribed to PGRMC1 are dependent on cell surface expression of mPRα. Collectively, the results suggest PGRMC1 and mPRα are components of a membrane progesterone receptor protein complex. Increased expression of estrogen receptor β was also observed in the membranes of PGRMC1-transfected cells, suggesting that PGRMC1 can act as an adaptor protein for multiple classes of steroid receptors. PMID:24424068

  8. Adaptors in toll-like receptor signaling and their potential as therapeutic targets.

    PubMed

    Ve, Thomas; Gay, Nicholas J; Mansell, Ashley; Kobe, Bostjan; Kellie, Stuart

    2012-10-01

    To initiate the innate immune response, Toll-like receptors (TLRs) associate with cytoplasmic adaptor proteins through TIR (Toll/interleukin-1 receptor) domain interactions. The four principal signaling adaptor proteins include MyD88, MAL, TRIF and TRAM, and the fifth protein SARM, involved in negative regulation of TLR pathways, is usually considered a part of the TIR domain-containing adaptor protein group. Other TIR domain-containing proteins have also been shown to regulate these signaling pathways, including ST2 and SIGIRR, as well as several bacterial and viral TIR domain-containing proteins that modulate these pathways as virulence factors. TLR pathways and the adaptor proteins are associated with a number of diseases, including infection, sepsis, inflammatory, allergic and autoimmune diseases and cancer. We review our current understanding of the structure and function of adaptor proteins and their regulatory proteins, their association with disease and their potential as therapeutic targets in human disease. PMID:22664090

  9. Phospholipase Cgamma2 dosage is critical for B cell development in the absence of adaptor protein BLNK.

    PubMed

    Xu, Shengli; Huo, Jianxin; Chew, Weng-Keong; Hikida, Masaki; Kurosaki, Tomohiro; Lam, Kong-Peng

    2006-04-15

    B cell linker (BLNK) protein and phospholipase Cgamma2 (PLCgamma2) are components of the BCR signalosome that activate calcium signaling in B cells. Mice lacking either molecule have a severe but incomplete block in B lymphopoiesis. In this study, we generated BLNK-/- PLCgamma2-/- mice to examine the effect of simultaneous disruption of both molecules on B cell development. We showed that BLNK-/- PLCgamma2-/- mice had compounded defects in B cell maturation compared with either single mutant, suggesting that these two molecules cooperatively or synergistically signaled B lymphopoiesis. However, Ig H chain allelic exclusion was maintained in single and double mutants, indicating that signals propagated by BLNK and PLCgamma2 were not involved in this process. Interestingly, in the absence of BLNK, B cell development was dependent on plcgamma2 gene dosage. This was evidenced by the proportionate decrease in splenic B cell population and increase in bone marrow surface pre-BCR+ cells in PLCgamma2-diploid, -haploid, and -null animals. Intracellular calcium signaling and ERK activation in response to BCR engagement were also proportionately decreased and delayed, respectively, with stepwise reduction of plcgamma2 dosage in a BLNK(null) background. Thus, these data indicate the importance of BLNK not only as a conduit to specifically channel BCR-signaling pathways and as a scaffold for the assembling of macromolecular complex, but also as an efficient aggregator or concentrator of PLCgamma2 molecules to effect optimal signaling for B cell generation and activation. PMID:16585562

  10. 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. PMID:23510778

  11. REGULATION OF PROCESS RETRACTION AND CELL MIGRATION BY EPHA3 IS MEDIATED BY THE ADAPTOR PROTEIN NCK1†

    PubMed Central

    Hu, Tianjing; Shi, Guanfang; Larose, Louise; Rivera, Gonzalo M.; Mayer, Bruce J.; Zhou, Renping

    2009-01-01

    The Eph family of tyrosine kinase receptors and their ligands, the ephrins, participates in the regulation of a wide variety of biological functions under normal and pathological conditions. During embryonic development, interactions between the ligands and receptors define tissue boundaries, guide migrating axons, and regulate angiogenesis, as well as bone morphogenesis. These molecules have also been shown to modify neural activity in the adult nervous system, and influence tumor progression. However, the molecular mechanisms underlying these diverse functions are not completely understood. In the present study, a yeast two-hybrid screen has been conducted to identify molecules that physically interact with Eph receptors using the cytoplasmic domain of EphA3 as “bait”. This study identified Nck1 as a strong binding partner of EphA3 as assayed using both GST-fusion protein pull down and co-immunoprecipitation techniques. The interaction is mediated through binding of the Nck1 SH2 domain to the phosphotyrosine residue at position 602 (Y602) of EphA3 receptor. The removal of the SH2 domain or the mutation of the Y602 residue abolishes the interaction. It is further demonstrated that EphA3 activation inhibits cell migration and process outgrowth, and these inhibiting effects are partially alleviated by dominant-negative Nck1 mutants that lack functional SH2 or SH3 domains, but not by the wild type Nck1 gene. These results suggest that Nck1 interacts with EphA3 to regulate cell migration and process retraction. PMID:19505147

  12. Reduced photoreceptor death and improved retinal function during retinal degeneration in mice lacking innate immunity adaptor protein MyD88

    PubMed Central

    Syeda, Sarah; Patel, Amit K.; Lee, Tinthu; Hackam, Abigail S.

    2015-01-01

    The injury inflammatory response mediated by the innate immune system is an important contributor to neurodegeneration in the central nervous system (CNS) and retina. A major branch of the innate immune system is regulated by the Toll-like receptors (TLRs), which are receptors for endogenous damage associated molecules released from injured cells as well as pathogen-derived molecules, and interleukin-1 receptors (IL-1R), which are activated by IL-1α, IL-1β and IL-18 cytokines. TLRs and IL-1R are expressed on immune and non-immune cell types and act as first responders to cell damage, which results in tissue repair, or inflammation and apoptosis. Both TLR and IL-1R require the adaptor protein myeloid differentiation primary response gene 88 (MyD88) for signaling. Although inflammation is implicated in neuronal death in the retina, the role of MyD88-dependent TLR and IL-1R signaling in retinal degeneration is unknown. Therefore, the purpose of this study was to investigate the role of MyD88-mediated signaling in neuronal degeneration in the retinal degeneration 1 (rd1) mouse model, which exhibits a phenotype of rapid photoreceptor death and inflammation. To generate rd1 mice lacking the MyD88 gene, rd1 were bred with MyD88 knockout mice (MyD88-/-) for several generations to produce rd1/MyD88+/+ and rd1/MyD88-/- genotypes. Chemokine mRNA expression levels were analyzed by qRT-PCR, and recruitment of activated microglia was quantified by immunodetection of the IBA-1 protein. Retinal outer nuclear layer cell counts were performed to quantify photoreceptor degeneration, and retinal function was assessed using electroretinograms (ERG). Our results revealed that retinal expression of Ccl2, Ccl4, Ccl7 and Cxcl10 was reduced by 2 to 8-fold in rd1/MyD88-/- mice compared with rd1/MyD88+/+ mice (p<0.05), which coincided with attenuated microglial activation, higher numbers of photoreceptors and higher retina responses to photopic and scotopic stimuli. At later ages, rd1/MyD88

  13. Allosteric Coupling in the Bacterial Adhesive Protein FimH*

    PubMed Central

    Rodriguez, Victoria B.; Kidd, Brian A.; Interlandi, Gianluca; Tchesnokova, Veronika; Sokurenko, Evgeni V.; Thomas, Wendy E.

    2013-01-01

    The protein FimH is expressed by the majority of commensal and uropathogenic strains of Escherichia coli on the tips of type 1 fimbriae and mediates adhesion via a catch bond to its ligand mannose. Crystal structures of FimH show an allosteric conformational change, but it remains unclear whether all of the observed structural differences are part of the allosteric mechanism. Here we use the protein structural analysis tool RosettaDesign combined with human insight to identify and synthesize 10 mutations in four regions that we predicted would stabilize one of the conformations of that region. The function of each variant was characterized by measuring binding to the ligand mannose, whereas the allosteric state was determined using a conformation-specific monoclonal antibody. These studies demonstrated that each region investigated was indeed part of the FimH allosteric mechanism. However, the studies strongly suggested that some regions were more tightly coupled to mannose binding and others to antibody binding. In addition, we identified many FimH variants that appear locked in the low affinity state. Knowledge of regulatory sites outside the active and effector sites as well as the ability to make FimH variants locked in the low affinity state may be crucial to the future development of novel antiadhesive and antimicrobial therapies using allosteric regulation to inhibit FimH. PMID:23821547

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

    PubMed Central

    Lundmark, Richard; Carlsson, Sven R

    2002-01-01

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

  15. 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. PMID:25331956

  16. 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.'

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

    PubMed

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

    2015-09-15

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

  18. Biomimetic soy protein nanocomposites with calcium carbonate crystalline arrays for use as wood adhesive.

    PubMed

    Liu, Dagang; Chen, Huihuang; Chang, Peter R; Wu, Qinglin; Li, Kaifu; Guan, Litao

    2010-08-01

    Despite the biodegradability, non-toxicity, and renewability, commercially available soy protein-based adhesives still have not been widely adopted by industry, partially due to their disappointing performances, i.e., low glue strength in the dry state and no glue strength in the wet state. In this study, biomimetic soy protein/CaCO(3) hybrid wood glue was devised and an attempt made to improve the adhesion strength. The structure and morphology of the adhesive and its fracture bonding interface and adhesion strength were investigated. Results showed that the compact rivets or interlocking links, and ion crosslinking of calcium, carbonate, hydroxyl ions in the adhesive greatly improving the water-resistance and bonding strength of soy protein adhesives. Glue strength of soy protein hybrid adhesive was higher than 6 MPa even after three water-immersion cycles. This green and sustainable proteinous hybrid adhesive, with high glue strength and good water-resistance, is a good substitute for formaldehyde wood glues. PMID:20307978

  19. p38 mitogen-activated protein kinase interacts with vinculin at focal adhesions during fatty acid-stimulated cell adhesion

    PubMed Central

    George, Margaret D.; Wine, Robert N.; Lackford, Brad; Kissling, Grace E.; Akiyama, Steven K.; Olden, Kenneth; Roberts, John D.

    2014-01-01

    Arachidonic acid stimulates cell adhesion by activating α2β1 integrins in a process that depends on protein kinases, including p38 mitogen activated protein kinase. Here, we describe the interaction of cytoskeletal components with key signaling molecules that contribute to spreading of, and morphological changes in, arachidonic acid-treated MDA-MB-435 human breast carcinoma cells. Arachidonic acid-treated cells showed increased attachment and spreading on collagen type IV as measured by electric cell-substrate impedance sensing. Fatty acid-treated cells displayed short cortical actin filaments associated with an increased number of β1 integrin-containing pseudopodia whereas untreated cells displayed elongated stress fibers and fewer clusters of β1 integrins. Confocal microscopy of arachidonic acid-treated cells showed that vinculin and phospho-p38 both appeared enriched in pseudopodia and at the tips of actin filaments, and fluorescence ratio imaging indicated the increase was specific for the phospho-(active) form of p38. Immunoprecipitates of phospho-p38 from extracts of arachidonic acid-treated cells contained vinculin, and GST-vinculin fusion proteins carrying the central region of vinculin bound phospho-p38, whereas fusion proteins expressing the terminal portions of vinculin did not. These data suggest that phospho-p38 associates with particular domains on critical focal adhesion proteins that are involved in tumor cell adhesion and spreading and that this association can be regulated by factors in the tumor microenvironment. PMID:24219282

  20. Focal adhesion linker proteins expression of fibroblast related to adhesion in response to different transmucosal abutment surfaces

    PubMed Central

    Moon, Yeon-Hee; Yoon, Mi-Kyeong; Moon, Jung-Sun; Kang, Jee-Hae; Kim, Sun-Hun; Yang, Hong-Seo

    2013-01-01

    PURPOSE To evaluate adherence of human gingival fibroblasts (HGFs) to transmucosal abutment of dental implant with different surface conditions with time and to investigate the roles of focal adhesion linker proteins (FALPs) involved in HGFs adhesion to abutment surfaces. MATERIALS AND METHODS Morphologies of cultured HGFs on titanium and ceramic discs with different surface were observed by scanning electron microscopy. Biocompatibility and focal adhesion were evaluated by ultrasonic wave application and cell viability assay. FALPs expression levels were assessed by RT-PCR and western blot. RESULTS There seemed to be little difference in biocompatibility and adhesion strength of HGFs depending on the surface conditions and materials. In all experimental groups, the number of cells remaining on the disc surface after ultrasonic wave application increased more than 2 times at 3 days after seeding compared to 1-day cultured cells and this continued until 7 days of culture. FALPs expression levels, especially of vinculin and paxillin, also increased in 5-day cultured cells compared to 1-day cultured fibroblasts on the disc surface. CONCLUSION These results might suggest that the strength of adhesion of fibroblasts to transmucosal abutment surfaces increases with time and it seemed to be related to expressions of FALPs. PMID:24049577

  1. Protein Recovery from Secondary Paper Sludge and Its Potential Use as Wood Adhesive

    NASA Astrophysics Data System (ADS)

    Pervaiz, Muhammad

    Secondary sludge is an essential part of biosolids produced through the waste treatment plant of paper mills. Globally paper mills generate around 3.0 million ton of biosolids and in the absence of beneficial applications, the handling and disposal of this residual biomass poses a serious environmental and economic proposition. Secondary paper sludges were investigated in this work for recovery of proteins and their use as wood adhesive. After identifying extracellular polymeric substances as adhesion pre-cursors through analytical techniques, studies were carried out to optimize protein recovery from SS and its comprehensive characterization. A modified physicochemical protocol was developed to recover protein from secondary sludge in substantial quantities. The combined effect of French press and sonication techniques followed by alkali treatment resulted in significant improvement of 44% in the yield of solubilized protein compared to chemical methods. The characterization studies confirmed the presence of common amino acids in recovered sludge protein in significant quantities and heavy metal concentration was reduced after recovery process. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed the presence of both low and high molecular weight protein fractions in recovered sludge protein. After establishing the proof-of-concept in the use of recovered sludge protein as wood adhesive, the bonding mechanism of protein adhesives with cellulose substrate was further elucidated in a complementary protein-modification study involving soy protein isolate and its glycinin fractions. The results of this study validated the prevailing bonding theories by proving that surface wetting, protein structure, and type of wood play important role in determining final adhesive strength. Recovered sludge protein was also investigated for its compatibility to formulate hybrid adhesive blends with formaldehyde and bio-based polymers. Apart from chemical

  2. Molecular architecture of a complex between an adhesion protein from the malaria parasite and intracellular adhesion molecule 1.

    PubMed

    Brown, Alan; Turner, Louise; Christoffersen, Stig; Andrews, Katrina A; Szestak, Tadge; Zhao, Yuguang; Larsen, Sine; Craig, Alister G; Higgins, Matthew K

    2013-02-22

    The adhesion of Plasmodium falciparum-infected erythrocytes to human tissues or endothelium is central to the pathology caused by the parasite during malaria. It contributes to the avoidance of parasite clearance by the spleen and to the specific pathologies of cerebral and placental malaria. The PfEMP1 family of adhesive proteins is responsible for this sequestration by mediating interactions with diverse human ligands. In addition, as the primary targets of acquired, protective immunity, the PfEMP1s are potential vaccine candidates. PfEMP1s contain large extracellular ectodomains made from CIDR (cysteine-rich interdomain regions) and DBL (Duffy-binding-like) domains and show extensive variation in sequence, size, and domain organization. Here we use biophysical methods to characterize the entire ∼300-kDa ectodomain from IT4VAR13, a protein that interacts with the host receptor, intercellular adhesion molecule-1 (ICAM-1). We show through small angle x-ray scattering that IT4VAR13 is rigid, elongated, and monomeric. We also show that it interacts with ICAM-1 through the DBLβ domain alone, forming a 1:1 complex. These studies provide a first low resolution structural view of a PfEMP1 ectodomain in complex with its ligand. They show that it combines a modular domain arrangement consisting of individual ligand binding domains, with a defined higher order architecture that exposes the ICAM-1 binding surface to allow adhesion. PMID:23297413

  3. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review.

    PubMed

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-02-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  4. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

    PubMed Central

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-01-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  5. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths

    NASA Astrophysics Data System (ADS)

    Thormann, Esben; MizunoPresent Address: Nihon L'Oreal, Research; Innovation Center, 3-2-1 Sakado, Takatsu, Kawasaki, Kanagawa, Japan., Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M. Soledad; Arias, José Luis; Rutland, Mark W.; PaiPresent Address: CenterFunctional Nanomaterials, Brookhaven National Laboratory, 735 Brookhaven Avenue, Upton, New York 11973., Ranjith Krishna; Bergström, Lennart

    2012-06-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude.The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO3. The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30536d

  6. Specific Adhesion of Lipid Membranes Can Simultaneously Produce Two Types of Lipid and Protein Heterogeneities

    NASA Astrophysics Data System (ADS)

    Shindell, Orrin; Micah, Natalie; Ritzer, Max; Gordon, Vernita

    2015-03-01

    Living cells adhere to one another and their environment. Adhesion is associated with re-organization of the lipid and protein components of the cell membrane. The resulting heterogeneities are functional structures involved in biological processes. We use artificial lipid membranes that contain a single type of binding protein. Before adhesion, the lipid, protein, and dye components in the membrane are well-mixed and constitute a single disordered-liquid phase (Ld) . After adhesion, two distinct types of heterogeneities coexist in the adhesion zone: a central domain of ordered lipid phase that excludes both binding proteins and membrane dye, and a peripheral domain of disordered lipid phase that is densely packed with adhesion proteins and enriched in membrane dye relative to the non-adhered portion of the vesicle. Thus, we show that adhesion that is mediated by only one type of protein can organize the lipid and protein components of the membranes into heterogeneities that resemble those found in biology, for example the immune synapse.

  7. Cloning and expression of recombinant adhesive protein MEFP-2 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-02-07

    The present invention includes a Mytilus edulis cDNA having a nucleotide sequence that encodes for the Mytilus edulis foot protein-2 (Mefp-2), an example of a mollusk foot protein. Mefp-2 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-2 gene will allow researchers to produce Mefp-2 protein using genetic engineering techniques. The discovery of Mefp-2 gene sequences will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  8. Cloning and expression of recombinant adhesive protein Mefp-1 of the blue mussel, Mytilus edulis

    DOEpatents

    Silverman, Heather G.; Roberto, Francisco F.

    2006-01-17

    The present invention comprises a Mytilus edulis cDNA sequenc having a nucleotide sequence that encodes for the Mytilus edulis foot protein-1 (Mefp-1), an example of a mollusk foot protein. Mefp-1 is an integral component of the blue mussels' adhesive protein complex, which allows the mussel to attach to objects underwater. The isolation, purification and sequencing of the Mefp-1 gene will allow researchers to produce Mefp-1 protein using genetic engineering techniques. The discovery of Mefp-1 gene sequence will also allow scientists to better understand how the blue mussel creates its waterproof adhesive protein complex.

  9. Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

    SciTech Connect

    Baeyens, Nicolas; Latrache, Iman; Yerna, Xavier; Noppe, Gauthier; Horman, Sandrine; Morel, Nicole

    2013-11-22

    Highlights: •The three ERM proteins are expressed in vascular smooth muscle cell. •ERM depletion inhibited PDGF-evoked migration redundantly. •ERM depletion increased cell adhesion redundantly. •ERM depletion did not affect PDGF-evoked Ca signal, Rac1 activation, proliferation. •ERM proteins control PDGF-induced migration by regulating adhesion. -- Abstract: Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca{sup 2+} signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

  10. Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

    PubMed

    Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

    2013-12-14

    Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. PMID:23890721

  11. Adhesion of Mussel Foot Protein Mefp-5 to Mica: An Underwater Superglue†

    PubMed Central

    Danner, Eric W.; Kan, Yajing; Hammer, Malte U.; Israelachvili, Jacob N.; Waite, J. Herbert

    2012-01-01

    Mussels have a remarkable ability to attach their holdfast, or byssus, opportunistically to a variety of substrata that are wet, saline, corroded, and/or fouled by biofilms. Mytilus edulis foot protein-5 (Mefp-5) is one of several proteins in the byssal adhesive plaque of the mussel M. edulis. The high content of 3,4 dihydroxyphenylalanine (Dopa) (~30 mol%) and its localization near the plaque-substrate interface have often prompted speculation that Mefp-5 plays a key role in adhesion. Using the surface forces apparatus, we show that on mica surfaces Mefp-5 achieves an adhesion energy approaching Ead = ~− 14 mJ/m2. This exceeds the adhesion energy of another interfacial protein, Mefp-3, by a factor of 4–5 and is greater than the adhesion between highly oriented monolayers of biotin and streptavidin. The adhesion to mica is notable for its dependence on Dopa, which is most stable under reducing conditions and acidic pH. Mefp-5 also exhibits strong protein-protein interactions with itself as well as with Mefp-3 from M. edulis. PMID:22873939

  12. Effect of adhesion proteins and surface chemistry on the procoagulant state of adherent platelets

    NASA Astrophysics Data System (ADS)

    Grunkemeier, John Mark

    Poor hemocompatibility of a blood contacting device can lead to blood clotting, reduced blood flow, and depletion of platelets from the blood. Improved understanding of the processes by which blood-material contact leads to these responses could result in more hemocompatible materials. Platelets accelerate blood clotting by adhesion, aggregation, secretion of proteins and agonists and acceleration of thrombin generation. Platelets are said to be "procoagulant" after phosphatidylserine residues flip from the cytosolic to the extracellular face of the lipid bilayer. This then allows for the assembly of the prothrombinase complex (Xa, Va and calcium) on the platelet membrane, which can rapidly convert prothrombin to thrombin. In this study, three different methods confirmed that adhesion causes platelets to become procoagulant: shortening of clotting times of recalcified plasma, binding of FITC-annexin V, and generation of thrombin in the presence of Va, Xa and prothrombin by adherent platelets. Adherent platelets were 10--23 times more activated than bulk phase unactivated platelets and 10--24 times less activated than bulk phase platelets activated by calcium ionophore. The role of adsorbed fibrinogen, vWF, mixtures of fibrinogen and vWF, fibronectin, whole and dilute plasma, and plasma deficient in adhesion proteins in stimulating platelet procoagulant activity was investigated. The results of these experiments suggested that adhesion proteins affect procoagulant activation to varying degrees and that surfaces preadsorbed with mixtures of adhesion proteins are more activating that surfaces preadsorbed with single adhesion proteins. The hypothesis that materials that affect tightness of binding of adsorbed adhesion proteins affect platelet procoagulant activity was investigated. These studies showed that increasing fluorine content of RFGD polymerized films caused reduced platelet adhesion, but increased procoagulant activity, possibly due to their ability to adsorb

  13. Strong underwater adhesives made by self-assembling multi-protein nanofibres

    NASA Astrophysics Data System (ADS)

    Zhong, Chao; Gurry, Thomas; Cheng, Allen A.; Downey, Jordan; Deng, Zhengtao; Stultz, Collin M.; Lu, Timothy K.

    2014-10-01

    Many natural underwater adhesives harness hierarchically assembled amyloid nanostructures to achieve strong and robust interfacial adhesion under dynamic and turbulent environments. Despite recent advances, our understanding of the molecular design, self-assembly and structure-function relationships of these natural amyloid fibres remains limited. Thus, designing biomimetic amyloid-based adhesives remains challenging. Here, we report strong and multi-functional underwater adhesives obtained from fusing mussel foot proteins (Mfps) of Mytilus galloprovincialis with CsgA proteins, the major subunit of Escherichia coli amyloid curli fibres. These hybrid molecular materials hierarchically self-assemble into higher-order structures, in which, according to molecular dynamics simulations, disordered adhesive Mfp domains are exposed on the exterior of amyloid cores formed by CsgA. Our fibres have an underwater adhesion energy approaching 20.9 mJ m-2, which is 1.5 times greater than the maximum of bio-inspired and bio-derived protein-based underwater adhesives reported thus far. Moreover, they outperform Mfps or curli fibres taken on their own and exhibit better tolerance to auto-oxidation than Mfps at pH ≥ 7.0.

  14. Adhesive Proteins of Stalked and Acorn Barnacles Display Homology with Low Sequence Similarities

    PubMed Central

    Jonker, Jaimie-Leigh; Abram, Florence; Pires, Elisabete; Varela Coelho, Ana; Grunwald, Ingo; Power, Anne Marie

    2014-01-01

    Barnacle adhesion underwater is an important phenomenon to understand for the prevention of biofouling and potential biotechnological innovations, yet so far, identifying what makes barnacle glue proteins ‘sticky’ has proved elusive. Examination of a broad range of species within the barnacles may be instructive to identify conserved adhesive domains. We add to extensive information from the acorn barnacles (order Sessilia) by providing the first protein analysis of a stalked barnacle adhesive, Lepas anatifera (order Lepadiformes). It was possible to separate the L. anatifera adhesive into at least 10 protein bands using SDS-PAGE. Intense bands were present at approximately 30, 70, 90 and 110 kilodaltons (kDa). Mass spectrometry for protein identification was followed by de novo sequencing which detected 52 peptides of 7–16 amino acids in length. None of the peptides matched published or unpublished transcriptome sequences, but some amino acid sequence similarity was apparent between L. anatifera and closely-related Dosima fascicularis. Antibodies against two acorn barnacle proteins (ab-cp-52k and ab-cp-68k) showed cross-reactivity in the adhesive glands of L. anatifera. We also analysed the similarity of adhesive proteins across several barnacle taxa, including Pollicipes pollicipes (a stalked barnacle in the order Scalpelliformes). Sequence alignment of published expressed sequence tags clearly indicated that P. pollicipes possesses homologues for the 19 kDa and 100 kDa proteins in acorn barnacles. Homology aside, sequence similarity in amino acid and gene sequences tended to decline as taxonomic distance increased, with minimum similarities of 18–26%, depending on the gene. The results indicate that some adhesive proteins (e.g. 100 kDa) are more conserved within barnacles than others (20 kDa). PMID:25295513

  15. Embedded proteins and sacrificial bonds provide the strong adhesive properties of gastroliths.

    PubMed

    Thormann, Esben; Mizuno, Hiroyasu; Jansson, Kjell; Hedin, Niklas; Fernández, M Soledad; Arias, José Luis; Rutland, Mark W; Pai, Ranjith Krishna; Bergström, Lennart

    2012-07-01

    The adhesive properties of gastroliths from a freshwater crayfish (Cherax quadricarinatus) were quantified by colloidal probe atomic force microscopy (AFM) between heavily demineralized gastrolith microparticles and gastrolith substrates of different composition. Combined AFM and transmission electron microscopy studies demonstrated that the sequential detachment and large adhesion energies that characterise the adhesive behaviour of a native gastrolith substrate are dominated by sacrificial bonds between chitin fibres and between chitin fibres and CaCO(3). The sacrificial bonds were shown to be strongly related to the gastrolith proteins and when the majority of these proteins were removed by ethylenediaminetetraacetic acid (EDTA), the sequential detachment disappeared and the adhesive energy was reduced by more than two orders of magnitude. PMID:22653376

  16. Adhesion mechanism in a DOPA-deficient foot protein from green mussels†

    PubMed Central

    Hwang, Dong Soo; Zeng, Hongbo; Lu, Qingye; Israelachvili, Jacob; Waite, J. Herbert

    2012-01-01

    The holdfast or byssus of Asian green mussels, Perna viridis, contains a foot protein, pvfp-1, that differs in two respects from all other known adhesive mussel foot proteins (mfp): (1) instead of the hallmark L-3,4-dihydroxyphenylalanine (DOPA) residues in mfp-1, for example, pvfp-1 contains C2-mannosyl-7-hydroxytryptophan (Man7OHTrp). (2) In addition, pvfp-1 chains are not monomeric like mfp-1 but trimerized by collagen and coiled-coil domains near the carboxy terminus after a typical domain of tandemly repeated decapeptides. Here, the contribution of these peculiarities to adhesion was examined using a surface forces apparatus (SFA). Unlike previously studied mfp-1s, pvfp-1 showed significant adhesion to mica and, in symmetric pvfp-1 films, substantial cohesive interactions were present at pH 5.5. The role of Man7OHTrp in adhesion is not clear, and a DOPA-like role for Man7OHTrp in metal complexation (e.g., Cu2+, Fe3+) was not observed. Instead, cation–π interactions with low desolvation penalty between Man7OHTrp and lysyl side chains and conformational changes (raveling and unraveling of collagen helix and coiled-coil domains) are the best explanations for the strong adhesion between pvfp-1 monomolecular films. The strong adhesion mechanism induced by cation–π interactions and conformational changes in pvfp-1 provides new insights for the development of biomimetic underwater adhesives. PMID:23105946

  17. A 130-kDa Protein 4.1B Regulates Cell Adhesion, Spreading, and Migration of Mouse Embryo Fibroblasts by Influencing Actin Cytoskeleton Organization*

    PubMed Central

    Wang, Jie; Song, Jinlei; An, Chao; Dong, Wenji; Zhang, Jingxin; Yin, Changcheng; Hale, John; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2014-01-01

    Protein 4.1B is a member of protein 4.1 family, adaptor proteins at the interface of membranes and the cytoskeleton. It is expressed in most mammalian tissues and is known to be required in formation of nervous and cardiac systems; it is also a tumor suppressor with a role in metastasis. Here, we explore functions of 4.1B using primary mouse embryonic fibroblasts (MEF) derived from wild type and 4.1B knock-out mice. MEF cells express two 4.1B isoforms: 130 and 60-kDa. 130-kDa 4.1B was absent from 4.1B knock-out MEF cells, but 60-kDa 4.1B remained, suggesting incomplete knock-out. Although the 130-kDa isoform was predominantly located at the plasma membrane, the 60-kDa isoform was enriched in nuclei. 130-kDa-deficient 4.1B MEF cells exhibited impaired cell adhesion, spreading, and migration; they also failed to form actin stress fibers. Impaired cell spreading and stress fiber formation were rescued by re-expression of the 130-kDa 4.1B but not the 60-kDa 4.1B. Our findings document novel, isoform-selective roles for 130-kDa 4.1B in adhesion, spreading, and migration of MEF cells by affecting actin organization, giving new insight into 4.1B functions in normal tissues as well as its role in cancer. PMID:24381168

  18. Anionic deep cavitands enable the adhesion of unmodified proteins at a membrane bilayer.

    PubMed

    Ghang, Yoo-Jin; Perez, Lizeth; Morgan, Melissa A; Si, Fang; Hamdy, Omar M; Beecher, Consuelo N; Larive, Cynthia K; Julian, Ryan R; Zhong, Wenwan; Cheng, Quan; Hooley, Richard J

    2014-12-28

    An anionic self-folding deep cavitand is capable of immobilizing unmodified proteins and enzymes at a supported lipid bilayer interface, providing a simple, soft bioreactive surface that allows enzymatic function under mild conditions. The adhesion is based on complementary charge interactions, and the hosts are capable of binding enzymes such as trypsin at the bilayer interface: the catalytic activity is retained upon adhesion, allowing selective reactions to be performed at the membrane surface. PMID:25366572

  19. The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

    PubMed Central

    Ophir, Michael J.; Liu, Beiyun C.

    2013-01-01

    The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

  20. Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts.

    PubMed Central

    Vaudaux, P E; François, P; Proctor, R A; McDevitt, D; Foster, T J; Albrecht, R M; Lew, D P; Wabers, H; Cooper, S L

    1995-01-01

    We used an ex vivo canine arteriovenous shunt model, previously developed to study plasma protein adsorption and thrombogenesis on polymeric biomaterials, to define the role of host proteins in promoting adhesion of Staphylococcus aureus. Either polyethylene or polyvinyl chloride tubings were exposed to canine blood for 5, 15, or 60 min at a flow rate of 300 ml/min and then were flushed in phosphate-buffered saline (PBS), cut into 1.5-cm segments, and stored at -70 degrees C. After thawing, each segment was preincubated in 0.5% albumin in PBS to prevent nonspecific staphylococcal attachment to surfaces that were not exposed to blood. Each segment was then incubated with 4 x 10(6) CFU of [3H]thymidine-labelled S. aureus per ml for 60 min at 37 degrees C in an in vitro adhesion assay. Two site-specific mutants of S. aureus were tested: one specifically defective in adhesion to surface-bound fibronectin (FnAd-def) and the other defective in adhesion to fibrinogen (FgAD-def) [corrected]. Compared with their respective parental strains, the FgAd-def, but not the FnAd-def, mutant of S. aureus showed a strong (> 80%) decrease in attachment to ex vivo tubings. The adhesion of each strain of S. aureus onto polyethylene was consistently more than twofold higher than the adhesion onto polyvinyl chloride segments exposed to flowing blood for 5 or 15 min, but adhesion became similar to that on polyvinyl chloride after 60 min of exposure. In conclusion, the specific adhesion-defective mutants of S. aureus suggested that fibrinogen was the most active adhesion-promoting protein in a short-term blood-material interaction. The experimental approach described in this study should prove useful for screening materials thought to be resistant to protein-mediated staphylococcal adhesion and colonization. PMID:7822026

  1. Cell adhesion-dependent inactivation of a soluble protein kinase during fertilization in Chlamydomonas.

    PubMed Central

    Zhang, Y; Luo, Y; Emmett, K; Snell, W J

    1996-01-01

    Within seconds after the flagella of mt+ and mt- Chlamydomonas gametes adhere during fertilization, their flagellar adenylyl cyclase is activated several fold and preparation for cell fusion is initiated. Our previous studies indicated that early events in this pathway, including control of adenylyl cyclase, are regulated by phosphorylation and dephosphorylation. Here, we describe a soluble, flagellar protein kinase activity that is regulated by flagellar adhesion. A 48-kDa, soluble flagellar protein was consistently phosphorylated in an in vitro assay in flagella isolated from nonadhering mt+ and mt- gametes, but not in flagella isolated from mt+ and mt- gametes that had been adhering for 1 min. Although the 48-kDa protein was present in the flagella isolated from adhering gametes, we demonstrate that its protein kinase was inactivated by flagellar adhesion. Immunoblot analysis and inhibitor studies indicate that the 48-kDa protein in nonadhering gametes is phosphorylated by a protein tyrosine kinase. In vivo experiments showing that the protein tyrosine phosphatase inhibitor sodium orthovanadate inhibits fertilization suggest that protein dephosphorylation may be required for signal transduction. The 48-kDa protein and its protein kinase may be among the first elements of a novel signalling pathway that couples interaction of flagellar adhesion molecules to gamete activation. Images PMID:8730096

  2. Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

    PubMed

    Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

    2015-01-01

    We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding. PMID:25351253

  3. Secreted Frizzled-related protein 1 (sFRP1) regulates spermatid adhesion in the testis via dephosphorylation of focal adhesion kinase and the nectin-3 adhesion protein complex

    PubMed Central

    Wong, Elissa W. P.; Lee, Will M.; Cheng, C. Yan

    2013-01-01

    Development of spermatozoa in adult mammalian testis during spermatogenesis involves extensive cell migration and differentiation. Spermatogonia that reside at the basal compartment of the seminiferous epithelium differentiate into more advanced germ cell types that migrate toward the apical compartment until elongated spermatids are released into the tubule lumen during spermiation. Apical ectoplasmic specialization (ES; a testis-specific anchoring junction) is the only cell junction that anchors and maintains the polarity of elongating/elongated spermatids (step 8–19 spermatids) in the epithelium. Little is known regarding the signaling pathways that trigger the disassembly of the apical ES at spermiation. Here, we show that secreted Frizzled-related protein 1 (sFRP1), a putative tumor suppressor gene that is frequently down-regulated in multiple carcinomas, is a crucial regulatory protein for spermiation. The expression of sFRP1 is tightly regulated in adult rat testis to control spermatid adhesion and sperm release at spermiation. Down-regulation of sFRP1 during testicular development was found to coincide with the onset of the first wave of spermiation at approximately age 45 d postpartum, implying that sFRP1 might be correlated with elongated spermatid adhesion conferred by the apical ES before spermiation. Indeed, administration of sFRP1 recombinant protein to the testis in vivo delayed spermiation, which was accompanied by down-regulation of phosphorylated (p)-focal adhesion kinase (FAK)-Tyr397 and retention of nectin-3 adhesion protein at the apical ES. To further investigate the functional relationship between p-FAK-Tyr397 and localization of nectin-3, we overexpressed sFRP1 using lentiviral vectors in the Sertoli-germ cell coculture system. Consistent with the in vivo findings, overexpression of sFRP1 induced down-regulation of p-FAK-Tyr397, leading to a decline in phosphorylation of nectin-3. In summary, this report highlights the critical role of s

  4. Single Adhesive Nanofibers from a Live Diatom Have the Signature Fingerprint of Modular Proteins

    PubMed Central

    Dugdale, T. M.; Dagastine, R.; Chiovitti, A.; Mulvaney, P.; Wetherbee, R.

    2005-01-01

    The adhesive and mechanical properties of a cell-substratum adhesive secreted by live diatom cells were examined in situ using atomic force microscopy. The resulting force curves have a regular saw-tooth pattern, the characteristic fingerprint of modular proteins, and when bridged between tip and surface can repeatedly be stretched and relaxed resulting in precisely overlaying saw-tooth curves (up to ∼600 successive cycles). The average rupture force of the peaks is 0.794 ± 0.007 (mean ± SE) nN at a loading rate of 0.8 μm/s and the average persistence length is 0.026 ± <0.001 (mean ± SE) nm (fit using the worm-like chain model). We propose that we are pulling on single adhesive nanofibers, each a cohesive unit composed of a set number of modular proteins aligned in register. Furthermore, we can observe and differentiate when up to three adhesive nanofibers are pulled based upon multimodal distributions of force and persistence length. The high force required for bond rupture, high extensibility (∼1.2 μm), and the accurate and rapid refolding upon relaxation, together provide strong and flexible properties ideally suited for the cell-substratum adhesion of this fouling diatom and allow us to understand the mechanism responsible for the strength of adhesion. PMID:16169972

  5. Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

    PubMed Central

    Koirala, Sajjan; Guo, Qian; Kalia, Raghav; Bui, Huyen T.; Eckert, Debra M.; Frost, Adam; Shaw, Janet M.

    2013-01-01

    Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity. PMID:23530241

  6. Modification of human platelet adhesion on biomaterial surfaces by protein preadsorption under static and flow conditions.

    PubMed

    Otto, Mike; Franzen, Arno; Hansen, Torsten; Kirkpatrick, Charles James

    2004-01-01

    Biomaterial-induced thrombosis remains one of the main complications of vascular implant devices. Preadsorbed proteins on the biomaterial/blood interface will modify the adhesion and activation of platelets (PTLs) during the initial contact-phase. Our results clearly show that PTL-adherence on biomaterials is influenced not only by protein preadsorption, but also by flow conditions. The covalent coating of TCPS and glass by phosphorylcholine (PC) induces a significant decrease of PTL adhesion but leads to a slight, but nevertheless significant activation of PTL, which was detected by the induction of P-selectin expression using FACS analysis. Methodologically, the visualization of PTL adhesion gave more reliable results for measurement of PTL adhesion than the cell-enzyme immunoassay (EIA) for P-selectin. Human citrated plasma caused an inhibition of PTL. It is probable, that the contained sodium citrate may inhibit PTL adhesion by its calcium ion-binding capacity. The flow experiment as dynamic system is in our view absolutely essential for the evaluation of biomaterials for vascular prosthesis, and is in accordance with the international standards. The results of the experiments also suggest that investigations under static and flow conditions are needed to determine the influence of protein adsorption on mixed blood cell populations, for example, on PTL and PMN mixtures/co-cultures in order to achieve a better simulation of the in vivo situation. PMID:15338589

  7. A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis

    PubMed Central

    Daley, William P.; Kohn, Joshua M.; Larsen, Melinda

    2011-01-01

    Cleft formation is the initial step of branching morphogenesis in many organs. We previously demonstrated that ROCK 1 regulates a non-muscle myosin II-dependent mechanochemical checkpoint to transition initiated clefts to progressing clefts in developing submandibular salivary glands. Here, we report that ROCK-mediated integrin activation and subsequent formation of focal adhesion complexes comprise this mechanochemical checkpoint. Inhibition of ROCK1 and non-muscle myosin II activity decreased integrin β1 activation in the cleft region and interfered with localization and activation of focal adhesion complex proteins, such as focal adhesion kinase (FAK). Inhibition of FAK activity also prevented cleft progression, by disrupting recruitment of the focal adhesion proteins talin and vinculin and subsequent fibronectin assembly in the cleft region while decreasing ERK1/2 activation. These results demonstrate that inside-out integrin signaling leading to a localized recruitment of active FAK-containing focal adhesion protein complexes generates a mechanochemical checkpoint that facilitates progression of branching morphogenesis. PMID:22016182

  8. Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

    PubMed Central

    Gerritsen, M. E.; Carley, W. W.; Ranges, G. E.; Shen, C. P.; Phan, S. A.; Ligon, G. F.; Perry, C. A.

    1995-01-01

    Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking. Images Figure 7 Figure 8 Figure 11 PMID:7543732

  9. Corneal Cell Adhesion to Contact Lens Hydrogel Materials Enhanced via Tear Film Protein Deposition

    PubMed Central

    Elkins, Claire M.; Qi, Qin M.; Fuller, Gerald G.

    2014-01-01

    Tear film protein deposition on contact lens hydrogels has been well characterized from the perspective of bacterial adhesion and viability. However, the effect of protein deposition on lens interactions with the corneal epithelium remains largely unexplored. The current study employs a live cell rheometer to quantify human corneal epithelial cell adhesion to soft contact lenses fouled with the tear film protein lysozyme. PureVision balafilcon A and AirOptix lotrafilcon B lenses were soaked for five days in either phosphate buffered saline (PBS), borate buffered saline (BBS), or Sensitive Eyes Plus Saline Solution (Sensitive Eyes), either pure or in the presence of lysozyme. Treated contact lenses were then contacted to a live monolayer of corneal epithelial cells for two hours, after which the contact lens was sheared laterally. The apparent cell monolayer relaxation modulus was then used to quantify the extent of cell adhesion to the contact lens surface. For both lens types, lysozyme increased corneal cell adhesion to the contact lens, with the apparent cell monolayer relaxation modulus increasing up to an order of magnitude in the presence of protein. The magnitude of this increase depended on the identity of the soaking solution: lenses soaked in borate-buffered solutions (BBS, Sensitive Eyes) exhibited a much greater increase in cell attachment upon protein addition than those soaked in PBS. Significantly, all measurements were conducted while subjecting the cells to moderate surface pressures and shear rates, similar to those experienced by corneal cells in vivo. PMID:25144576

  10. The role of serum proteins in Staphylococcus aureus adhesion to ethylene glycol coated surfaces.

    PubMed

    Schuster, Swen; Yu, Wenqi; Nega, Mulugeta; Chu, Ya-Yun; Zorn, Stefan; Zhang, Fajun; Götz, Friedrich; Schreiber, Frank

    2014-11-01

    Bacterial adhesion on implants is a first step in the development of chronic foreign body associated infections. Finding strategies to minimize bacterial adhesion may contribute to minimize such infections. It is known that surfaces with oligo-ethylene-glycol (EG3OMe) or poly-ethylene-glycol (PEG2k) terminations decrease unspecific protein adsorption and bacterial adhesion. However, little is known about the influence of serum and its components on bacterial adhesion. We therefore prepared two coatings on gold surface with HS-(CH2)11EG3OMe (EG3OMe) and PEG2k-thiol and studied the role of bovine serum albumin (BSA), γ-globulins, and serum on Staphylococcus aureus adhesion. While BSA and lysozyme showed no adherence even when applied at very high concentrations (100 mg/ml), γ-globulins adsorbed already from 10 mg/ml on. The adsorption of γ-globulins was, however, significantly decreased when it was mixed with BSA in a ratio of 3:1, as it is in the serum. Pretreatment of EG3OMe and PEG2k coatings with γ-globulins or serum strongly promoted adherence of S. aureus when resuspended in buffer, suggesting that γ-globulins play a pivotal role in promoting S. aureus adhesion by its IgG binding proteins; the finding that a spa-deletion mutant, lacking the IgG binding protein A, showed decreased adherence corroborated this. Similarly, when S. aureus was pretreated with serum or γ-globulins its adherence was also significantly decreased. Our findings show that particularly γ-globulins bind to the coated surfaces thus mediating adherence of S. aureus via its protein A. As pretreatment of S. aureus with serum or γ-globulins significantly decreased adherence, treatment of patients with γ-globulins before implant surgery might lower the risk of implant-associated infections. PMID:24980510

  11. Reduced bacterial adhesion to hydrocephalus shunt catheters mediated by cerebrospinal fluid proteins.

    PubMed Central

    Brydon, H L; Bayston, R; Hayward, R; Harkness, W

    1996-01-01

    BACKGROUND--Prosthetic infections are a major problem, requiring complex and lengthy management. The role of blood proteins in the pathogenesis of implant infection has been investigated, but research into the role of CSF protein in shunt infections has not been undertaken, even though a high CSF protein has been assumed to increase the risk of such infections. METHODS--New shunt catheters were exposed to either CSF or individual protein solutions, and the numbers of radiolabelled staphylococci that adhered to them were compared with controls that had been exposed to saline only. RESULTS--A significant reduction in bacteria adhering to the test catheter was found in each instance. Furthermore, the CSF with the highest protein content, from a patient with intraventricular haemorrhage, had the greatest inhibitory effect on bacterial adhesion. The effect of the solutions on the hydrophobicity of the silicone rubber was also investigated. The silicone rubber was more hydrophilic, and bacterial adhesion was less, with solutions containing a higher protein content, and these findings were in keeping with the current theories on the mechanism of bacterial adhesion to polymers. CONCLUSIONS--A high CSF protein content does not predispose to the development of shunt infections. PMID:8648336

  12. Low-cost Soybean Protein Products as Extenders in Plywood Adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean flour and meal were evaluated as alternate protein extenders in plywood adhesives. This research is part of our laboratory’s efforts to develop new uses for the proteinaceous co-products from soybean and cereal processing. Ground soybean meal was tested as replacement for wheat flour in glu...

  13. Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces

    PubMed Central

    Barnes, L.-M.; Lo, M. F.; Adams, M. R.; Chamberlain, A. H. L.

    1999-01-01

    Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon. PMID:10508087

  14. Low-Cost Soybean Protein Products as Extenders in Plywood Adhesives

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean flour and meal were evaluated as alternate protein extenders in plywood adhesives. This research is part of our laboratory’s efforts to develop new uses for the proteinaceous co-products from soybean and cereal processing. Ground soybean meal was tested as replacement for wheat flour in gl...

  15. Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

    PubMed Central

    Wu, Zeyu; Zhao, Gang; Peng, Lin; Du, Jialin; Wang, Sanming; Huang, Yijie; Ou, Jinrui; Jian, Zhixiang

    2013-01-01

    Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ) in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them. PMID:24039784

  16. Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

    PubMed Central

    Guo, Wei-hui

    2007-01-01

    Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell. PMID:17804814

  17. Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

    PubMed

    Zuchowska, Agnieszka; Kwiatkowski, Piotr; Jastrzebska, Elzbieta; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2016-02-01

    PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer). PMID:26311334

  18. Thrombolytic protein from cobra venom with anti-adhesive properties.

    PubMed

    Chanda, Chandrasekhar; Sarkar, Angshuman; Chakrabarty, Dibakar

    2016-01-15

    A metalloproteinase anticoagulant toxin of molecular weight 66 kDa has been purified from the venom of Indian monocled cobra (Naja kaouthia). This toxin named as NKV 66 cleaved fibrinogen in a dose and time dependent manner. The digestion process was specific to Aα chain and cleaved fibrinogen to peptide fragments. NKV 66 completely liquefied the fibrin clots developed in vitro in 18 h. Plasma recalcification time and thrombin time were significantly prolonged following treatment of plasma with NKV 66. NKV 66 significantly inhibited ADP and collagen induced platelet aggregation in a dose dependent manner. It showed disintegrin like activity on A549 cells cultured in vitro. About 40% inhibition of adherence of A549 cells to matrix was observed following NKV 66 treatment also NKV 66 treated A549 cells were drastically inhibited from passing through the matrix in cell invasion assays in vitro, suggesting anti-adhesive properties of NKV 66. PMID:26558696

  19. Immune adaptor ADAP in T cells regulates HIV-1 transcription and cell-cell viral spread via different co-receptors

    PubMed Central

    2013-01-01

    Background Immune cell adaptor protein ADAP (adhesion and degranulation-promoting adaptor protein) mediates aspects of T-cell adhesion and proliferation. Despite this, a connection between ADAP and infection by the HIV-1 (human immunodeficiency virus-1) has not been explored. Results In this paper, we show for the first time that ADAP and its binding to SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) regulate HIV-1 infection via two distinct mechanisms and co-receptors. siRNA down-regulation of ADAP, or expression of a mutant that is defective in associating to its binding partner SLP-76 (termed M12), inhibited the propagation of HIV-1 in T-cell lines and primary human T-cells. In one step, ADAP and its binding to SLP-76 were needed for the activation of NF-κB and its transcription of the HIV-1 long terminal repeat (LTR) in cooperation with ligation of co-receptor CD28, but not LFA-1. In a second step, the ADAP-SLP-76 module cooperated with LFA-1 to regulate conjugate formation between T-cells and dendritic cells or other T-cells as well as the development of the virological synapse (VS) and viral spread between immune cells. Conclusions These findings indicate that ADAP regulates two steps of HIV-1 infection cooperatively with two distinct receptors, and as such, serves as a new potential target in the blockade of HIV-1 infection. PMID:24047317

  20. Identification of proteins associated with adhesive prints from Holothuria dofleinii Cuvierian tubules.

    PubMed

    Peng, Yong Y; Glattauer, Veronica; Skewes, Timothy D; McDevitt, Andrew; Elvin, Christopher M; Werkmeister, Jerome A; Graham, Lloyd D; Ramshaw, John A M

    2014-12-01

    Cuvierian tubules are expelled as a defence mechanism against predators by various species within the family Holothuridae. When the tubules are expelled, they become sticky almost immediately and ensnare the predator. The mechanism of this rapid adhesion is not clear, but proteins on the surface of the expelled tubules are widely believed to be involved. This study has examined such proteins from Holothuria dofleinii, sourced from adhesive prints left on glass after the removal of adhered tubules. Gel electrophoresis showed that seven strongly staining protein bands were consistently present in all samples, with molecular masses ranging from 89 to 17 kDa. N-terminal sequence data was obtained from two bands, while others seemed blocked. Tandem mass spectrometry-based sequencing of tryptic peptides derived from individual protein bands indicated that the proteins were unlikely to be homopolymers. PCR primers designed using the peptide sequences enabled us to amplify, clone and sequence cDNA segments relating to four gel bands; for each, the predicted translation product contained other peptide sequences observed for that band that had not been used in primer design. Database searches using the peptide and cDNA-encoded sequences suggest that two of the seven proteins are novel and one is a C-type lectin, while-surprisingly-at least three of the other four are closely related to enzymes associated with the pentose phosphate cycle and glycolysis. We discuss precedents in which lectins and metabolic enzymes are involved in attachment and adhesion phenomena. PMID:25086572

  1. Bicaudal D Family of Motor Adaptors: Linking Dynein Motility to Cargo Binding.

    PubMed

    Hoogenraad, Casper C; Akhmanova, Anna

    2016-05-01

    Transport of different intracellular cargoes along cytoskeleton filaments is essential for the morphogenesis and function of a broad variety of eukaryotic cells. Intracellular transport is mediated by cytoskeletal motors including myosin, kinesin, and dynein, which are typically linked to various cargoes by adaptor proteins. Recent studies suggest that adaptor proteins can also act as essential transport cofactors, which control motor activity and coordination. Characterization of the evolutionary conserved Bicaudal D (BICD) family of dynein adaptor proteins has provided important insights into the fundamental mechanisms governing cargo trafficking. This review highlights the advances in the current understanding of how BICD adaptors regulate microtubule-based transport and how they contribute to developmental processes and human disease. PMID:26822037

  2. Heat shock protein 60 acts as a receptor for the Listeria adhesion protein in Caco-2 cells.

    PubMed

    Wampler, Jennifer L; Kim, Kwang-Pyo; Jaradat, Ziad; Bhunia, Arun K

    2004-02-01

    The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion. PMID:14742538

  3. A novel interaction between the SH2 domain of signaling adaptor protein Nck-1 and the upstream regulator of the Rho family GTPase Rac1 engulfment and cell motility 1 (ELMO1) promotes Rac1 activation and cell motility.

    PubMed

    Zhang, Guo; Chen, Xia; Qiu, Fanghua; Zhu, Fengxin; Lei, Wenjing; Nie, Jing

    2014-08-15

    Nck family proteins function as adaptors to couple tyrosine phosphorylation signals to actin cytoskeleton reorganization. Several lines of evidence indicate that Nck family proteins involve in regulating the activity of Rho family GTPases. In the present study, we characterized a novel interaction between Nck-1 with engulfment and cell motility 1 (ELMO1). GST pull-down and co-immunoprecipitation assay demonstrated that the Nck-1-ELMO1 interaction is mediated by the SH2 domain of Nck-1 and the phosphotyrosine residues at position 18, 216, 395, and 511 of ELMO1. A R308K mutant of Nck-1 (in which the SH2 domain was inactive), or a 4YF mutant of ELMO1 lacking these four phosphotyrosine residues, diminished Nck-1-ELMO1 interaction. Conversely, tyrosine phosphatase inhibitor treatment and overexpression of Src family kinase Hck significantly enhanced Nck-1-ELMO1 interaction. Moreover, wild type Nck-1, but not R308K mutant, significantly augmented the interaction between ELMO1 and constitutively active RhoG (RhoG(V12A)), thus promoted Rac1 activation and cell motility. Taken together, the present study characterized a novel Nck-1-ELMO1 interaction and defined a new role for Nck-1 in regulating Rac1 activity. PMID:24928514

  4. 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

  5. Rapidly light-activated surgical protein glue inspired by mussel adhesion and insect structural crosslinking.

    PubMed

    Jeon, Eun Young; Hwang, Byeong Hee; Yang, Yun Jung; Kim, Bum Jin; Choi, Bong-Hyuk; Jung, Gyu Yong; Cha, Hyung Joon

    2015-10-01

    Currently approved surgical tissue glues do not satisfy the requirements for ideal bioadhesives due to limited adhesion in wet conditions and severe cytotoxicity. Herein, we report a new light-activated, mussel protein-based bioadhesive (LAMBA) inspired by mussel adhesion and insect dityrosine crosslinking chemistry. LAMBA exhibited substantially stronger bulk wet tissue adhesion than commercially available fibrin glue and good biocompatibility in both in vitro and in vivo studies. Besides, the easily tunable, light-activated crosslinking enabled an effective on-demand wound closure and facilitated wound healing. Based on these outstanding properties, LAMBA holds great potential as an ideal surgical tissue glue for diverse medical applications, including sutureless wound closures of skin and internal organs. PMID:26197411

  6. Influence of Aae Autotransporter Protein on Adhesion and Biofilm Formation by Aggregatibacter actinomycetemcomitans.

    PubMed

    Nunes, Ana Carla Robatto; Longo, Priscila Larcher; Mayer, Marcia Pinto Alves

    2016-01-01

    The periodontopathogen Aggregatibacter actinomycetemcomitans colonizes oral cavity by binding to and invading epithelial cells as well as by participating in biofilms formed on hard surfaces. Aae, an autotransporter protein, is implicated in bacterial adhesion to epithelial cells. Due to the multiple functions of bacterial autotransporter proteins, this study aimed to evaluate the role of aae in A. actinomycetemcomitans ability to adhere to both saliva-coated hydroxyapatite (SHA) and biofilm. An aae null mutant was constructed. Its hydrophobic properties as well as its ability to adhere to epithelial cells, SHA and to form biofilm were evaluated and compared with the parental strain, A. actinomycetemcomitans VT1169. The aae null mutant showed reduced hydrophobicity, as well as decreased binding to SHA and biofilm formation compared to the parental strain. These data suggest that aae mediates A. actinomycetemcomitans adhesion to epithelial cells and may be involved in biofilm formation and interaction with adsorbed salivary proteins. PMID:27224556

  7. Mechanical and water soaking properties of medium density fiberboard with wood fiber and soybean protein adhesive.

    PubMed

    Li, Xin; Li, Yonghui; Zhong, Zhikai; Wang, Donghai; Ratto, Jo A; Sheng, Kuichuan; Sun, Xiuzhi Susan

    2009-07-01

    Soybean protein is a renewable and abundant material that offers an alternative to formaldehyde-based resins. In this study, soybean protein was modified with sodium dodecyl sulfate (SDS) as an adhesive for wood fiber medium density fiberboard (MDF) preparation. Second-order response surface regression models were used to study the effects and interactions of initial moisture content (IMC) of coated wood fiber, press time (PT) and temperature on mechanical and water soaking properties of MDF. Results showed that IMC of coated fiber was the dominant influencing factor. Mechanical and soaking properties improved as IMC increased and reached their highest point at an IMC of 35%. Press time and temperature also had a significant effect on mechanical and water soaking properties of MDF. Second-order regression results showed that there were strong relationships between mechanical and soaking properties of MDF and processing parameters. Properties of MDF made using soybean protein adhesive are similar to those of commercial board. PMID:19329303

  8. Adhesive strength and curing rate of marine mussel protein extracts on porcine small intestinal submucosa*

    PubMed Central

    Ninan, Lal; Stroshine, R L; Wilker, J.J.; Shi, Riyi

    2008-01-01

    An adhesive protein extracted from marine mussel (Mytilus edulis) was used to bond strips of connective tissue for the purpose of evaluating the use of curing agents to improve adhesive curing. Specifically, mussel adhesive protein solution (MAPS, 0.5 mM dihydroxyphenylalanine) was applied, with or without the curing agents, to the ends of two overlapping strips of porcine small intestinal submucosa. The bond strength of this lap joint was determined after curing for 1 h at room temperature (25°C). The strength of joints formed using only MAPS or with only the ethyl, butyl or octyl cyanoacrylate adhesives were determined. Although joints bonded using ethyl cyanoacrylate were strongest, those using MAPS were stronger than those using butyl and octyl cyanoacrylates. The addition of 25 mM solutions of the transition metal ions V5+, Fe3+ and Cr6+, which are all oxidants, increased the bond strength of the MAPS joints. The V5+ gave the strongest bonds and the Fe3+ the second strongest. In subsequent tests with V5+ and Fe3+ solutions, the bond strength increased with V5+ concentration, but it did not increase with Fe3+ concentration. Addition of 250 mM V5+ gave a very strong bond. PMID:17434815

  9. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    NASA Astrophysics Data System (ADS)

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-03-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments.

  10. Ubiquitous distribution of salts and proteins in spider glue enhances spider silk adhesion

    PubMed Central

    Amarpuri, Gaurav; Chaurasia, Vishal; Jain, Dharamdeep; Blackledge, Todd A.; Dhinojwala, Ali

    2015-01-01

    Modern orb-weaving spiders use micron-sized glue droplets on their viscid silk to retain prey in webs. A combination of low molecular weight salts and proteins makes the glue viscoelastic and humidity responsive in a way not easily achieved by synthetic adhesives. Optically, the glue droplet shows a heterogeneous structure, but the spatial arrangement of its chemical components is poorly understood. Here, we use optical and confocal Raman microscopy to show that salts and proteins are present ubiquitously throughout the droplet. The distribution of adhesive proteins in the peripheral region explains the superior prey capture performance of orb webs as it enables the entire surface area of the glue droplet to act as a site for prey capture. The presence of salts throughout the droplet explains the recent Solid-State NMR results that show salts directly facilitate protein mobility. Understanding the function of individual glue components and the role of the droplet's macro-structure can help in designing better synthetic adhesives for humid environments. PMID:25761668

  11. Mussel adhesive protein provides cohesive matrix for collagen type-1α

    PubMed Central

    Martinez Rodriguez, Nadine R.; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N.; Waite, J. Herbert

    2015-01-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant loadbearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m2) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  12. Mussel adhesive protein provides cohesive matrix for collagen type-1α.

    PubMed

    Martinez Rodriguez, Nadine R; Das, Saurabh; Kaufman, Yair; Wei, Wei; Israelachvili, Jacob N; Waite, J Herbert

    2015-05-01

    Understanding the interactions between collagen and adhesive mussel foot proteins (mfps) can lead to improved medical and dental adhesives, particularly for collagen-rich tissues. Here we investigated interactions between collagen type-1, the most abundant load-bearing animal protein, and mussel foot protein-3 (mfp-3) using a quartz crystal microbalance and surface forces apparatus (SFA). Both hydrophilic and hydrophobic variants of mfp-3 were exploited to probe the nature of the interaction between the protein and collagen. Our chief findings are: 1) mfp-3 is an effective chaperone for tropocollagen adsorption to TiO2 and mica surfaces; 2) at pH 3, collagen addition between two mfp-3 films (Wc = 5.4 ± 0.2 mJ/m(2)) increased their cohesion by nearly 35%; 3) oxidation of Dopa in mfp-3 by periodate did not abolish the adhesion between collagen and mfp-3 films, and 4) collagen bridging between both hydrophilic and hydrophobic mfp-3 variant films is equally robust, suggesting that hydrophobic interactions play a minor role. Extensive H-bonding, π-cation and electrostatic interactions are more plausible to explain the reversible bridging of mfp-3 films by collagen. PMID:25770997

  13. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum

    PubMed Central

    SUZUKI, Kenta; NISHIYAMA, Keita; MIYAJIMA, Hiroki; OSAWA, Ro; YAMAMOTO, Yuji; MUKAI, Takao

    2015-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10−8 M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  14. Adhesion properties of a putative polymorphic fimbrial subunit protein from Bifidobacterium longum subsp. longum.

    PubMed

    Suzuki, Kenta; Nishiyama, Keita; Miyajima, Hiroki; Osawa, Ro; Yamamoto, Yuji; Mukai, Takao

    2016-01-01

    In our previous study, we found that the open reading frame bl0675 in the genome of Bifidobacterium longum subsp. longum isolated from human feces encoded a novel putative fimbrial protein, was highly polymorphic, and had five variants (A, B, C, D, and E types). The aim of this study was to evaluate the affinity of these variants to porcine colonic mucins (PCMs). Protein-binding properties were examined using the recombinant BL0675 protein containing a C-terminal 6 × His tag (His-BL0675). Surface plasmon resonance analysis demonstrated that the His-BL0675 A type had strong affinity to PCMs (KD = 9.82 × 10(-8) M), whereas the B, C, D, and E types exhibited little or no binding. In a competitive enzyme-linked immunosorbent assay, His-BL0675 A type binding was reduced by addition of mucin oligosaccharides, suggesting that the binding occurs via carbohydrate chains of PCMs. The localization of BL0675 to the B. longum subsp. longum cell surface was confirmed by western blot analysis using A type polyclonal antibodies. Bacterial adhesion of B. longum subsp. longum to PCMs was also blocked by A type-specific antibodies; however, its adhesion properties were strain specific. Our results suggest that the BL0675 variants significantly contribute to the adhesion of B. longum subsp. longum strains. The expression and the adhesive properties of this protein are affected by genetic polymorphisms and are specific for B. longum subsp. longum strains. However, further studies are required on the properties of binding of these putative fimbrial proteins to the human gastrointestinal tract. PMID:26858927

  15. Loss of Apm1, the μ1 Subunit of the Clathrin-Associated Adaptor-Protein-1 Complex, Causes Distinct Phenotypes and Synthetic Lethality with Calcineurin Deletion in Fission Yeast

    PubMed Central

    Kita, Ayako; Sugiura, Reiko; Shoji, Hiromi; He, Yi; Deng, Lu; Lu, Yabin; Sio, Susie O.; Takegawa, Kaoru; Sakaue, Motoyoshi; Shuntoh, Hisato; Kuno, Takayoshi

    2004-01-01

    Calcineurin is a highly conserved regulator of Ca2+ signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl- homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1+ gene that encodes a homolog of the mammalian μ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Δapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Δapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Δapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events. PMID:15047861

  16. Phosphorylation of Adaptor Protein Containing Pleckstrin Homology Domain, Phosphotyrosine Binding Domain, and Leucine Zipper Motif 1 (APPL1) at Ser430 Mediates Endoplasmic Reticulum (ER) Stress-induced Insulin Resistance in Hepatocytes*

    PubMed Central

    Liu, Meilian; Zhou, Lijun; Wei, Li; Villarreal, Ricardo; Yang, Xin; Hu, Derong; Riojas, Ramon A.; Holmes, Bekke M.; Langlais, Paul R.; Lee, Hakjoo; Dong, Lily Q.

    2012-01-01

    APPL1 is an adaptor protein that plays a critical role in regulating adiponectin and insulin signaling. However, how APPL1 is regulated under normal and pathological conditions remains largely unknown. In this study, we show that APPL1 undergoes phosphorylation at Ser430 and that this phosphorylation is enhanced in the liver of obese mice displaying insulin resistance. In cultured mouse hepatocytes, APPL1 phosphorylation at Ser430 is stimulated by phorbol 12-myristate 13-acetate, an activator of classic PKC isoforms, and by the endoplasmic reticulum (ER) stress inducer, thapsigargin. Overexpression of wild-type but not dominant negative PKCα increases APPL1 phosphorylation at Ser430 in mouse hepatocytes. In addition, suppressing PKCα expression by shRNA in hepatocytes reduces ER stress-induced APPL1 phosphorylation at Ser430 as well as the inhibitory effect of ER stress on insulin-stimulated Akt phosphorylation. Consistent with a negative regulatory role of APPL1 phosphorylation at Ser430 in insulin signaling, overexpression of APPL1S430D but not APPL1S430A impairs the potentiating effect of APPL1 on insulin-stimulated Akt phosphorylation at Thr308. Taken together, our results identify APPL1 as a novel target in ER stress-induced insulin resistance and PKCα as the kinase mediating ER stress-induced phosphorylation of APPL1 at Ser430. PMID:22685300

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2012-12-01

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

  1. Application of tung oil to improve adhesion strength and water resistance of cottonseed meal and protein adhesives on maple veneer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cottonseed meal-based products show promise in serving as environment-friendly wood adhesives. However, their practical utilization is currently limited due to low durability and water resistant properties. In this research, we tested the improvement of adhesion strength and water resistance of cott...

  2. Protein Adhesion and Ion Substitution (on/in)to Minerals

    NASA Astrophysics Data System (ADS)

    Charlet, L.; Fernandez Martinez, A.; Chapron, Y.; Sahai, N.; Cuello, G.; Brendle, J.; Marichal, C.

    2008-12-01

    Arsenic and pathogenic prion protein-scrapie (PrPsc) are important contaminants which may soil and water for decades, unless they are removed by sorption. Two sorption mechanisms will be discussed, namely the organics (Prp and single aminoacid) adsorption on clay and the arsenic substitution in gypsum. The elucidation of these contrasted mechanisms will be shown to request complementary molecular-mechanical simulations with experimental spectroscopic investigations. As first example, structural studies performed at ILL/ESRF on As-doped gypsum (CaSO4 2H2O) using neutron and X-ray diffraction data and EXAFS were performed to determine how As fits into the bulk of gypsum structure. The combined Rietveld analysis of neutron and X-ray diffraction data shows an expansion of the unit cell volume proportional to the As concentration within the samples. to-sulfate substitution mechanisms were used as simulation starting hypotheses. DFT-based simulations (Mulliken analysis) were used to interpret charge distribution and to show that among the possible mechanisms, a sulphate substitution by either protonated, or fully deprotonated, arsenate ion, only the protonated arsenate substitution could best fit the EXAFS data. In the second example, we used Molecular Dynamics to understand the mechanism of strong binding of the pathogenic PrP peptide with clay mineral surfaces. We modeled only the infectious moiety, PrP92-138, of the whole PrPsc structure, with explicitly solvating water molecules in contact with the cleavage plane of pyrophillite, as a model for montmorillonite without any cationic substitution. Partial residual negative charges on the cleavage plane were balanced with K+ ions. The peptide anchored to the clay surface via up to 10 hydrogen bonds from lysine and histidine residues to oxygen atoms of the siloxane cavities, and a total adsorption energy of 3465 KJ.mol-1 was obtained. Our results were compared to the one obtained by chemical and thermal analysis, 23Na, 1H

  3. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  4. Loss of PDZ-adaptor protein NHERF2 affects membrane localization and cGMP- and [Ca2+]- but not cAMP-dependent regulation of Na+/H+ exchanger 3 in murine intestine

    PubMed Central

    Chen, Mingmin; Sultan, Ayesha; Cinar, Ayhan; Yeruva, Sunil; Riederer, Brigitte; Singh, Anurag Kumar; Li, Junhua; Bonhagen, Janina; Chen, Gang; Yun, Chris; Donowitz, Mark; Hogema, Boris; deJonge, Hugo; Seidler, Ursula

    2010-01-01

    Trafficking and regulation of the epithelial brush border membrane (BBM) Na+/H+ exchanger 3 (NHE3) in the intestine involves interaction with four different members of the NHERF family in a signal-dependent and possibly segment-specific fashion. The aim of this research was to study the role of NHERF2 (E3KARP) in intestinal NHE3 BBM localization and second messenger-mediated and receptor-mediated inhibition of NHE3. Immunolocalization of NHE3 in WT mice revealed predominant microvillar localization in jejunum and colon, a mixed distribution in the proximal ileum but localization near the terminal web in the distal ileum. The terminal web localization of NHE3 in the distal ileum correlated with reduced acid-activated NHE3 activity (fluorometrically assessed). NHERF2 ablation resulted in a shift of NHE3 to the microvilli and higher basal fluid absorption rates in the ileum, but no change in overall NHE3 protein or mRNA expression. Forskolin-induced NHE3 inhibition was preserved in the absence of NHERF2, whereas Ca2+ ionophore- or carbachol-mediated inhibition was abolished. Likewise, Escherichia coli heat stable enterotoxin peptide (STp) lost its inhibitory effect on intestinal NHE3. It is concluded that in native murine intestine, the NHE3 adaptor protein NHERF2 plays important roles in tethering NHE3 to a position near the terminal web and in second messenger inhibition of NHE3 in a signal- and segment-specific fashion, and is therefore an important regulator of intestinal fluid transport. PMID:20962002

  5. Phosphorylation of the beta-subunit of CD11/CD18 integrins by protein kinase C correlates with leukocyte adhesion.

    PubMed

    Valmu, L; Autero, M; Siljander, P; Patarroyo, M; Gahmberg, C G

    1991-11-01

    Adhesion of activated leukocytes to cells is of critical functional importance. The adhesion is known to be mediated mainly by the CD11/CD18 integrins, also known as leukocytic cell adhesion molecules, or Leu-CAM. We have now studied the phosphorylation of Leu-CAM by protein kinase C and the correlation of phosphorylation with the generation of the adhesive phenotype among human peripheral blood mononuclear leukocytes during cell activation. We here show that a good correlation exists between the phosphorylation of the beta subunit of Leu-CAM (CD18), and the extent of cell-to-cell adhesion. The phosphorylated CD18 subunit was associated with both CD11a and CD11b. Purified protein kinase C was able to phosphorylate the beta subunit of isolated Leu-CAM in vitro. The phosphorylation occurred mainly on serine residues. PMID:1682156

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

    PubMed Central

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

    2013-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. In vitro investigation of protein adsorption and platelet adhesion on inorganic biomaterial surfaces

    NASA Astrophysics Data System (ADS)

    Huang, Yan; Lü, Xiaoying; Jingwu, Ma; Huang, Nan

    2008-11-01

    The aim of this paper was to study the surface properties, protein adsorption and platelet adhesion behaviors of diamond-like carbon (DLC) and titanium (Ti) films. The surface energy and microstructures of these films were characterized by contact angle measurement and atomic force microscopy (AFM). A modified Coomassie brilliant blue (CBB) protein assay was used to study the amount of adsorbed proteins. Platelet adhesion was assessed by scanning electron microscopy (SEM). The AFM results show that the DLC film is smoother than Ti. Protein adsorption results from CBB protein assay show that the ratio of adsorbed albumin (Alb) to IgG ( RA/I) on DLC is larger than Ti, which coincide with the sequence of the ratio of interfacial tension between solid surface and Alb ( γS,Alb) to interfacial tension between surface and IgG ( γS,IgG) ( γS,Alb/ γS,IgG). The DLC film has a preferential adsorption for Alb. The results suggest that the ratio of γS,Alb/ γS,IgG may indicate an Alb/IgG affinity ratio of materials. More platelets adhere on Ti film than on DLC, which may correspond to the surface roughness of materials. The conclusion is the blood compatibility of DLC seems to be better than Ti.

  9. Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

    PubMed Central

    Chamma, Ingrid; Letellier, Mathieu; Butler, Corey; Tessier, Béatrice; Lim, Kok-Hong; Gauthereau, Isabel; Choquet, Daniel; Sibarita, Jean-Baptiste; Park, Sheldon; Sainlos, Matthieu; Thoumine, Olivier

    2016-01-01

    The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures. PMID:26979420

  10. 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. PMID:24481819

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

    PubMed Central

    Hung, Chao-Wei; Duncan, Mara C.

    2016-01-01

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

  12. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors.

    PubMed

    Hamann, Jörg; Aust, Gabriela; Araç, Demet; Engel, Felix B; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A; Harty, Breanne L; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C; Monk, Kelly R; Peeters, Miriam C; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W; Xu, Lei; Langenhan, Tobias; Schiöth, Helgi B

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential. PMID:25713288

  13. In Vivo Detection of Vascular Adhesion Protein-1 in Experimental Inflammation

    PubMed Central

    Jaakkola, Kimmo; Nikula, Tuomo; Holopainen, Riikka; Vähäsilta, Tommi; Matikainen, Marja-Terttu; Laukkanen, Marja-Leena; Huupponen, Risto; Halkola, Lauri; Nieminen, Lauri; Hiltunen, Jukka; Parviainen, Sakari; Clark, Michael R.; Knuuti, Juhani; Savunen, Timo; Kääpä, Pekka; Voipio-Pulkki, Liisa Maria; Jalkanen, Sirpa

    2000-01-01

    Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation. PMID:10934150

  14. Proximity biotinylation provides insight into the molecular composition of focal adhesions at the nanometer scale.

    PubMed

    Dong, Jing-Ming; Tay, Felicia Pei-Ling; Swa, Hannah Lee-Foon; Gunaratne, Jayantha; Leung, Thomas; Burke, Brian; Manser, Ed

    2016-01-01

    Focal adhesions are protein complexes that link metazoan cells to the extracellular matrix through the integrin family of transmembrane proteins. Integrins recruit many proteins to these complexes, referred to as the "adhesome." We used proximity-dependent biotinylation (BioID) in U2OS osteosarcoma cells to label proteins within 15 to 25 nm of paxillin, a cytoplasmic focal adhesion protein, and kindlin-2, which directly binds β integrins. Using mass spectrometry analysis of the biotinylated proteins, we identified 27 known adhesome proteins and 8 previously unknown components close to paxillin. However, only seven of these proteins interacted directly with paxillin, one of which was the adaptor protein Kank2. The proteins in proximity to β integrin included 15 of the adhesion proteins identified in the paxillin BioID data set. BioID also correctly established kindlin-2 as a cell-cell junction protein. By focusing on this smaller data set, new partners for kindlin-2 were found, namely, the endocytosis-promoting proteins liprin β1 and EFR3A, but, contrary to previous reports, not the filamin-binding protein migfilin. A model adhesome based on both data sets suggests that focal adhesions contain fewer components than previously suspected and that paxillin lies away from the plasma membrane. These data not only illustrate the power of using BioID and stable isotope-labeled mass spectrometry to define macromolecular complexes but also enable the correct identification of therapeutic targets within the adhesome. PMID:27303058

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

    PubMed Central

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

    2014-01-01

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

  16. Redox Capacity of an Extracellular Matrix Protein Associated with Adhesion in Mytilus californianus.

    PubMed

    Nicklisch, Sascha C T; Spahn, Jamie E; Zhou, Hongjun; Gruian, Cristina M; Waite, J Herbert

    2016-04-01

    Adhesive mussel foot proteins (Mfps) rely in part on DOPA (3,4-dihydroxyphenyl-l-alanine) side chains to mediate attachment to mineral surfaces underwater. Oxidation of DOPA to Dopaquinone (Q) effectively abolishes the adsorption of Mfps to these surfaces. The thiol-rich mussel foot protein-6 (Mfp-6) rescues adhesion compromised by adventitious DOPA oxidation by reducing Q back to DOPA. The redox chemistry and kinetics of foot-extracted Mfp-6 were investigated by using a nonspecific chromogenic probe to equilibrate with the redox pool. Foot-extracted Mfp-6 has a reducing capacity of ~17 e(-) per protein; half of this comes from the cysteine residues, whereas the other half comes from other constituents, probably a cohort of four or five nonadhesive, redox-active DOPA residues in Mfp-6 with an anodic peak potential ~500 mV lower than that for oxidation of cysteine to cystine. At higher pH, DOPA redox reversibility is lost possibly due to Q scavenging by Cys thiolates. Analysis by one- and two-dimensional proton nuclear magnetic resonance identified a pronounced β-sheet structure with a hydrophobic core in foot-extracted Mfp-6 protein. The structure endows redox-active side chains in Mfp-6, i.e., cysteine and DOPA, with significant reducing power over a broad pH range, and this power is measurably diminished in recombinant Mfp-6. PMID:26998552

  17. Partial characterization of a human submandibular/sublingual salivary adhesion-promoting protein.

    PubMed

    Akintoye, S O; Dasso, M; Hay, D I; Ganeshkumar, N; Spielman, A I

    2002-05-01

    Human submandibular/sublingual saliva contains a protein that promotes adhesion of Streptococcus mutans JBP serotype-c to spheroidal hydroxyapatite in vitro. A high molecular-weight (250,000-300,000 Da) adhesion-promoting protein (APP) was purified by Trisacryl 2000 M gel-filtration chromatography and gel electroelution before it was partially characterized. Lectin blotting identified that the terminal carbohydrates include N-acetyl glucosamine-beta 1-4-N-acetylglucosamine, galactose and galactose-beta 1-3-N-acetyl galactosamine. Antibodies to APP demonstrated no difference in the immunoreactive pattern of APP from saliva of caries-active or caries-resistant individuals belonging to four different ethnic groups: Asian, African-American, Hispanic or Caucasian. No immunological similarities to salivary mucins or parotid agglutinins were detected by Western blotting using immuno-cross-reactivity as a criterion. APP appears to be a unique protein found in submandibular/sublingual saliva. Understanding such a protein could help prevent S. mutans attachment to the enamel surface. PMID:12015214

  18. Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C.elegans

    PubMed Central

    McShea, Molly A.; Schmidt, Kristopher L.; Dubuke, Michelle L.; Baldiga, Christina E.; Sullender, Meagan E.; Reis, Andrea L.; Zhang, Subaiou; O'Toole, Sean M.; Jeffers, Mary C.; Warden, Rachel M.; Kenney, Allison H.; Gosselin, Jennifer; Kuhlwein, Mark; Hashmi, Sana K.; Stringham, Eve G.; Ryder, Elizabeth F.

    2012-01-01

    Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or ‘canals’ of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, we conducted a yeast two hybrid screen using isoform MIG-10A as bait and isolated Abelson-interactor protein-1 (ABI-1). ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-mmunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large

  19. MyD88 adaptor-like (Mal) functions in the epithelial barrier and contributes to intestinal integrity via protein kinase C.

    PubMed

    Corr, S C; Palsson-McDermott, E M; Grishina, I; Barry, S P; Aviello, G; Bernard, N J; Casey, P G; Ward, J B J; Keely, S J; Dandekar, S; Fallon, P G; O'Neill, L A J

    2014-01-01

    MyD88 adapter-like (Mal)-deficient mice displayed increased susceptibility to oral but not intraperitoneal infection with Salmonella Typhimurium. Bone marrow chimeras demonstrated that mice with Mal-deficient non-hematopoietic cells were more susceptible to infection, indicating a role for Mal in non-myeloid cells. We observed perturbed barrier function in Mal(-/-) mice, as indicated by reduced electrical resistance and increased mucosa blood permeability following infection. Altered expression of occludin, Zonula occludens-1, and claudin-3 in intestinal epithelia from Mal(-/-) mice suggest that Mal regulates tight junction formation, which may in part contribute to intestinal integrity. Mal interacted with several protein kinase C (PKC) isoforms in a Caco-2 model of intestinal epithelia and inhibition of Mal or PKC increased permeability and bacterial invasion via a paracellular route, while a pan-PKC inhibitor increased susceptibility to oral infection in mice. Mal signaling is therefore beneficial to the integrity of the intestinal barrier during infection. PMID:23612054

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

    PubMed

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

    2015-12-01

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

  1. Antiadhesive properties of arabinogalactan protein from ribes nigrum seeds against bacterial adhesion of Helicobacter pylori.

    PubMed

    Messing, Jutta; Niehues, Michael; Shevtsova, Anna; Borén, Thomas; Hensel, Andreas

    2014-01-01

    Fruit extracts from black currants (Ribes nigrum L.) are traditionally used for treatment of gastritis based on seed polysaccharides that inhibit the adhesion of Helicobacter pylori to stomach cells. For detailed investigations an arabinogalactan protein (F2) was isolated from seeds and characterized concerning molecular weight, carbohydrate, amino acid composition, linkage, configuration and reaction with β-glucosyl Yariv. Functional testing of F2 was performed by semiquantitative in situ adhesion assay on sections of human gastric mucosa and by quantitative in vitro adhesion assay with FITC-labled H. pylori strain J99 and human stomach AGS cells. Bacterial adhesins affected were identified by overlay assay with immobilized ligands. ¹²⁵I-radiolabeled F2 served for binding studies to H. pylori and interaction experiments with BabA and SabA. F2 had no cytotoxic effects against H. pylori and AGS cells; but inhibited bacterial binding to human gastric cells. F2 inhibited the binding of BabA and fibronectin-binding adhesin to its specific ligands. Radiolabeled F2 bound non-specifically to different strains of H. pylori; and to BabA deficient mutant. F2 did not lead to subsequent feedback regulation or increased expression of adhesins or virulence factors. From these data the non-specific interactions between F2 and the H. pylori lead to moderate antiadhesive effects. PMID:24662083

  2. Serum protein layers on parylene-C and silicon oxide: Effect on cell adhesion

    PubMed Central

    Delivopoulos, Evangelos; Ouberai, Myriam M.; Coffey, Paul D.; Swann, Marcus J.; Shakesheff, Kevin M.; Welland, Mark E.

    2015-01-01

    Among the range of materials used in bioengineering, parylene-C has been used in combination with silicon oxide and in presence of the serum proteins, in cell patterning. However, the structural properties of adsorbed serum proteins on these substrates still remain elusive. In this study, we use an optical biosensing technique to decipher the properties of fibronectin (Fn) and serum albumin adsorbed on parylene-C and silicon oxide substrates. Our results show the formation of layers with distinct structural and adhesive properties. Thin, dense layers are formed on parylene-C, whereas thicker, more diffuse layers are formed on silicon oxide. These results suggest that Fn acquires a compact structure on parylene-C and a more extended structure on silicon oxide. Nonetheless, parylene-C and silicon oxide substrates coated with Fn host cell populations that exhibit focal adhesion complexes and good cell attachment. Albumin adopts a deformed structure on parylene-C and a globular structure on silicon oxide, and does not support significant cell attachment on either surface. Interestingly, the co-incubation of Fn and albumin at the ratio found in serum, results in the preferential adsorption of albumin on parylene-C and Fn on silicon oxide. This finding is supported by the exclusive formation of focal adhesion complexes in differentiated mouse embryonic stem cells (CGR8), cultured on Fn/albumin coated silicon oxide, but not on parylene-C. The detailed information provided in this study on the distinct properties of layers of serum proteins on substrates such as parylene-C and silicon oxide is highly significant in developing methods for cell patterning. PMID:25555155

  3. Wiskott–Aldrich syndrome protein is involved in αIIbβ3-mediated cell adhesion

    PubMed Central

    Tsuboi, Shigeru; Nonoyama, Shigeaki; Ochs, Hans D

    2006-01-01

    The Wiskott–Aldrich syndrome (WAS) is an X-chromosome-linked immunodeficiency disorder. The most common symptom seen in WAS patients is bleeding. One of the main causes of bleeding is defective platelet aggregation. The causative gene of WAS encodes WAS protein (WASP). Here, we show that WASP binds to the calcium- and integrin-binding protein (CIB) in platelets. CIB was originally identified as a protein binding to the αIIb cytoplasmic tail of platelet integrin αIIbβ3, which has a primary role in platelet aggregation. We also show that the WASP–CIB complex is important in αIIbβ3-mediated cell adhesion, and that in patients mutant forms of WASP are expressed at reduced levels or show lower affinities for CIB than wild-type WASP. Our results indicate that impaired complex formation between mutant WASPs and CIB reduces αIIbβ3-mediated cell adhesion and causes defective platelet aggregation, resulting in bleeding. PMID:16582881

  4. Amigo Adhesion Protein Regulates Development of Neural Circuits in Zebrafish Brain*

    PubMed Central

    Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A.; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

    2014-01-01

    The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. PMID:24904058

  5. Characterizing the modification of surface proteins with poly(ethylene glycol) to interrupt platelet adhesion

    PubMed Central

    Xu, Haiyan; Kaar, Joel L.; Russell, Alan J.; Wagner, William R.

    2010-01-01

    Surface protein modification with poly(ethylene glycol) (PEG) can inhibit acute thrombosis on damaged vascular and biomaterial surfaces by blocking surface protein–platelet interactions. However, the feasibility of employing protein reactive PEGs to limit intravascular and biomaterial thrombosis in vivo is contingent upon rapid and extensive surface protein modification. To characterize the factors controlling this potential therapeutic approach, the model protein bovine serum albumin was adsorbed onto polyurethane surfaces and modified with PEG-carboxymethyl succinimidyl ester (PEG-NHS), PEG-isocyanate (PEG-ISO), or PEG-diisocyanate (PEG-DISO) in aqueous buffer at varying concentrations and contact times. It was found that up to 5 PEGs could be attached per albumin molecule within one min and that adsorbed albumin PEGylation approached maximal levels by 6 min. The lability of reactive PEGs in aqueous buffer reduced total protein modification by 50% when the PEG solution was incubated for 7 min prior to application. For fibrinogen PEGylation (performed in the solution phase), PEG-NHS was more reactive than PEG-ISO or PEG-DISO. The γ peptide of fibrinogen, which contains several key platelet-binding motifs, was highly modified. A marked reduction in platelet adhesion was observed on fibrinogen-adsorbed polyurethane treated with PEG-NHS or PEG-DISO. Relative differences in platelet adhesion on PEG-NHS and PEG-DISO modified surfaces could be attributed to differences in reactivity towards fibrinogen and the size of the polymer backbone. Taken together, these findings provide insight and guidance for applying protein reactive PEGs for the interruption of acute thrombotic deposition. PMID:16457880

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

    PubMed

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

    2016-02-26

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

  7. Paradigms lost-an emerging role for over-expression of tight junction adhesion proteins in cancer pathogenesis.

    PubMed

    Leech, Astrid O; Cruz, Rodrigo G B; Hill, Arnold D K; Hopkins, Ann M

    2015-08-01

    Tight junctions (TJ) are multi-protein complexes located at the apicalmost tip of the lateral membrane in polarised epithelial and endothelial cells. Their principal function is in mediating intercellular adhesion and polarity. Accordingly, it has long been a paradigm that loss of TJ proteins and consequent deficits in cell-cell adhesion are required for tumour cell dissemination in the early stages of the invasive/metastatic cascade. However it is becoming increasingly apparent that TJ proteins play important roles in not just adhesion but also intracellular signalling events, activation of which can contribute to, or even drive, tumour progression and metastasis. In this review, we shall therefore highlight cases wherein the gain of TJ proteins has been associated with signals promoting tumour progression. We will also discuss the potential of overexpressed TJ proteins to act as therapeutic targets in cancer treatment. The overall purpose of this review is not to disprove the fact that loss of TJ-based adhesion contributes to the progression of several cancers, but rather to introduce the growing body of evidence that gain of TJ proteins may have adhesion-independent consequences for promoting progression in other cancers. PMID:26366401

  8. Structural Basis for Membrane Binding and Remodeling by the Exomer Secretory Vesicle Cargo Adaptor

    PubMed Central

    Paczkowski, Jon E.; Fromme, J. Christopher

    2014-01-01

    Summary Cargo adaptor subunits of vesicle coat protein complexes sort transmembrane proteins to distinct membrane compartments in eukaryotic cells. The exomer complex is the only cargo adaptor known to sort proteins at the trans-Golgi network into secretory vesicles. Exomer function is regulated by the Arf1 GTPase, a master regulator of trafficking at the Golgi. We report the structure of exomer bound to two copies of Arf1. Exomer interacts with each Arf1 molecule via two surfaces; one is a non-canonical interface that regulates GTP hydrolysis. The structure uncovers an unexpected membrane-proximal hydrophobic element that exomer uses in cooperation with Arf1 to remodel membranes. Given the constrained motion of the exomer hinge region, we envision that exomer dynamically positions multiple membrane insertion elements to drive membrane fission. In contrast to other known cargo adaptors, exomer therefore couples two functions, cargo sorting and membrane fission, into a single complex. PMID:25203211

  9. Lack of Protein 4.1G Causes Altered Expression and Localization of the Cell Adhesion Molecule Nectin-Like 4 in Testis and Can Cause Male Infertility ▿

    PubMed Central

    Yang, Shaomin; Weng, Haibo; Chen, Lixiang; Guo, Xinhua; Parra, Marilyn; Conboy, John; Debnath, Gargi; Lambert, Amy J.; Peters, Luanne L.; Baines, Anthony J.; Mohandas, Narla; An, Xiuli

    2011-01-01

    Protein 4.1G is a member of the protein 4.1 family, which in general serves as adaptors linking transmembrane proteins to the cytoskeleton. 4.1G is thought to be widely expressed in many cells and tissues, but its function remains largely unknown. To explore the function of 4.1G in vivo, we generated 4.1G−/− mice and bred the mice in two backgrounds: C57BL/6 (B6) and 129/Sv (129) hybrids (B6-129) and inbred B6. Although the B6 4.1G−/− mice showed no obvious abnormalities, deficiency of 4.1G in B6-129 hybrids was associated with male infertility. Histological examinations of these 4.1G−/− mice revealed atrophy, impaired cell-cell contact and sloughing off of spermatogenic cells in seminiferous epithelium, and lack of mature spermatids in the epididymis. Ultrastructural examination revealed enlarged intercellular spaces between spermatogenic and Sertoli cells as well as the spermatid deformities. At the molecular level, 4.1G is associated with the nectin-like 4 (NECL4) adhesion molecule. Importantly, the expression of NECL4 was decreased, and the localization of NECL4 was altered in 4.1G−/− testis. Thus, our findings imply that 4.1G plays a role in spermatogenesis by mediating cell-cell adhesion between spermatogenic and Sertoli cells through its interaction with NECL4 on Sertoli cells. Additionally, the finding that infertility is present in B6-129 but not on the B6 background suggests the presence of a major modifier gene(s) that influences 4.1G function and is associated with male infertility. PMID:21482674

  10. 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.

  11. Focal adhesion kinase regulates expression of thioredoxin-interacting protein (TXNIP) in cancer cells.

    PubMed

    Ho, Baotran; Huang, Grace; Golubovskaya, Vita M

    2014-01-01

    Focal Adhesion Kinase (FAK) plays an important role in cancer cell survival. Previous microarray gene profiling study detected inverse regulation between expression of thioredoxin-interacting protein (TXNIP) and FAK, where down-regulation of FAK by siRNA in MCF-7 cells caused up-regulation of TXNIP mRNA level, and in contrast up-regulation of doxycyclin- induced FAK caused repression of TXNIP. In the present report, we show that overexpression of FAK in MCF-7 cells repressed TXNIP promoter activity. Treatment of MCF-7 cells with 1alpha, 25-dihydroxyvitamin D3 (1,25D) down-regulated endogenous FAK and up-regulated TXNIP protein level, and treatment with 5-FU decreased FAK protein expression and up-regulated TXNIP protein expression in 293 cells. Moreover, silencing of FAK with siRNA increased TXNIP protein expression, while overexpression of FAK inhibited TXNIP protein expression in 293 cells. In addition, treatment of DBTRG glioblastoma cells with FAK inhibitor Y15 increased TXNIP mRNA, decreased cancer cell viability and increased apoptosis. These results for the first time demonstrate FAK-regulated TXNIP expression which is important for apoptotic, survival and oxidative stress signaling pathways in cancer cells. PMID:23387972

  12. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy.

    PubMed

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives. PMID:27217558

  13. Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

    NASA Astrophysics Data System (ADS)

    Huang, Yujian; Wang, Yongzhong; Tan, Li; Sun, Leming; Petrosino, Jennifer; Cui, Mei-Zhen; Hao, Feng; Zhang, Mingjun

    2016-06-01

    Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

  14. Understanding Marine Mussel Adhesion

    SciTech Connect

    H. G. Silverman; F. F. Roberto

    2007-12-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are waterimpervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion.

  15. Understanding marine mussel adhesion.

    PubMed

    Silverman, Heather G; Roberto, Francisco F

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  16. Understanding Marine Mussel Adhesion

    PubMed Central

    Roberto, Francisco F.

    2007-01-01

    In addition to identifying the proteins that have a role in underwater adhesion by marine mussels, research efforts have focused on identifying the genes responsible for the adhesive proteins, environmental factors that may influence protein production, and strategies for producing natural adhesives similar to the native mussel adhesive proteins. The production-scale availability of recombinant mussel adhesive proteins will enable researchers to formulate adhesives that are water-impervious and ecologically safe and can bind materials ranging from glass, plastics, metals, and wood to materials, such as bone or teeth, biological organisms, and other chemicals or molecules. Unfortunately, as of yet scientists have been unable to duplicate the processes that marine mussels use to create adhesive structures. This study provides a background on adhesive proteins identified in the blue mussel, Mytilus edulis, and introduces our research interests and discusses the future for continued research related to mussel adhesion. PMID:17990038

  17. Inhibition of fibroblast adhesion by covalently immobilized protein repellent polymer coatings studied by single cell force spectroscopy.

    PubMed

    Aliuos, Pooyan; Sen, Aromita; Reich, Uta; Dempwolf, Wibke; Warnecke, Athanasia; Hadler, Christoph; Lenarz, Thomas; Menzel, Henning; Reuter, Guenter

    2014-01-01

    Cochlea implants (CI) restore the hearing in patients with sensorineural hearing loss by electrical stimulation of the auditory nerve via an electrode array. The increase of the impedance at the electrode-tissue interface due to a postoperative connective tissue encapsulation leads to higher power consumption of the implants. Therefore, reduced adhesion and proliferation of connective tissue cells around the CI electrode array is of great clinical interest. The adhesion of cells to substrate surfaces is mediated by extracellular matrix (ECM) proteins. Protein repellent polymers (PRP) are able to inhibit unspecific protein adsorption. Thus, a reduction of cell adhesion might be achieved by coating the electrode carriers with PRPs. The aim of this study was to investigate the effects of two different PRPs, poly(dimethylacrylamide) (PDMAA) and poly(2-ethyloxazoline) (PEtOx), on the strength and the temporal dynamics of the initial adhesion of fibroblasts. Polymers were immobilized onto glass plates by a photochemical grafting onto method. Water contact angle measurements proved hydrophilic surface properties of both PDMAA and PEtOx (45 ± 1° and 44 ± 1°, respectively). The adhesion strength of NIH3T3 fibroblasts after 5, 30, and 180 s of interaction with surfaces was investigated by using single cell force spectroscopy. In comparison to glass surfaces, both polymers reduced the adhesion of fibroblasts significantly at all different interaction times and lower dynamic rates of adhesion were observed. Thus, both PDMAA and PEtOx represented antiadhesive properties and can be used as implant coatings to reduce the unspecific ECM-mediated adhesion of fibroblasts to surfaces. PMID:23596088

  18. A standardized bamboo leaf extract inhibits monocyte adhesion to endothelial cells by modulating vascular cell adhesion protein-1

    PubMed Central

    Choi, Sunga; Park, Myoung Soo; Lee, Yu Ran; Lee, Young Chul; Kim, Tae Woo; Do, Seon-Gil; Kim, Dong Seon

    2013-01-01

    Bamboo leaves (Phyllostachys pubescens Mazel ex J. Houz (Poacea)) have a long history of food and medical applications in Asia, including Japan and Korea. They have been used as a traditional medicine for centuries. We investigated the mechanism of anti-inflammatory activity of a bamboo leaf extract (BLE) on tumor necrosis factor-alpha (TNF-α)-induced monocyte adhesion in human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to BLE did not inhibit cell viability or cause morphological changes at concentrations ranging from 1 µg/ml to 1 mg/ml. Treatment with 0.1 mg/ml BLE caused 63% inhibition of monocyte adhesion in TNF-α-activated HUVECs, which was associated with 38.4% suppression of vascular cell adhesion molecule-1 expression. Furthermore, TNF-α-induced reactive oxygen species generation was decreased to 47.9% in BLE treated TNF-α-activated HUVECs. BLE (0.05 mg/ml) also caused about 50% inhibition of interleukin-6 secretion from lipopolysaccharide-stimulated monocyte. The results indicate that BLE may be clinically useful as an anti-inflammatory or anti-oxidant for human cardiovascular disease including atherosclerosis. PMID:23422838

  19. Protein-mediated adhesion of the dissimilatory Fe(III)-reducing bacterium Shewanella alga BrY to hydrous ferric oxide

    SciTech Connect

    Caccavo, F. Jr.

    1999-11-01

    The rate and extent of bacterial Fe(III) mineral reduction are governed by molecular-scale interactions between the bacterial cell surface and the mineral surface. These interactions are poorly understood. This study examined the role of surface proteins in the adhesion of Shewanella alga BrY to hydrous ferric oxide (HFO). Enzymatic degradation of cell surface polysaccharides had no effect on cell adhesion to HFO. The proteolytic enzymes Streptomyces griseus protease and chymotrypsin inhibited the adhesion of S. alga BrY cells to HFO through catalytic degradation of surface proteins. Trypsin inhibited S. alga BrY adhesion solely through surface-coating effects. Protease and chymotrypsin also mediated desorption of adhered S. alga BrY cells from HFO while trypsin did not mediate cell desorption. Protease removed a single peptide band that represented a protein with an apparent molecular mass of 50 kDa. Chymotrypsin removed two peptide bands that represented proteins with apparent molecular masses of 60 and 31 kDa. These proteins represent putative HGO adhesion molecules. A. alga BrY adhesion was inhibited by up to 46% when cells were cultured at sub-MICs of chloramphenicol, suggesting that protein synthesis is necessary for adhesion. Proteins extracted from the surface of S. alga BrY cells inhibited adhesion to HFO by up to 41%. A number of these proteins bound specifically to HFO, suggesting that a complex system of surface proteins mediates S. alga BrY adhesion to HFO.

  20. The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.

    PubMed Central

    Reinhard, M; Giehl, K; Abel, K; Haffner, C; Jarchau, T; Hoppe, V; Jockusch, B M; Walter, U

    1995-01-01

    Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation. Images PMID:7737110

  1. Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion

    PubMed Central

    Altman, Stuart M.; Dixit, Neha; Simon, Scott I.

    2014-01-01

    Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype. PMID:24504956

  2. Recombinant mussel adhesive protein fp-5 (MAP fp-5) as a bulk bioadhesive and surface coating material.

    PubMed

    Choi, Yoo Seong; Kang, Dong Gyun; Lim, Seonghye; Yang, Yun Jung; Kim, Chang Sup; Cha, Hyung Joon

    2011-08-01

    Mussel adhesive proteins (MAPs) attach to all types of inorganic and organic surfaces, even in wet environments. MAP of type 5 (fp-5), in particular, has been considered as a key adhesive material. However, the low availability of fp-5 has hampered its biochemical characterization and practical applications. Here, soluble recombinant fp-5 is mass-produced in Escherichia coli. Tyrosinase-modified recombinant fp-5 showed ∼1.11 MPa adhesive shear strength, which is the first report of a bulk-scale adhesive force measurement for purified recombinant of natural MAP type. Surface coatings were also performed through simple dip-coating of various objects. In addition, complex coacervate using recombinant fp-5 and hyaluronic acid was prepared as an efficient adhesive formulation, which greatly improved the bulk adhesive strength. Collectively, it is expected that this work will enhance basic understanding of mussel adhesion and that recombinant fp-5 can be successfully used as a realistic bulk-scale bioadhesive and an efficient surface coating material. PMID:21770718

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

    PubMed Central

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

    2012-01-01

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

  4. Peptide-presenting two-dimensional protein matrix on supported lipid bilayers: an efficient platform for cell adhesion.

    PubMed

    Bérat, Rémi; Rémy-Zolghadry, Murielle; Gounou, Céline; Manigand, Claude; Tan, Sisareuth; Saltó, Carmen; Arenas, Ernest; Bordenave, Laurence; Brisson, Alain R

    2007-12-01

    Understanding and controlling cell adhesion to biomaterials and synthetic materials are important issues in basic research and applied sciences. Supported lipid bilayers (SLBs) functionalized with cell adhesion peptides linked to lipid molecules are popular platforms of cell adhesion. In this paper, an alternative approach of peptide presentation is presented in which peptides are stereo-selectively linked to proteins self-assembling in a rigid two-dimensional (2D) matrix on SLBs. Annexin-A5 (Anx5) was used as prototype protein for its known properties of forming stable and rigid 2D matrices on lipid surfaces. Two types of Anx5-peptide complexes, containing either a RGD or an IKVAV sequence, were synthesized. The authors show that both Anx5-peptide complexes present the same properties of binding and 2D organization on lipid surfaces as Anx5, when investigated by quartz crystal microbalance with dissipation monitoring, atomic force microscopy, and transmission electron microscopy techniques. Anx5-RGD and Anx5-IKVAV 2D matrices were found to promote specific adhesion of human saphenous vein endothelial cells and mouse embryonic stem cells, respectively. The influence of the surface density of exposed peptides on cell adhesion was investigated, showing that cells attach to Anx5-peptide matrices when the average distance between peptides is smaller than about 60 nm. This cell adhesion platform provides control of the orientation and density of cell ligands, opening interesting possibilities for future applications. PMID:20408654

  5. Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment.

    PubMed

    Abazeed, Mohamed E; Fuller, Robert S

    2008-11-01

    Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN-PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the beta subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Delta membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Delta mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

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

    PubMed Central

    Abazeed, Mohamed E.

    2008-01-01

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

  7. 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)

  8. Vasoconstrictor-induced endocytic recycling regulates focal adhesion protein localization and function in vascular smooth muscle

    PubMed Central

    Poythress, Ransom H.; Gallant, Cynthia; Vetterkind, Susanne

    2013-01-01

    Turnover of focal adhesions (FAs) is known to be critical for cell migration and adhesion of proliferative vascular smooth muscle (VSM) cells. However, it is often assumed that FAs in nonmigratory, differentiated VSM (dVSM) cells embedded in the wall of healthy blood vessels are stable structures. Recent work has demonstrated agonist-induced actin polymerization and Src-dependent FA phosphorylation in dVSM cells, suggesting that agonist-induced FA remodeling occurs. However, the mechanisms and extent of FA remodeling are largely unknown in dVSM. Here we show, for the first time, that a distinct subpopulation of dVSM FA proteins, but not the entire FA, remodels in response to the α-agonist phenylephrine. Vasodilator-stimulated phosphoprotein and zyxin displayed the largest redistributions, while β-integrin and FA kinase showed undetectable redistribution. Vinculin, metavinculin, Src, Crk-associated substrate, and paxillin displayed intermediate degrees of redistribution. Redistributions into membrane fractions were especially prominent, suggesting endosomal mechanisms. Deconvolution microscopy, quantitative colocalization analysis, and Duolink proximity ligation assays revealed that phenylephrine increases the association of FA proteins with early endosomal markers Rab5 and early endosomal antigen 1. Endosomal disruption with the small-molecule inhibitor primaquine inhibits agonist-induced redistribution of FA proteins, confirming endosomal recycling. FA recycling was also inhibited by cytochalasin D, latrunculin B, and colchicine, indicating that the redistribution is actin- and microtubule-dependent. Furthermore, inhibition of endosomal recycling causes a significant inhibition of the rate of development of agonist-induced dVSM contractions. Thus these studies are consistent with the concept that FAs in dVSM cells, embedded in the wall of the aorta, remodel during the action of a vasoconstrictor. PMID:23703522

  9. Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

    NASA Astrophysics Data System (ADS)

    Dobrowsky, Terrence M.; Rabi, S. Alireza; Nedellec, Rebecca; Daniels, Brian R.; Mullins, James I.; Mosier, Donald E.; Siliciano, Robert F.; Wirtz, Denis

    2013-10-01

    In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.

  10. Fabrication of three-dimensional multi-protein microstructures for cell migration and adhesion enhancement

    PubMed Central

    Da Sie, Yong; Li, Yi-Cheng; Chang, Nan-Shan; Campagnola, Paul J.; Chen, Shean-Jen

    2015-01-01

    In this study, three-dimensional (3D) multi-component microstructures were precisely fabricated via multiphoton excited photochemistry using a femtosecond laser direct-writing system with proposed repetition positioning and vector scanning techniques. Extracellular matrix (ECM) proteins, such as fibronectin (FN), are difficult to stack and form 3D structures larger than several-hundred microns in height due to the nature of their protein structure. Herein, to fabricate complex 3D microstructures with FN, a 3D scaffold was designed and formed from bovine serum albumin (BSA), after which human FN was inserted at specific locations on the BSA scaffold; in this manner, the fabricated ECM microstructure can guide cells in a 3D environment. A human breast cancer cell line, MDA-MB-231, was used to investigate the behavior of cell migration and adhesion on the fabricated human FN and BSA protein structures. Experimental results indicate that many cells are not able to attach or climb on a 3D structure’s inclined plane without FN support; hence, the influence of cell growth in a 3D context with FN should being taken into consideration. This 3D multi-protein fabrication technique holds potential for cell studies in designed complex 3D ECM scaffolds. PMID:25780738

  11. Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton*

    PubMed Central

    Tudor, Cicerone; te Riet, Joost; Eich, Christina; Harkes, Rolf; Smisdom, Nick; Bouhuijzen Wenger, Jessica; Ameloot, Marcel; Holt, Matthew; Kanger, Johannes S.; Figdor, Carl G.; Cambi, Alessandra; Subramaniam, Vinod

    2014-01-01

    Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side. PMID:24662291

  12. Evaluation of Serum Vascular Adhesion Protein-1 as a Potential Biomarker in Thyroid Cancer

    PubMed Central

    Zhao, Pengxin; Zhang, Kaili

    2016-01-01

    Vascular adhesion protein-1 (VAP-1) is a glycoprotein that mediates tissue-selective lymphocyte adhesion. The prognostic value of VAP-1 has been determined in gastric cancer. The aim of this study was to evaluate the changes and the predictive value of serum VAP-1 in patients with thyroid cancer. A total of 126 patients with thyroid nodules and 53 healthy controls participated in this study. The patients were further divided into subgroup 1 (69 cases with benign thyroid nodules) and subgroup 2 (57 cases with thyroid cancer). Serum VAP-1 was measured by time-resolved immunofluorometric assay. Diagnostic value of presurgical VAP-1 for thyroid cancer was conducted by receiver operating characteristic (ROC) curves. Serum levels of VAP-1 were significantly lower in thyroid cancer group than in healthy control and benign thyroid nodule groups. VAP-1 concentrations negatively correlated with serum thyroglobulin (Tg) levels in thyroid cancer patients (r = −0.81; p < 0.001). The optimum cut-off value of VAP-1 was 456.6 ng/mL with a 77.4% specificity and 66.7% sensitivity for thyroid cancer diagnosis. Serum VAP-1 decreased in thyroid cancer patients and VAP-1 could be a potential useful adjunct biomarker in the diagnosis of thyroid cancer. PMID:27446209

  13. Anterior Gradient Protein-2 Is a Regulator of Cellular Adhesion in Prostate Cancer

    PubMed Central

    Chanda, Diptiman; Lee, Joo Hyoung; Sawant, Anandi; Hensel, Jonathan A.; Isayeva, Tatyana; Reilly, Stephanie D.; Siegal, Gene P.; Smith, Claire; Grizzle, William; Singh, Raj; Ponnazhagan, Selvarangan

    2014-01-01

    Anterior Gradient Protein (AGR-2) is reported to be over-expressed in many epithelial cancers and promotes metastasis. A clear-cut mechanism for its observed function(s) has not been previously identified. We found significant upregulation of AGR-2 expression in a bone metastatic prostate cancer cell line, PC3, following culturing in bone marrow-conditioned medium. Substantial AGR-2 expression was also confirmed in prostate cancer tissue specimens in patients with bone lesions. By developing stable clones of PC3 cells with varying levels of AGR-2 expression, we identified that abrogation of AGR-2 significantly reduced cellular attachment to fibronectin, collagen I, collagen IV, laminin I and fibrinogen. Loss of cellular adhesion was associated with sharp decrease in the expression of α4, α5, αV, β3 and β4 integrins. Failure to undergo apoptosis following detachment is a hallmark of epithelial cancer metastasis. The AGR-2-silenced PC3 cells showed higher resistance to Tumor necrosis factor-related apoptosis- inducing ligand (TRAIL) induced apoptosis in vitro. This observation was also supported by significantly reduced Caspase-3 expression in AGR-2-silenced PC3 cells, which is a key effector of both extrinsic and intrinsic death signaling pathways. These data suggest that AGR-2 influence prostate cancer metastasis by regulation of cellular adhesion and apoptosis. PMID:24587138

  14. GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow

    PubMed Central

    Sarratt, Kendra L.; Chen, Hong; Zutter, Mary M.; Santoro, Samuel A.; Hammer, Daniel A.; Kahn, Mark L.

    2005-01-01

    The roles of the 2 major platelet-collagen receptors, glycoprotein VI (GPVI) and integrin α2β1, have been intensely investigated using a variety of methods over the past decade. In the present study, we have used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under flow conditions. Our studies demonstrate that both GPVI and integrin α2β1 play significant roles for platelet adhesion to collagen under flow and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) but not by the transmembrane adaptor linker for activation of T cells (LAT) is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail-bleeding times in mice lacking one or both collagen receptors. These studies establish platelet-collagen responses under physiologic flow as the consequence of a close partnership between 2 structurally distinct receptors and suggest that both receptors play significant hemostatic roles in vivo. PMID:15886326

  15. Characterization of the protein fraction of the temporary adhesive secreted by the tube feet of the sea star Asterias rubens.

    PubMed

    Hennebert, Elise; Wattiez, Ruddy; Waite, J Herbert; Flammang, Patrick

    2012-01-01

    Sea stars are able to make firm but temporary attachments to various substrata by secretions released by their tube feet. After tube foot detachment, the adhesive secretions remain on the substratum as a footprint. Proteins presumably play a key role in sea star adhesion, as evidenced by the removal of footprints from surfaces after a treatment with trypsin. However, until now, characterisation was hampered by their high insolubility. In this study, a non-hydrolytic method was used to render most of the proteins constituting the adhesive footprints soluble. After analysis by SDS-PAGE, the proteins separated into about 25 bands, which ranged from 25 to 450 kDa in apparent molecular weight. Using mass spectrometry and a homology-database search, it was shown that several of the proteins are known intracellular proteins, presumably resulting from contamination of footprint material with tube foot epidermal cells. However, 11 protein bands, comprising the most abundant proteins, were not identified and might correspond to novel adhesive proteins. They were named 'Sea star footprint proteins' (Sfps). Tandem mass spectrometry analysis of the protein bands yielded 43 de novo-generated peptide sequences. Most of them were shared by several, if not all, Sfps. Polyclonal antibodies were raised against one of the peptides (HEASGEYYR from Sfp-115) and were used in immunoblotting. They specifically labelled Sfp-115 and other bands with lower apparent molecular weights. The different results suggest that all Sfps might belong to a single family of related proteins sharing similar motifs or, alternatively, they are the products of polymerization and/or degradation processes. PMID:22439774

  16. Novel Pyridazinone Inhibitors for Vascular Adhesion Protein-1 (VAP-1): Old target – New Inhibition Mode

    PubMed Central

    Bligt-Lindén, Eva; Pihlavisto, Marjo; Szatmári, István; Otwinowski, Zbyszek; Smith, David J.; Lázár, László; Fülöp, Ferenc; Salminen, Tiina A.

    2014-01-01

    Vascular adhesion protein-1 (VAP-1) is a primary amine oxidase and a drug target for inflammatory and vascular diseases. Despite extensive attempts to develop potent, specific and reversible inhibitors of its enzyme activity, the task has proven challenging. Here we report the synthesis, inhibitory activity and molecular binding mode of novel pyridazinone inhibitors, which show specificity for VAP-1 over monoamine and diamine oxidases. The crystal structures of three inhibitor-VAP-1 complexes show that these compounds bind reversibly into a unique binding site in the active site channel. Though they are good inhibitors of human VAP-1, they do not inhibit rodent VAP-1 well. To investigate this further, we used homology modeling and structural comparison to identify amino acid differences, which explain the species-specific binding properties. Our results prove the potency and specificity of these new inhibitors and the detailed characterization of their binding mode is of importance for further development of VAP-1 inhibitors. PMID:24304424

  17. Investigation of alginate binding to germanium and polystyrene substrata conditioned with mussel adhesive protein

    SciTech Connect

    Suci, P.A.; Geesey, G.G.

    1995-06-15

    Binding of alginate from Macrocystis pyrifera (kelp) to germanium and polystyrene substrata conditioned with mussel adhesive protein (MAP) from Mytilis edulis, to germanium substrata conditioned with bovine serum albumin (BSA) and polylysine, and to germanium substrata coated with aminopropyltriethoxysilane (APS) was investigated using attenuated total reflection Fourier transform infrared spectrometry. Binding of alginate to MAP appears to be proportional to surface coverage for levels tested. Distinct spectral features appear in the region associated with pyranose ring vibrations upon binding of alginate to MAP, polylysine, and APS, indicating that lysine residues play a prominent role in promoting irreversible adsorption with perturbation of pyranose ring atoms. BSA does not appear to enhance alginate adsorption over that observed on clean germanium and no new spectral features appear as a result of binding. The level of irreversible binding of alginate to germanium and polystyrene substrata conditioned with MAP is similar.

  18. Adhesion G protein-coupled receptors in nervous system development and disease.

    PubMed

    Langenhan, Tobias; Piao, Xianhua; Monk, Kelly R

    2016-09-01

    Members of the adhesion G protein-coupled receptor (aGPCR) class have emerged as crucial regulators of nervous system development, with important implications for human health and disease. In this Review, we discuss the current understanding of aGPCR functions during key steps in neural development, including cortical patterning, dendrite and synapse formation, and myelination. We focus on aGPCR modulation of cell-cell and cell-matrix interactions and signalling to control these varied aspects of neural development, and we discuss how impaired aGPCR function leads to neurological disease. We further highlight the emerging hypothesis that aGPCRs can be mechanically activated and the implications of this property in the nervous system. PMID:27466150

  19. 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...

  20. 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...

  1. 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...

  2. 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...

  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, 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...

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

    ERIC Educational Resources Information Center

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

    2007-01-01

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

  6. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 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...

  7. 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...

  8. 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...

  9. 21 CFR 870.3620 - Pacemaker lead adaptor.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  10. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  11. Effect of dispersion method and CNT loading on the quality and performance of nanocomposite soy protein/CNTs adhesive for wood application

    NASA Astrophysics Data System (ADS)

    Afolabi, Ayo Samuel; Oluwafolakemi Sadare, Olawumi; Olawale Daramola, Michael

    2016-09-01

    In this article the effect of dispersion method and carbon nanotubes (CNTs) loading on the quality and performance of a nanocomposite adhesive is reported. The nanocomposite soy protein isolate adhesive was successfully developed by incorporating CNTs into the soy protein isolate (SPI) for enhanced bond strength and water resistance. Dispersion methods, namely mechanical (shear) mixing and mechanical/sonication were employed to aid good dispersion and interfacial interaction between soy protein matrix and the carbon nanofillers during the preparation of the adhesive. The concentration of the CNT was varied from 0.1–0.7 wt% in the nanocomposite adhesive. The morphology and the surface chemistry of the adhesives were checked with SEM and FTIR, respectively. The shear strength of the developed adhesives was investigated according to European standard (EN-204) for interior wood application on a tensile testing machine. The morphological structure of the nanocomposite adhesive obtained from SEM images showed homogeneous dispersion of CNTs in SPI using the two dispersion methods; shear mixing and sonication/shear mixing. Fourier transform infrared spectra showed chemical functionalities and successful interaction between CNTs and SPI adhesive. Thermogravimetric profile of the adhesive samples showed that the newly developed nanocomposite adhesive was thermally stable at a temperature up to about 600 °C at a higher percentage loading of 0.5 wt% CNTs. The result showed that sonication method of dispersion of CNTs into the SPI adhesive had a higher shear strength compared to the mechanical method of dispersion both at dry and wet state.

  12. Diamagnetic levitation causes changes in the morphology, cytoskeleton, and focal adhesion proteins expression in osteocytes.

    PubMed

    Qian, A R; Wang, L; Gao, X; Zhang, W; Hu, L F; Han, J; Li, J B; Di, S M; Shang, Peng

    2012-01-01

    Diamagnetic levitation technology is a novel simulated weightless technique and has recently been applied in life-science research. We have developed a superconducting magnet platform with large gradient high magnetic field (LG-HMF), which can provide three apparent gravity levels, namely, μg (diamagnetic levitation), 1g, and 2g for diamagnetic materials. In this study, the effects of LG-HMF on the activity, morphology, and cytoskeleton (actin filament, microtubules, and vimentin intermediate filaments) in osteocyte - like cell line MLO-Y4 were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) methods, hematoxylin-eosin (HE) staining, and laser scanning confocal microscopy (LSCM), respectively. The changes induced by LG-HMF in distribution and expression of focal adhesion (FA) proteins, including vinculin, paxillin, and talin in MLO-Y4 were determined by LSCM and Western blotting. The results showed that LG-HMF produced by superconducting magnet had no lethal effects on MLO-Y4. Compared to control, diamagnetic levitation (μg) affected MLO-Y4 morphology, nucleus size, cytoskeleton architecture, and FA proteins distribution and expression. The study indicates that osteocytes are sensitive to altered gravity and FA proteins (vinculin, paxillin, and talin) may be involved in osteocyte mechanosensation. The diamagnetic levitation may be a novel ground-based space-gravity simulator and can be used for biological experiment at cellular level. PMID:21216704

  13. Medium-density particleboards from modified rice husks and soybean protein concentrate-based adhesives.

    PubMed

    Ciannamea, Emiliano M; Stefani, Pablo M; Ruseckaite, Roxana A

    2010-01-01

    The main goal of this work was to evaluate the technical feasibility of using rice husk (RH) as wood substitute in the production of environmentally sound medium-density particleboards using adhesives from soybean protein concentrate (SPC). Chemical modification of rice husk with sodium hydroxide and sodium hydroxide followed by hydrogen peroxide (bleaching) were undertaken to evaluate the effect of such treatments on the composition and topology of rice husk and the performance of produced panels. Both treatments were efficient in partially eliminating hemicelluloses, lignin and silica from RH, as evidenced by thermo-gravimetric analysis (TGA). Scanning electron microscopy observations suggested that alkaline treatment resulted in a more damaged RH substrate than bleaching. The dependence of mechanical properties (modulus of rupture, modulus of elasticity, and internal bond) and the physical properties (water absorption and thickness swelling) on chemical treatments performed on both, rice husk and SPC was studied. Bleached-rice husk particleboards bonded with alkaline-treated soybean protein concentrate displayed the best set of final properties. Particleboards with this formulation met the minimum requirements of internal bond, modulus of elasticity and modulus of rupture recommended by the US Standard ANSI/A208.1 specifications for M1, MS and M2-grade medium-density particleboards, but failed to achieve the thickness swelling value recommended for general use panels. This limitation of soybean protein concentrate-bonded rice husk particleboards was counterbalanced by the advantage of being formaldehyde-free which makes them a suitable alternative for indoor applications. PMID:19766482

  14. [AMP-activated protein kinase activation regulates adhesion of monocytes to vascular endothelial cells and the underlying mechanism].

    PubMed

    Bai, Hong-Bo; Wang, Yun; Zhang, Yu-Hua; Zhang, Yuan

    2016-02-25

    The present study was aimed to explore the effect of AMP-activated protein kinase (AMPK) on monocyte adhesion to vascular endothelial cells and underlying molecular mechanism. Tumor necrosis factor α (TNFα)-activated human aortic endothelial cells (HAECs) were treated with different concentrations of AMPK agonist 5-Aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) or AMPK inhibitor compound C. And other HAECs were overexpressed with constitutive active or dominant negative AMPK protein and then treated with TNFα. The rates of monocytes adhering to endothelial cells were detected by fluorescent staining. Intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) mRNA levels and protein secretions were detected by quantitative PCR and ELISA, respectively. Acetylation of NF-κB p65 at lysine 221 site was assessed by Western blot. NF-κB p65 DNA binding activity was analyzed by an ELISA-based method. By using small interfering RNA based strategy, p300 expression in HAECs was down-regulated and then cells were incubated with TNFα. NF-κB p65 DNA binding activity, ICAM-1 and VCAM-1 expressions and adhesion rates were detected, respectively. The activity of p300 was also detected by ELISA. The results showed that AICAR treatment significantly reduced monocyte-endothelial adhesion rate, as well as ICAM-1 and VCAM-1 mRNA levels and protein secretions, in TNFα-activated HAECs. Moreover, transfection of constitutive active AMPKα but not dominant negative AMPKα strongly diminished TNFα-induced upregulation of ICAM-1 and VCAM-1 mRNA expressions and secretions, as well as monocyte-endothelial adhesion. Furthermore, AMPK activation decreased TNFα-mediated acetylation of NF-κB p65 at Lys221 site and reduced NF-κB p65 DNA binding activity. Silencing p300 by siRNA significantly abolished the effect of TNFα- induced adhesion molecules expression and monocyte-endothelial adhesion. Blocking AMPK activation by compound C almost

  15. The Terminal A Domain of the Fibrillar Accumulation-Associated Protein (Aap) of Staphylococcus epidermidis Mediates Adhesion to Human Corneocytes▿

    PubMed Central

    Macintosh, Robin L.; Brittan, Jane L.; Bhattacharya, Ritwika; Jenkinson, Howard F.; Derrick, Jeremy; Upton, Mathew; Handley, Pauline S.

    2009-01-01

    The opportunistic pathogen Staphylococcus epidermidis colonizes indwelling medical devices by biofilm formation but is primarily a skin resident. In many S. epidermidis strains biofilm formation is mediated by a cell wall-anchored protein, the accumulation-associated protein (Aap). Here, we investigate the role of Aap in skin adhesion. Aap is an LPXTG protein with a domain architecture including a terminal A domain and a B-repeat region. S. epidermidis NCTC 11047 expresses Aap as localized, lateral tufts of fibrils on one subpopulation of cells (Fib+), whereas a second subpopulation does not express these fibrils of Aap (Fib−). Flow cytometry showed that 72% of NCTC 11047 cells expressed Aap and that 28% of cells did not. Aap is involved in the adhesion of Fib+ cells to squamous epithelial cells from the hand (corneocytes), as the recombinant A-domain protein partially blocked binding to corneocytes. To confirm the role of the Aap A domain in corneocyte attachment, Aap was expressed on the surface of Lactococcus lactis MG1363 as sparsely distributed, peritrichous fibrils. The expression of Aap increased corneocyte adhesion 20-fold compared to L. lactis carrying Aap without an A domain. S. epidermidis isolates from catheters, artificial joints, skin, and the nose also used the A domain of Aap to adhere to corneocytes, emphasizing the role of Aap in skin adhesion. In addition, L. lactis expressing Aap with different numbers of B repeats revealed a positive correlation between the number of B repeats and adhesion to corneocytes, suggesting an additional function for the B region in enhancing A-domain-dependent attachment to skin. Therefore, in addition to its established role in biofilm formation, Aap can also promote adhesion to corneocytes and is likely to be an important adhesin in S. epidermidis skin colonization. PMID:19749046

  16. Energy metabolism regulates clathrin adaptors at the trans-Golgi network and endosomes

    PubMed Central

    Aoh, Quyen L.; Hung, Chao-wei; Duncan, Mara C.

    2013-01-01

    Glucose is a master regulator of cell behavior in the yeast Saccharomyces cerevisiae. It acts as both a metabolic substrate and a potent regulator of intracellular signaling cascades. Glucose starvation induces the transient delocalization and then partial relocalization of clathrin adaptors at the trans-Golgi network and endosomes. Although these localization responses are known to depend on the protein kinase A (PKA) signaling pathway, the molecular mechanism of this regulation is unknown. Here we demonstrate that PKA and the AMP-regulated kinase regulate adaptor localization through changes in energy metabolism. We show that genetic and chemical manipulation of intracellular ATP levels cause corresponding changes in adaptor localization. In permeabilized cells, exogenous ATP is sufficient to induce adaptor localization. Furthermore, we reveal distinct energy-dependent steps in adaptor localization: a step that requires the ADP-ribosylation factor ARF, an ATP-dependent step that requires the phosphatidyl-inositol-4 kinase Pik1, and third ATP-dependent step for which we provide evidence but for which the mechanism is unknown. We propose that these energy-dependent mechanisms precisely synchronize membrane traffic with overall proliferation rates and contribute a crucial aspect of energy conservation during acute glucose starvation. PMID:23345590

  17. A protein interaction map for cell-cell adhesion regulators identifies DUSP23 as a novel phosphatase for β-catenin

    PubMed Central

    Gallegos, Lisa Leon; Ng, Mei Rosa; Sowa, Mathew E.; Selfors, Laura M.; White, Anne; Zervantonakis, Ioannis K.; Singh, Pragya; Dhakal, Sabin; Harper, J. Wade; Brugge, Joan S.

    2016-01-01

    Cell-cell adhesion is central to morphogenesis and maintenance of epithelial cell state. We previously identified 27 candidate cell-cell adhesion regulatory proteins (CCARPs) whose down-regulation disrupts epithelial cell-cell adhesion during collective migration. Using a protein interaction mapping strategy, we found that 18 CCARPs link to core components of adherens junctions or desmosomes. We further mapped linkages between the CCARPs and other known cell-cell adhesion proteins, including hits from recent screens uncovering novel components of E-cadherin adhesions. Mechanistic studies of one novel CCARP which links to multiple cell-cell adhesion proteins, the phosphatase DUSP23, revealed that it promotes dephosphorylation of β-catenin at Tyr 142 and enhances the interaction between α- and β-catenin. DUSP23 knockdown specifically diminished adhesion to E-cadherin without altering adhesion to fibronectin matrix proteins. Furthermore, DUSP23 knockdown produced “zipper-like” cell-cell adhesions, caused defects in transmission of polarization cues, and reduced coordination during collective migration. Thus, this study identifies multiple novel connections between proteins that regulate cell-cell interactions and provides evidence for a previously unrecognized role for DUSP23 in regulating E-cadherin adherens junctions through promoting the dephosphorylation of β-catenin. PMID:27255161

  18. Non-cleavable talin rescues defect in the T-cell conjugation of T-cells deficient in the immune adaptor SKAP1

    PubMed Central

    Lim, Daina; Lu, Yuning; Rudd, Christopher E.

    2016-01-01

    While the cytoskeletal protein talin binds to the β-chain of LFA-1, the immune cell adaptor SKAP1 (SKAP-55) binds to the α-chain of the same integrin via RapL. Whereas calpain protease cleavage of talin is important for LFA-1 activation, it has been unclear whether SKAP1 can alter the function of talin or its associated adaptor RIAM in T-cells. In this paper, we report that Skap1−/− T-cells showed a reduction in the translocation of talin and RIAM to the contact interface of T-cells with antigenic beads or dendritic cells (DCs) presenting OVA peptide to OT-1 T-cells. In addition, Skap1−/− T-cells show an altered pattern of talin cleavage, while the expression of a cleavage resistant form of talin (L432G) restored the impaired adhesion of OT1 transgenic Skap1−/− T-cells with DCs. SKAP1 therefore can affect the function of talin in T-cells needed for optimal T-cell/DC conjugation. PMID:26905930

  19. Dihydromunduletone Is a Small-Molecule Selective Adhesion G Protein-Coupled Receptor Antagonist.

    PubMed

    Stoveken, Hannah M; Bahr, Laura L; Anders, M W; Wojtovich, Andrew P; Smrcka, Alan V; Tall, Gregory G

    2016-09-01

    Adhesion G protein-coupled receptors (aGPCRs) have emerging roles in development and tissue maintenance and is the most prevalent GPCR subclass mutated in human cancers, but to date, no drugs have been developed to target them in any disease. aGPCR extracellular domains contain a conserved subdomain that mediates self-cleavage proximal to the start of the 7-transmembrane domain (7TM). The two receptor protomers, extracellular domain and amino terminal fragment (NTF), and the 7TM or C-terminal fragment remain noncovalently bound at the plasma membrane in a low-activity state. We recently demonstrated that NTF dissociation liberates the 7TM N-terminal stalk, which acts as a tethered-peptide agonist permitting receptor-dependent heterotrimeric G protein activation. In many cases, natural aGPCR ligands are extracellular matrix proteins that dissociate the NTF to reveal the tethered agonist. Given the perceived difficulty in modifying extracellular matrix proteins to create aGPCR probes, we developed a serum response element (SRE)-luciferase-based screening approach to identify GPR56/ADGRG1 small-molecule inhibitors. A 2000-compound library comprising known drugs and natural products was screened for GPR56-dependent SRE activation inhibitors that did not inhibit constitutively active Gα13-dependent SRE activation. Dihydromunduletone (DHM), a rotenoid derivative, was validated using cell-free aGPCR/heterotrimeric G protein guanosine 5'-3-O-(thio)triphosphate binding reconstitution assays. DHM inhibited GPR56 and GPR114/ADGRG5, which have similar tethered agonists, but not the aGPCR GPR110/ADGRF1, M3 muscarinic acetylcholine, or β2 adrenergic GPCRs. DHM inhibited tethered peptide agonist-stimulated and synthetic peptide agonist-stimulated GPR56 but did not inhibit basal activity, demonstrating that it antagonizes the peptide agonist. DHM is a novel aGPCR antagonist and potentially useful chemical probe that may be developed as a future aGPCR therapeutic. PMID:27338081

  20. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.

    PubMed Central

    Takeshita, S; Kikuno, R; Tezuka, K; Amann, E

    1993-01-01

    A cDNA library prepared from the mouse osteoblastic cell line MC3T3-E1 was screened for the presence of specifically expressed genes by employing a combined subtraction hybridization/differential screening approach. A cDNA was identified and sequenced which encodes a protein designated osteoblast-specific factor 2 (OSF-2) comprising 811 amino acids. OSF-2 has a typical signal sequence, followed by a cysteine-rich domain, a fourfold repeated domain and a C-terminal domain. The protein lacks a typical transmembrane region. The fourfold repeated domain of OSF-2 shows homology with the insect protein fasciclin I. RNA analyses revealed that OSF-2 is expressed in bone and to a lesser extent in lung, but not in other tissues. Mouse OSF-2 cDNA was subsequently used as a probe to clone the human counterpart. Mouse and human OSF-2 show a high amino acid sequence conservation except for the signal sequence and two regions in the C-terminal domain in which 'in-frame' insertions or deletions are observed, implying alternative splicing events. On the basis of the amino acid sequence homology with fasciclin I, we suggest that OSF-2 functions as a homophilic adhesion molecule in bone formation. Images Figure 3 Figure 4 Figure 5 Figure 6 PMID:8363580

  1. Cell adhesion and growth enabled by biomimetic oligopeptide modification of a polydopamine-poly(ethylene oxide) protein repulsive surface.

    PubMed

    Musilkova, Jana; Kotelnikov, Ilya; Novotna, Katarina; Pop-Georgievski, Ognen; Rypacek, Frantisek; Bacakova, Lucie; Proks, Vladimir

    2015-11-01

    Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses. PMID:26449443

  2. Adsorption and adhesion of common serum proteins to nanotextured gallium nitride

    NASA Astrophysics Data System (ADS)

    Bain, Lauren E.; Hoffmann, Marc P.; Bryan, Isaac; Collazo, Ramón; Ivanisevic, Albena

    2015-01-01

    , particularly when considering biological systems. It is well known that thin films and nanostructures feature different optical, electrical, and mechanical properties from their bulk composites; however, interactions taking place at the interface between nanomaterials and their surroundings are less understood. Here, we explore interactions between common serum proteins - serum albumin, fibrinogen, and immunoglobulin G - and a nanotextured gallium nitride surface. Atomic force microscopy with a carboxyl-terminated colloid tip is used to probe the `activity' of proteins adsorbed onto the surface, including both the accessibility of the terminal amine to the tip as well as the potential for protein extension. By evaluating the frequency of tip-protein interactions, we can establish differences in protein behaviour on the basis of both the surface roughness as well as morphology, providing an assessment of the role of surface texture in dictating protein-surface interactions. Unidirectional surface features - either the half-unit cell steppes of as-grown GaN or those produced by mechanical polishing - appear to promote protein accessibility, with a higher frequency of protein extension events taking place on these surfaces when compared with less ordered surface features. Development of a full understanding of the factors influencing surface-biomolecule interactions can pave the way for specific surface modification to tailor the bio-material interface, offering a new path for device optimization. Electronic supplementary information (ESI) available: Additional figures demonstrating the adhesion force magnitude (Fig. S1) and lateral steppe surface topography (Fig. S2). See DOI: 10.1039/c4nr06353h

  3. Adsorption of parotid saliva proteins and adhesion of Streptococcus mutans ATCC 21752 to dental fiber-reinforced composites.

    PubMed

    Tanner, Johanna; Carlén, Anette; Söderling, Eva; Vallittu, Pekka K

    2003-07-15

    The use of fiber-reinforced composites (FRC) in dentistry has increased during recent years. In marginal areas of crowns and removable partial dentures the fibers may become exposed and come into contact with oral tissues, saliva, and microbes. To date, few articles have been published on oral microbial adhesion to FRCs. The aim of this study was to compare different FRCs, their components, and conventional restorative materials with respect to S. mutans ATCC 21752 adhesion and adsorption of specific S. mutans binding proteins. Surface roughness of the materials was also determined. Four different FRCs, a restorative composite, and a high-leucite ceramic material were studied. Polyethylene FRC was found to be significantly rougher than all other materials. Aramid FRC also showed higher surface roughness in comparison with all materials but polyethylene FRC. Without a saliva pellicle, adhesion of S. mutans coincided with surface roughness and polyethylene and aramid FRC promoted S. mutans adhesion better than the other smoother materials. In the presence of salivary pellicle, ceramic and polyethylene FRC bound more bacteria than the other materials studied. Higher quantities of S. mutans binding proteins in the pellicles may in part account for the higher S. mutans adhesion to saliva-coated ceramic and polyethylene FRC. PMID:12808599

  4. Shugoshins: Tension-Sensitive Pericentromeric Adaptors Safeguarding Chromosome Segregation

    PubMed Central

    2014-01-01

    The shugoshin/Mei-S332 family are proteins that associate with the chromosomal region surrounding the centromere (the pericentromere) and that play multiple and distinct roles in ensuring the accuracy of chromosome segregation during both mitosis and meiosis. The underlying role of shugoshins appears to be to serve as pericentromeric adaptor proteins that recruit several different effectors to this region of the chromosome to regulate processes critical for chromosome segregation. Crucially, shugoshins undergo changes in their localization in response to the tension that is exerted on sister chromosomes by the forces of the spindle that will pull them apart. This has led to the idea that shugoshins provide a platform for activities required at the pericentromere only when sister chromosomes lack tension. Conversely, disassembly of the shugoshin pericentromeric platform may provide a signal that sister chromosomes are under tension. Here the functions and regulation of these important tension-sensitive pericentromeric proteins are discussed. PMID:25452306

  5. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    PubMed

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states. PMID:19189961

  6. Overexpression of vascular adhesion protein-1 is associated with poor prognosis of astrocytomas.

    PubMed

    Kostoro, Joanna; Chang, Shu-Jyuan; Clark Lai, Yen-Chang; Wu, Chun-Chieh; Chai, Chee-Yin; Kwan, Aij-Lie

    2016-06-01

    Vascular adhesion protein-1 (VAP-1) is one of the endothelial adhesion molecules that is believed to play a role in tumor progression and metastasis, supporting cancer cell extravasation. Very few studies have been performed on analyzing the contribution of VAP-1 in brain tumor. Astrocytomas are the most common type of brain tumors, which are classified by World Health Organization (WHO) into four grades according to the degree of malignancy. This study was designed to investigate VAP-1 expression level in different astrocytoma grades and its correlation with clinicopathological features as well as prognosis of astrocytoma patients. Eighty-seven patients with different grades of astrocytoma (WHO Grade I-Grade IV) were enrolled in this study. The expression of VAP-1 was assayed by immunohistochemistry. The correlation between VAP-1 expression and clinicopathological features was evaluated by Chi-square test, and overall survival was analyzed by Kaplan-Meier method. Cox regression analysis was applied to analyze the independent influence of each parameter on overall survival. The expression level of VAP-1 was significantly higher in diffuse astrocytoma than those of pilocytic astrocytoma (p < 0.0001). In the subgroup analysis, upregulated VAP-1 expression was frequently found in older age patients (≥50 years). The VAP-1 expression was found to be significantly correlated with the overall survival (p = 0.0002). There was a statistical correlation between VAP-1(high) tumors in diffuse astrocytoma and VAP-1(low) tumors in pilocytic astrocytoma (p < 0.0001). Multivariate Cox analysis indicated VAP-1 was an independent predictive marker for poorer prognosis (p = 0.0036). Therefore, VAP-1 could be a promising prognostic biomarker in astrocytoma. PMID:26935340

  7. Scanning-force techniques to monitor time-dependent changes in topography and adhesion force of proteins on surfaces.

    PubMed

    Mondon, M; Berger, S; Ziegler, C

    2003-04-01

    Scanning-force microscopy (SFM) investigations were conducted to probe the influences of the interactions of proteins with surfaces relevant in medicine. These interactions are an important feature in the area of biofilm formation. The adsorption of proteins leads to changes in topography, which was monitored for the build up of protein layers of hen egg-white lysozyme and bovine serum albumin (BSA) on mica in real time in phosphate-buffered aqueous solution over a time period of 10 min. Phase imaging was additionally applied to compare material contrasts and to evaluate this method for further application in this field. The adhesion forces that develop on a time scale below 20 s between a protein-modified SFM tip and titanium surfaces (TiO(2), TiAl6V4 and TiAl6Nb7) were investigated. The influences of the parameters loading force and interaction time between the protein and the surface were monitored as well as the influence of protein structure. The interaction time dependency of the adhesion force could be described with a kinetic model of two consecutive first-order reactions. For the maximal adhesion force a correlation to the ratio of the amino acids cysteine, proline and glycine has been proposed. PMID:12707750

  8. Platelet adhesion signalling and the regulation of thrombus formation.

    PubMed

    Gibbins, Jonathan M

    2004-07-15

    Platelets perform a central role in haemostasis and thrombosis. They adhere to subendothelial collagens exposed at sites of blood vessel injury via the glycoprotein (GP) Ib-V-IX receptor complex, GPVI and integrin alpha(2)beta(1). These receptors perform distinct functions in the regulation of cell signalling involving non-receptor tyrosine kinases (e.g. Src, Fyn, Lyn, Syk and Btk), adaptor proteins, phospholipase C and lipid kinases such as phosphoinositide 3-kinase. They are also coupled to an increase in cytosolic calcium levels and protein kinase C activation, leading to the secretion of paracrine/autocrine platelet factors and an increase in integrin receptor affinities. Through the binding of plasma fibrinogen and von Willebrand Factor to integrin alpha(IIb)beta(3), a platelet thrombus is formed. Although increasing evidence indicates that each of the adhesion receptors GPIb-V-IX and GPVI and integrins alpha(2)beta(1) and alpha(IIb)beta(3) contribute to the signalling that regulates this process, the individual roles of each are only beginning to be dissected. By contrast, adhesion receptor signalling through platelet endothelial cell adhesion molecule 1 (PECAM-1) is implicated in the inhibition of platelet function and thrombus formation in the healthy circulation. Recent studies indicate that understanding of platelet adhesion signalling mechanisms might enable the development of new strategies to treat and prevent thrombosis. PMID:15252124

  9. Early Growth Response Protein 1 Promotes Restenosis by Upregulating Intercellular Adhesion Molecule-1 in Vein Graft

    PubMed Central

    Zhang, Kui; Cao, Jian; Dong, Ran; Du, Jie

    2013-01-01

    Objectives. To verify the relationship between Egr-1 and vein graft restenosis and investigate the related mechanisms. Methods. Mouse vein graft models were established in Egr-1 knockout (KO) and wild-type (WT) mice. The vein grafts in the mice were taken for pathological examination and immunohistochemical analysis. The endothelial cells (ECs) were stimulated by using a computer-controlled cyclic stress unit. BrdU staining and PCR were used to detect ECs proliferation activity and Egr-1 and ICAM-1 mRNA expression, respectively. Western-blot analysis was also used to detect expression of Egr-1 and intercellular adhesion molecule-1 (ICAM-1) proteins. Results. The lumens of vein grafts in Egr-1 KO mice were wider than in WT mice. ECs proliferation after mechanical stretch stimulation was suppressed by Egr-1 knockout (P < 0.05). Both in vein grafts and ECs from WT mice after mechanical stretch stimulation, mRNA expression and protein of Egr-1 and ICAM-1 showed increases (P < 0.05). However, ICAM-1 expression was significantly suppressed in ECs from Egr-1 knockout mice (P < 0.05). Conclusions. Egr-1 may promote ECs proliferation and result in vein graft restenosis by upregulating the expression of ICAM-1. As a key factor of vein graft restenosis, it could be a target for the prevention of restenosis after CABG surgery. PMID:24386503

  10. Neural Cell Adhesion Protein CNTN1 Promotes the Metastatic Progression of Prostate Cancer.

    PubMed

    Yan, Judy; Ojo, Diane; Kapoor, Anil; Lin, Xiaozeng; Pinthus, Jehonathan H; Aziz, Tariq; Bismar, Tarek A; Wei, Fengxiang; Wong, Nicholas; De Melo, Jason; Cutz, Jean-Claude; Major, Pierre; Wood, Geoffrey; Peng, Hao; Tang, Damu

    2016-03-15

    Prostate cancer metastasis is the main cause of disease-related mortality. Elucidating the mechanisms underlying prostate cancer metastasis is critical for effective therapeutic intervention. In this study, we performed gene-expression profiling of prostate cancer stem-like cells (PCSC) derived from DU145 human prostate cancer cells to identify factors involved in metastatic progression. Our studies revealed contactin 1 (CNTN1), a neural cell adhesion protein, to be a prostate cancer-promoting factor. CNTN1 knockdown reduced PCSC-mediated tumor initiation, whereas CNTN1 overexpression enhanced prostate cancer cell invasion in vitro and promoted xenograft tumor formation and lung metastasis in vivo. In addition, CNTN1 overexpression in DU145 cells and corresponding xenograft tumors resulted in elevated AKT activation and reduced E-cadherin (CDH1) expression. CNTN1 expression was not readily detected in normal prostate glands, but was clearly evident on prostate cancer cells in primary tumors and lymph node and bone metastases. Tumors from 637 patients expressing CNTN1 were associated with prostate cancer progression and worse biochemical recurrence-free survival following radical prostatectomy (P < 0.05). Collectively, our findings demonstrate that CNTN1 promotes prostate cancer progression and metastasis, prompting further investigation into the mechanisms that enable neural proteins to become aberrantly expressed in non-neural malignancies. PMID:26795349

  11. Interfacial tension of complex coacervated mussel adhesive protein according to the Hofmeister series.

    PubMed

    Lim, Seonghye; Moon, Dustin; Kim, Hyo Jeong; Seo, Jeong Hyun; Kang, In Seok; Cha, Hyung Joon

    2014-02-01

    Complex coacervation is a liquid-liquid phase separation in a colloidal system of two oppositely charged polyelectrolytes or colloids. The interfacial tension of the coacervate phase is the key parameter for micelle formation and interactions with the encapsulating material. However, the relationship between interfacial tensions and various salt solutions is poorly understood in complex coacervation. In the present work, the complex coacervate dynamics of recombinant mussel adhesive protein (MAP) with hyaluronic acid (HA) were determined in the presence of Hofmeister series salt ions. Using measurements of absorbance, hydrodynamic diameter, capillary force, and receding contact angle in the bulk phase, the interfacial tensions of complex coacervated MAP/HA were determined to be 0.236, 0.256, and 0.287 mN/m in 250 mM NaHCOO, NaCl, and NaNO3 solutions, respectively. The sequences of interfacial tensions and contact angles of the complex coacervates in the presence of three sodium salts with different anions were found to follow the Hofmeister ordering. The tendency of interfacial tension between the coacervate and dilute phases in the presence of different types of Hofmeister salt ions could provide a better understanding of Hofmeister effects on complex coacervated materials based on the protein-polysaccharide system. This information can also be utilized for microencapsulation and adsorption by controlling intramolecular interactions. In addition, the injection molding dynamics of mussel byssus formation was potentially explained based on the measured interfacial tension of coacervated MAP. PMID:24490867

  12. 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.

  13. [Expression and purification of an adhesive protein of rabbit Pasteurella multocida C51-3 and detection of its antigenicity].

    PubMed

    Nazierbieke, Wulumuhan; Yan, Fang; He, Cui; Zhang, Lei; Borrathybay, Entomack

    2008-08-01

    The cp36 gene encoding an adhesive protein was amplified by PCR from genomic DNA of rabbit P. multocida C51-3 strain, and cloned into the pMD18-T vector and then sequenced. The mature adhesive protein without a signal peptide of cpm36 gene was amplified by PCR from the recombinant plasmid pMD18-cp36, then cloned into the prokaryotic expression vector pQE30 to provide a recombinant plasmid pQE30-cpm36. The recombinant protein of CPM36 was produced in Escherichia coli M15 harboring the recombinant plasmid pQE30-cpm36 by IPTG induction, and the recombinant protein purified by the affinity chromatography with Ni(2+)-NTA resin. The sequence analyses showed that the ORF of cp36 gene was 1032 bp in length, and DNA homology of the cp36 genes between the C51-3 strain and the previously reported different serotype strains of P. multocida in GenBank was 76.9 to 100%. The SDS-PAGE analyses revealed a single fusion protein band with a molecular weight of 37 kD, and the Western blotting analysis demonstrated that the recombinant protein CPM36 and native 36 kD protein of C51-3 were recognized specifically by an antiserum against the recombinant protein, suggesting that the recombinant protein is an antigenic protein. PMID:18998549

  14. Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

    PubMed Central

    Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

    2013-01-01

    Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

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

    PubMed Central

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

    2011-01-01

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

  16. Multiscale approaches to protein-mediated interactions between membranes—relating microscopic and macroscopic dynamics in radially growing adhesions

    NASA Astrophysics Data System (ADS)

    Bihr, Timo; Seifert, Udo; Smith, Ana-Sunčana

    2015-08-01

    Macromolecular complexation leading to coupling of two or more cellular membranes is a crucial step in a number of biological functions of the cell. While other mechanisms may also play a role, adhesion always involves the fluctuations of deformable membranes, the diffusion of proteins and the molecular binding and unbinding. Because these stochastic processes couple over a multitude of time and length scales, theoretical modeling of membrane adhesion has been a major challenge. Here we present an effective Monte Carlo scheme within which the effects of the membrane are integrated into local rates for molecular recognition. The latter step in the Monte Carlo approach enables us to simulate the nucleation and growth of adhesion domains within a system of the size of a cell for tens of seconds without loss of accuracy, as shown by comparison to 106 times more expensive Langevin simulations. To perform this validation, the Langevin approach was augmented to simulate diffusion of proteins explicitly, together with reaction kinetics and membrane dynamics. We use the Monte Carlo scheme to gain deeper insight to the experimentally observed radial growth of micron sized adhesion domains, and connect the effective rate with which the domain is growing to the underlying microscopic events. We thus demonstrate that our technique yields detailed information about protein transport and complexation in membranes, which is a fundamental step toward understanding even more complex membrane interactions in the cellular context.

  17. Human heat shock protein (Hsp) 90 interferes with Neisseria meningitidis adhesin A (NadA)-mediated adhesion and invasion.

    PubMed

    Montanari, Paolo; Bozza, Giuseppe; Capecchi, Barbara; Caproni, Elena; Barrile, Riccardo; Norais, Nathalie; Capitani, Mirco; Sallese, Michele; Cecchini, Paola; Ciucchi, Laura; Gao, Zhenai; Rappuoli, Rino; Pizza, Mariagrazia; Aricò, Beatrice; Merola, Marcello

    2012-03-01

    NadA (N eisseria meningitidisadhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. PMID:22066472

  18. An occludin-focal adhesion kinase protein complex at the blood-testis barrier: a study using the cadmium model.

    PubMed

    Siu, Erica R; Wong, Elissa W P; Mruk, Dolores D; Sze, K L; Porto, Catarina S; Cheng, C Yan

    2009-07-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII-IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl(2) to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  19. An Occludin-Focal Adhesion Kinase Protein Complex at the Blood-Testis Barrier: A Study Using the Cadmium Model

    PubMed Central

    Siu, Erica R.; Wong, Elissa W. P.; Mruk, Dolores D.; Sze, K. L.; Porto, Catarina S.; Cheng, C. Yan

    2009-01-01

    Several integral membrane proteins that constitute the blood-testis barrier (BTB) in mammalian testes, in particular rodents, are known to date. These include tight junction (TJ) proteins (e.g. occludin, junctional adhesion molecule-A, claudins), basal ectoplasmic specialization proteins (e.g. N-cadherin), and gap junction proteins (e.g. connexin43). However, the regulators (e.g. protein kinases and phosphatases) that affect these proteins, such as their interaction with the cytoskeletal actin, which in turn confer cell adhesion at the TJ, remain largely unknown. We report herein that focal adhesion kinase (FAK) is a putative interacting partner of occludin, but not claudin-11 or junctional adhesion molecule-A. Immunohistochemistry and fluorescence microscopy studies illustrated that the expression of FAK in the seminiferous epithelium of adult rat testes was stage specific. FAK colocalized with occludin at the BTB in virtually all stages of the seminiferous epithelial cycle but considerably diminished in stages VIII–IX, at the time of BTB restructuring to facilitate the transit of primary leptotene spermatocytes. Using Sertoli cells cultured in vitro with established TJ-permeability barrier and ultrastructures of TJ, basal ectoplasmic specialization and desmosome-like junction that mimicked the BTB in vivo, FAK was shown to colocalize with occludin and zonula occludens-1 (ZO-1) at the Sertoli-Sertoli cell interface. When these Sertoli cell cultures were treated with CdCl2 to perturb the TJ-barrier function, occludin underwent endocytic-mediated internalization in parallel with FAK and ZO-1. Thus, these findings demonstrate that FAK is an integrated regulatory component of the occludin-ZO-1 protein complex, suggesting that functional studies can be performed to study the role of FAK in BTB dynamics. PMID:19213829

  20. Actions of translocator protein ligands on neutrophil adhesion and motility induced by G-protein coupled receptor signaling.

    PubMed

    de Lima, Camila Bento; Tamura, Eduardo K; Montero-Melendez, Trindad; Palermo-Neto, João; Perretti, Mauro; Markus, Regina P; Farsky, Sandra Helena Poliselli

    2012-01-13

    The 18 kDa translocator protein (TSPO) also known as the peripheral benzodiazepine receptor (PBR), mediates the transportation of cholesterol and anions from the outer to the inner mitochondrial membrane in different cells types. Although recent evidences indicate a potential role for TSPO in the development of inflammatory processes, the mechanisms involved have not been elucidated. The present study investigated the ability of the specific TSPO ligands, the isoquinoline carboxamide PK11195 and benzodiazepine Ro5-4864, on neutrophil recruitment promoted by the N-formylmethionyl-leucyl-phenylalanine peptide (fMLP), an agonist of G-protein coupled receptor (GPCR). Pre-treatment with Ro5-4864 abrograted fMLP-induced leukocyte-endothelial interactions in mesenteric postcapillary venules in vivo. Moreover, in vitro Ro5-4864 treatment prevented fMLP-induced: (i) L-selectin shedding and overexpression of PECAM-1 on the neutrophil cell surface; (ii) neutrophil chemotaxis and (iii) enhancement of intracellular calcium cations (iCa(+2)). Intriguingly, the two latter effects were augmented by cell treatment with PK11195. An allosteric agonist/antagonist relation may be suggested, as the effects of Ro5-4864 on fMLP-stimulated neutrophils were reverted by simultaneous treatment with PK11195. Taken together, these data highlight TSPO as a modulator of pathways of neutrophil adhesion and locomotion induced by GPCR, connecting TSPO actions and the onset of an innate inflammatory response. PMID:22209795

  1. Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions

    PubMed Central

    Bouchet, Benjamin P; Gough, Rosemarie E; Ammon, York-Christoph; van de Willige, Dieudonnée; Post, Harm; Jacquemet, Guillaume; Altelaar, AF Maarten; Heck, Albert JR; Goult, Benjamin T; Akhmanova, Anna

    2016-01-01

    The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5β and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules. DOI: http://dx.doi.org/10.7554/eLife.18124.001 PMID:27410476

  2. Composites containing albumin protein or cyanoacrylate adhesives and biodegradable scaffolds: II. In vivo wound closure study in a rat model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Heintzelman, Douglas L.; Duffy, Mark T.; Bloom, Jeffrey N.; Soller, Eric C.; Gilmour, Travis M.; Hoffman, Grant T.; Edward, Deepak

    2004-07-01

    Our Scaffold-Enhanced Biological Adhesive (SEBA) system was investigated as an alternative to sutures or adhesives alone for repair of wounds. Two scaffold materials were investigated: (i) a synthetic biodegradable material fabricated from poly(L-lactic-co-glycolic acid); and (ii) a biologic material, small intestinal submucosa, manufactured by Cook BioTech. Two adhesive materials were also investigated: (i) a biologic adhesive composed of 50%(w/v) bovine serum albumin solder and 0.5mg/ml indocyanine green dye mixed in deionized water, and activated with an 808-nm diode laser; and (ii) Ethicon"s Dermabond, a 2-octyl-cyanoacrylate. The tensile strength and time-to-failure of skin incisions repaired in vivo in a rat model were measured at seven days postoperative. Incisions closed by protein solder alone, by Dermabond alone, or by suture, were also tested for comparison. The tensile strength of repairs formed using the SEBA system were 50% to 65% stronger than repairs formed by suture or either adhesive alone, with significantly less variations within each experimental group (average standard deviations of 15% for SEBA versus 38% for suture and 28% for adhesive alone). In addition, the time-to-failure curves showed a longevity not previously seen with the suture or adhesive alone techniques. The SEBA system acts to keep the dermis in tight apposition during the critical early phase of wound healing when tissue gaps are bridged by scar and granulation tissue. It has the property of being more flexible than either of the adhesives alone and may allow the apposed edges to move in conjunction with each other as a unit for a longer period of time and over a greater range of stresses than adhesives alone. This permits more rapid healing and establishment of integrity since the microgaps between the dermis edges are significantly reduced. By the time the scaffolds are sloughed from the wound site, there is greater strength and healing than that produced by adhesive alone or

  3. Lymphocyte binding to vascular endothelium in inflamed skin revisited: a central role for vascular adhesion protein-1 (VAP-1).

    PubMed

    Arvilommi, A M; Salmi, M; Kalimo, K; Jalkanen, S

    1996-04-01

    The binding of leukocytes to vascular endothelium and their migration into tissues is mediated by adhesion molecules on the endothelial cells and leukocytes. Vascular adhesion protein-1 (VAP-1) is a 170-180/90-kDa endothelial molecule expressed most prominently in high endothelial venules in peripheral lymph node (PLN) type lymphatic tissues. VAP-1 mediates lymphocyte binding to PLN, tonsil and synovium. The expression of VAP-1 is induced in inflammatory diseases such as arthritis and gut inflammation. We examined the expression, structure and function of VAP-1 in normal and inflamed skin and compared it to those of other adhesion molecules implicated in skin homing. In psoriasis lichen ruber planus, pemphigoid and allergic lesions, VAP-1 was markedly upregulated. The expression of VAP-1 was also increased in biopsies of healthy skin of the patients. The VAP-1 molecule induced in skin is decorated with abundant sialic acids. VAP-1 inflamed skin is functional, since inhibition with anti-VAP-1 monoclonal antibodies caused a 60% reduction in lymphocytes adhesion to vascular endothelium. Antibodies against E-selectin, which has been regarded as the major vascular addressin directing cutaneous lymphocyte traffic, and, surprisingly, against peripheral lymph node addressin (PNAd), caused inhibitions of 30% and 60%, respectively, in the frozen section adhesion assay. These findings suggest important roles also for VAP-1 and PNAd in lymphocyte homing into inflamed skin. PMID:8625974

  4. Different forms of human vascular adhesion protein-1 (VAP-1) in blood vessels in vivo and in cultured endothelial cells: implications for lymphocyte-endothelial cell adhesion models.

    PubMed

    Salmi, M; Jalkanen, S

    1995-10-01

    Vascular endothelium plays a pivotal role in controlling leukocyte extravasation from the blood into the tissues. Vascular adhesion protein-1 (VAP-1) is a novel endothelial cell molecule which mediates lymphocyte binding to the vascular lining (Salmi, M., and Jalkanen, S., Science 1992. 257:1407). In this study, we analyzed endothelial cell type-specific differences of VAP-1. In vivo, VAP-1 is a 90/170-kDa molecule which is mainly expressed on the lumenal surface and in cytoplasmic granules of peripheral lymph node-type postcapillary venules (high endothelial venules, HEV). In tonsil HEV, VAP-1 is modified with abundant sialic acids. VAP-1 is also detectable in the cytoplasm of human umbilical vein endothelial cells (HUVEC) and in an endothelial cell hybrid EaHy-926, although both cell types lack detectable surface VAP-1. Cultured endothelial cells do not express MECA-79-defined peripheral lymph node addressins either. VAP-1 was not translocated onto the endothelial cell surface after stimulation with multiple cytokines, mitogens or secretagogues which induced expression of other known endothelial adhesion molecules. Biochemical analyses revealed that VAP-1 is a approximately 180-kDa protein in these endothelial cell types. Digestions with neuraminidase, O-glycanase and N-glycanase, as well as treatment of cells with tunicamycin and benzyl-N-acetylgalactosaminide, did not alter the molecular mass of VAP-1 in EaHy-926. Pulse-chase experiments showed that VAP-1 is directly synthesized as a 180-kDa molecule without any detectable precursors. Thus, in cultured endothelial cells, VAP-1 is a 180-kDa protein which is devoid of post-translational modifications, and in particular, lacks the sialic acids crucial for the function of VAP-1 in tonsil vessels. Notably, the endothelial cell types commonly used as a model in studying lymphocyte-endothelial cell interactions lack surface expression of VAP-1 and peripheral node addressins, and hence are inherently of limited use in

  5. Focal adhesions are foci for tyrosine-based signal transduction via GIV/Girdin and G proteins

    PubMed Central

    Lopez-Sanchez, Inmaculada; Kalogriopoulos, Nicholas; Lo, I-Chung; Kabir, Firooz; Midde, Krishna K.; Wang, Honghui; Ghosh, Pradipta

    2015-01-01

    GIV/Girdin is a multimodular signal transducer and a bona fide metastasis-related protein. As a guanidine exchange factor (GEF), GIV modulates signals initiated by growth factors (chemical signals) by activating the G protein Gαi. Here we report that mechanical signals triggered by the extracellular matrix (ECM) also converge on GIV-GEF via β1 integrins and that focal adhesions (FAs) serve as the major hubs for mechanochemical signaling via GIV. GIV interacts with focal adhesion kinase (FAK) and ligand-activated β1 integrins. Phosphorylation of GIV by FAK enhances PI3K-Akt signaling, the integrity of FAs, increases cell–ECM adhesion, and triggers ECM-induced cell motility. Activation of Gαi by GIV-GEF further potentiates FAK-GIV-PI3K-Akt signaling at the FAs. Spatially restricted signaling via tyrosine phosphorylated GIV at the FAs is enhanced during cancer metastasis. Thus GIV-GEF serves as a unifying platform for integration and amplification of adhesion (mechanical) and growth factor (chemical) signals during cancer progression. PMID:26446841

  6. Myelin Basic Protein Cleaves Cell Adhesion Molecule L1 and Improves Regeneration After Injury.

    PubMed

    Lutz, David; Kataria, Hardeep; Kleene, Ralf; Loers, Gabriele; Chaudhary, Harshita; Guseva, Daria; Wu, Bin; Jakovcevski, Igor; Schachner, Melitta

    2016-07-01

    Myelin basic protein (MBP) is a serine protease that cleaves neural cell adhesion molecule L1 and generates a transmembrane L1 fragment which facilitates L1-dependent functions in vitro, such as neurite outgrowth, neuronal cell migration and survival, myelination by Schwann cells as well as Schwann cell proliferation, migration, and process formation. Ablation and blocking of MBP or disruption of its proteolytic activity by mutation of a proteolytically active serine residue abolish L1-dependent cellular responses. In utero injection of adeno-associated virus encoding proteolytically active MBP into MBP-deficient shiverer mice normalizes differentiation, myelination, and synaptogenesis in the developing postnatal spinal cord, in contrast to proteolytically inactive MBP. Application of active MBP to the injured wild-type spinal cord and femoral nerve augments levels of a transmembrane L1 fragment, promotes remyelination, and improves functional recovery after injury. Application of MBP antibody impairs recovery. Virus-mediated expression of active MBP in the lesion site after spinal cord injury results in improved functional recovery, whereas injection of virus encoding proteolytically inactive MBP fails to do so. The present study provides evidence for a novel L1-mediated function of MBP in the developing spinal cord and in the injured adult mammalian nervous system that leads to enhanced recovery after acute trauma. PMID:26081148

  7. Synergic interaction between amyloid precursor protein and neural cell adhesion molecule promotes neurite outgrowth

    PubMed Central

    Chen, Keping; Lu, Huixia; Gao, Tianli; Xue, Xiulei; Wang, Chunling; Miao, Fengqin

    2016-01-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases worldwide. The main features of AD are the pathological changes of density and distribution of intracellular neurofibrillary tangles (NFT) and extracellular amyloid plaques. The processing of amyloid beta precursor protein (APP) to β-amyloid peptide (Aβ) is one of the critical events in the pathogenesis of AD. In this study, we evaluated the role of the interaction of neural cell adhesion molecule (NCAM) and APP in neurite outgrowth using two different experimental systems: PC12E2 cells and hippocampal neurons that were isolated from wild type, APP knock-in and APP knock-out mice. PC12E2 cells or hippocampal neurons were co-cultured with NCAM-negative or NCAM-positive fibroblasts L929 cells. We found that APP promoted neurite outgrowth of PC12E2 cells and hippocampal neurons in either the presence or absence of NCAM. Secreted APP can rescue the neurite outgrowth in hippocampal neurons from APP knock-out mice. The interaction of APP and NCAM had synergic effect in promoting neurite outgrowth in both PC12E2 cells and hippocampal neurons. Our results suggested that the interaction of APP with NCAM played an important role in AD development and therefore could be a potential therapeutic target for AD treatment. PMID:26883101

  8. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis

    PubMed Central

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  9. Maximizing Fibroblast Adhesion on Protein-Coated Surfaces Using Microfluidic Cell Printing

    PubMed Central

    Davidoff, S.N.; Au, D.; Gale, B.K.; Brooks, B.D.; Brooks, A.E.

    2015-01-01

    translation of in vitro cell based assays to in vivo cellular response is imprecise at best. The advent of three-dimensional cell cultures in addition to bioreactor type microfluidics has improved the situation. However, these technical advances cannot be easily combined due to practical limitations. Development of a vertical microfluidic cell printer overcomes this obstacle, providing the ability to more closely recapitulate complex cellular environments and responses. As a proof of concept, we investigated the adhesion of fibroblasts under flow on protein-coated surfaces using a novel vertical microfluidic print head to isolate and manipulate both mechanical and biological factors as a model of fibroblast behavior during the foreign body response following implant insertion. A low flow rate with larger microfluidic channels onto a serum-coated surface has been determined to allow the highest density of viable fibroblasts to attach to the surface. While these insights into fibroblast surface attachment may lead to better material designs, the methods developed herein will certainly be useful as a biomaterials testing platform. PMID:26989480

  10. The Prion Protein Controls Polysialylation of Neural Cell Adhesion Molecule 1 during Cellular Morphogenesis.

    PubMed

    Mehrabian, Mohadeseh; Brethour, Dylan; Wang, Hansen; Xi, Zhengrui; Rogaeva, Ekaterina; Schmitt-Ulms, Gerold

    2015-01-01

    Despite its multi-faceted role in neurodegenerative diseases, the physiological function of the prion protein (PrP) has remained elusive. On the basis of its evolutionary relationship to ZIP metal ion transporters, we considered that PrP may contribute to the morphogenetic reprogramming of cells underlying epithelial-to-mesenchymal transitions (EMT). Consistent with this hypothesis, PrP transcription increased more than tenfold during EMT, and stable PrP-deficient cells failed to complete EMT in a mammalian cell model. A global comparative proteomics analysis identified the neural cell adhesion molecule 1 (NCAM1) as a candidate mediator of this impairment, which led to the observation that PrP-deficient cells fail to undergo NCAM1 polysialylation during EMT. Surprisingly, this defect was caused by a perturbed transcription of the polysialyltransferase ST8SIA2 gene. Proteomics data pointed toward β-catenin as a transcriptional regulator affected in PrP-deficient cells. Indeed, pharmacological blockade or siRNA-based knockdown of β-catenin mimicked PrP-deficiency in regards to NCAM1 polysialylation. Our data established the existence of a PrP-ST8SIA2-NCAM signaling loop, merged two mature fields of investigation and offer a simple model for explaining phenotypes linked to PrP. PMID:26288071

  11. β1 Integrin is an Adhesion Protein for Sperm Binding to Eggs

    PubMed Central

    Baessler, Keith A.; Lee, Younjoo; Sampson, Nicole S.

    2009-01-01

    We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment, but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different. PMID:19338281

  12. Inhibition of protein adsorption and cell adhesion on PNIPAAm-grafted polyurethane surface: effect of graft molecular weight.

    PubMed

    Zhao, Tieliang; Chen, Hong; Zheng, Jun; Yu, Qian; Wu, Zhaoqiang; Yuan, Lin

    2011-06-15

    In this work, the effect of molecular weight (MW) of surface grafted poly(N-isopropylacrylamide) (PNIPAAm) on protein adsorption and cell adhesion was investigated systematically. PNIPAAm-grafted polyurethane (PU) surfaces of varying graft MW were prepared via conventional radical polymerization. The MW was controlled by adjusting the monomer concentration. Fibrinogen (Fg) and human serum albumin (HSA) were selected as model proteins and their adsorption from phosphate-buffered saline (PBS, pH 7.4) and blood plasma at 37°C was measured using a radiolabeling method and immunoblot analysis respectively. It was found that in both media, as the MW increased, the adsorption of these two proteins decreased gradually reaching a plateau value at MW above 7.9×10(4). Compared to the unmodified PU, the surface grafted with PNIPAAm of MW 14.6×10(4) reduced the adsorption of Fg and HSA in PBS by 91% and 86%, respectively. Moreover, the surfaces with higher MW PNIPAAm showed minimal adhesion of L929 cells presumably due to the absence of cell-adhesive proteins on the surfaces. PMID:21093225

  13. Staphylococcus aureus Fibronectin-Binding Protein A Mediates Cell-Cell Adhesion through Low-Affinity Homophilic Bonds

    PubMed Central

    Herman-Bausier, Philippe; El-Kirat-Chatel, Sofiane; Foster, Timothy J.

    2015-01-01

    ABSTRACT Staphylococcus aureus is an important opportunistic pathogen which is a leading cause of biofilm-associated infections on indwelling medical devices. The cell surface-located fibronectin-binding protein A (FnBPA) plays an important role in the accumulation phase of biofilm formation by methicillin-resistant S. aureus (MRSA), but the underlying molecular interactions are not yet established. Here, we use single-cell and single-molecule atomic force microscopy to unravel the mechanism by which FnBPA mediates intercellular adhesion. We show that FnBPA is responsible for specific cell-cell interactions that involve the FnBPA A domain and cause microscale cell aggregation. We demonstrate that the strength of FnBPA-mediated adhesion originates from multiple low-affinity homophilic interactions between FnBPA A domains on neighboring cells. Low-affinity binding by means of FnBPA may be important for biofilm dynamics. These results provide a molecular basis for the ability of FnBPA to promote cell accumulation during S. aureus biofilm formation. We speculate that homophilic interactions may represent a generic strategy among staphylococcal cell surface proteins for guiding intercellular adhesion. As biofilm formation by MRSA strains depends on proteins rather than polysaccharides, our approach offers exciting prospects for the design of drugs or vaccines to inhibit protein-dependent intercellular interactions in MRSA biofilms. PMID:26015495

  14. MEASUREMENTS OF CONFORMATION CHANGES DURING ADHESION OF LIPID PROTEIN (POLYLYSINE AND S-LAYER) SURFACES

    EPA Science Inventory

    The adhesion forces between various surfaces were measured using the "surface forces apparatus" technique which allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. hree types of biologi...

  15. Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1

    PubMed Central

    Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

    2014-01-01

    T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130 000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1–calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion. PMID:24877199

  16. Organ-selective regulation of vascular adhesion protein-1 expression in man.

    PubMed

    Arvilommi, A M; Salmi, M; Jalkanen, S

    1997-07-01

    Vascular adhesion protein-1 (VAP-1) is an endothelial molecule which mediates lymphocyte binding to endothelium in peripheral lymph nodes and at certain sites of inflammation. The expression of VAP-1 in vivo is strongly up-regulated in inflamed tissues, such as gut and skin. The purpose of this work was to examine the factors responsible for this induction of VAP-1. Since the expression of VAP-1 could not be induced in cultured endothelial cells with a large panel of mediators, we used an organ culture technique for the investigation of the regulation of VAP-1 expression in a more physiological micromilieu. Indeed, we found that the expression of endothelial VAP-1 could be up-regulated in human tonsillar tissue with interleukin (IL)-1, IL-4, tumor necrosis factor (TNF-alpha), interferon (IFN)-gamma and lipopolysaccharide, whereas histamine, thrombin, dibutyryl cAMP, N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA) had no effect. The induced VAP-1 protein was similar in molecular weight to the non-induced VAP-1, suggesting that VAP-1 synthesized de novo carries appropriate carbohydrate moieties. In contrast to tonsil organ culture, similar inductions performed with human appendix showed no up-regulation of VAP-1 expression, indicating that the regulation of VAP-1 expression exhibits organ-selective characteristics. Furthermore, in these tissues the smooth muscle cells, which constitutively express VAP-1, could not be stimulated to alter their level of expression of this molecule. In conclusion, the expression of VAP-1 can be markedly up-regulated with several mediators in tonsil but not in appendix organ culture, whereas cultured endothelial cells cannot be induced to express VAP-1. These results indicate that the expression of VAP-1 is regulated in a tissue- and cell type-selective manner, and a correct micromilieu is required for the up-regulation to occur. PMID:9247594

  17. Thrombospondin-related adhesive protein (TRAP) of Plasmodium falciparum: expression during sporozoite ontogeny and binding to human hepatocytes.

    PubMed Central

    Robson, K J; Frevert, U; Reckmann, I; Cowan, G; Beier, J; Scragg, I G; Takehara, K; Bishop, D H; Pradel, G; Sinden, R

    1995-01-01

    Plasmodium sporozoites collected from oocysts, haemocoel and salivary glands of the mosquito show profound differences in their biological properties such as motility, ability to induce protective immune response and infectivity for vertebrate host cells. Sporozoites from salivary glands are much more infectious than those from oocysts and haemocoel. Differential expression of proteins, such as the circumsporozoite (CS) protein and the thrombospondin-related adhesive protein (TRAP), implicated in sporozoite recognition and entry into hepatocytes may account for the development of infectivity during ontogeny. We have carried out a series of experiments to: (i) analyse the expression and localization of TRAP in P.falciparum sporozoites during development in the mosquito; and (ii) elucidate the biochemical and adhesive properties of recombinant TRAP. Our data indicate that TRAP is not expressed in oocysts, whereas variable amounts of CS protein are found in this parasite developmental stage. Hemocoel sporozoites display the distinct phenotypes TRAP- CS protein+ and TRAP+ CS protein+ at a frequency of 98.5 and 1.5% respectively. Salivary gland sporozoites are all TRAP+ CS protein+. We also provide experimental evidence showing that recombinant TRAP binds to the basolateral cell membrane of hepatocytes in the Disse's space and that sulfated glycoconjugates function as TRAP ligands on human hepatocytes. Images PMID:7664729

  18. Cell adhesion defines the topology of endocytosis and signaling

    PubMed Central

    Grossier, Jean-Philippe; Xouri, Georgia; Goud, Bruno; Schauer, Kristine

    2014-01-01

    Preferred sites of endocytosis have been observed in various cell types, but whether they occur randomly or are linked to cellular cues is debated. Here, we quantified the sites of endocytosis of transferrin (Tfn) and epidermal growth factor (EGF) in cells whose adhesion geometry was defined by micropatterns. 3D probabilistic density maps revealed that Tfn was enriched in adhesive sites during uptake, whereas EGF endocytosis was restricted to the dorsal cellular surface. This spatial separation was not due to distributions of corresponding receptors but was regulated by uptake mechanisms. Asymmetric uptake of Tfn resulted from the enrichment of clathrin and adaptor protein 2 at adhesive areas. Asymmetry in EGF uptake was strongly dependent on the actin cytoskeleton and led to asymmetry in EGF receptor activation. Mild alteration of actin dynamics abolished asymmetry in EGF uptake and decreased EGF-induced downstream signaling, suggesting that cellular adhesion cues influence signal propagation. We propose that restriction of endocytosis at distinct sites allows cells to sense their environment in an “outside-in” mechanism. PMID:24366944

  19. 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.

  20. Negative staining and immunoelectron microscopy of adhesion-deficient mutants of Streptococcus salivarius reveal that the adhesive protein antigens are separate classes of cell surface fibril.

    PubMed Central

    Weerkamp, A H; Handley, P S; Baars, A; Slot, J W

    1986-01-01

    The subcellular distribution of the cell wall-associated protein antigens of Streptococcus salivarius HB, which are involved in specific adhesive properties of the cells, was studied. Mutants which had lost the adhesive properties and lacked the antigens at the cell surface were compared with the parent strain. Immunoelectron microscopy of cryosections of cells labeled with affinity-purified, specific antisera and colloidal gold-protein A complexes was used to locate the antigens. Antigen C (AgC), a glycoprotein involved in attachment to host surfaces, was mainly located in the fibrillar layer outside the cell wall. A smaller amount of label was also found throughout the cytoplasmic area in the form of small clusters of gold particles, which suggests a macromolecular association. Mutant HB-7, which lacks the wall-associated AgC, accumulated AgC reactivity intracellularly. Intracellular AgC was often found associated with isolated areas of increased electron density, but sometimes seemed to fill the entire interior of the cell. Antigen B (AgB), a protein responsible for interbacterial coaggregation, was also located in the fibrillar layer, although its distribution differed from that of the wall-associated AgC since AgB was found predominantly in the peripheral areas. A very small amount of label was also found in the cytoplasmic area as discrete gold particles. Mutant HB-V5, which lacks wall-associated AgB, was not labeled in the fibrillar coat, but showed the same weak intracellular label as the parent strain. Immunolabeling with serum against AgD, another wall-associated protein but of unknown function, demonstrated its presence in the fibrillar layer of strain HB. Negatively stained preparations of whole cells of wild-type S. salivarius and mutants that had lost wall-associated AgB or AgC revealed that two classes of short fibrils are carried on the cell surface at the same time. AgB and AgC are probably located on separate classes of short, protease

  1. Src Kinase Determines the Dynamic Exchange of the Docking Protein NEDD9 (Neural Precursor Cell Expressed Developmentally Down-regulated Gene 9) at Focal Adhesions*

    PubMed Central

    Bradbury, Peta; Bach, Cuc T.; Paul, Andre; O'Neill, Geraldine M.

    2014-01-01

    Dynamic exchange of molecules between the cytoplasm and integrin-based focal adhesions provides a rapid response system for modulating cell adhesion. Increased residency time of molecules that regulate adhesion turnover contributes to adhesion stability, ultimately determining migration speed across two-dimensional surfaces. In the present study we test the role of Src kinase in regulating dynamic exchange of the focal adhesion protein NEDD9/HEF1/Cas-L. Using either chemical inhibition or fibroblasts genetically null for Src together with fluorescence recovery after photobleaching (FRAP), we find that Src significantly reduces NEDD9 exchange at focal adhesions. Analysis of NEDD9 mutant constructs with the two major Src-interacting domains disabled revealed the greatest effects were due to the NEDD9 SH2 binding domain. This correlated with a significant change in two-dimensional migratory speed. Given the emerging role of NEDD9 as a regulator of focal adhesion stability, the time of NEDD9 association at the focal adhesions is key in modulating rates of migration and invasion. Our study suggests that Src kinase activity determines NEDD9 exchange at focal adhesions and may similarly modulate other focal adhesion-targeted Src substrates to regulate cell migration. PMID:25059660

  2. Phosphoinositides, kinases and adaptors coordinating endocytosis in Trypanosoma brucei

    PubMed Central

    Manna, Paul T; Field, Mark C

    2015-01-01

    In the kinetoplastid parasite Trypanosoma brucei clathrin-mediated endocytosis is essential for survival and aids immune evasion in the mammalian host. The formation of endocytic clathrin coated vesicles in T. brucei is via a unique mechanism owing to an evolutionarily recent loss of the adaptor protein (AP)2 complex, a central hub in endocytic vesicle assembly. Despite this loss, recent studies examining endocytic clathrin coat assembly have highlighted a high degree of conservation between trypanosomes and their mammalian hosts. In particular phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and its putative effectors, TbCALM and TbEpsinR, are central to clathrin-mediated endocytosis in the trypanosome, just as they are in animal cells. In addition to providing insights into the cell biology of T. brucei, these studies also suggest an ancient, possibly pan-eukaryotic connection between PtdIns(4,5)P2 and endocytosis. PMID:27064836

  3. Pregnancy-associated plasma protein A up-regulated by progesterone promotes adhesion and proliferation of trophoblastic cells.

    PubMed

    Wang, Jiao; Liu, Shuai; Qin, Hua-Min; Zhao, Yue; Wang, Xiao-Qi; Yan, Qiu

    2014-01-01

    Embryo implantation and development is a complex biological process for the establishment of the successful pregnancy. Progesterone is a critical factor in the regulation of embryo adhesion to uterine endometrium and proliferation. Although it has been reported that pregnancy-associated plasma protein A (PAPPA) is increased in pregnant women, the relationship between progesterone and PAPPA, and the effects of PAPPA on embryo adhesion and proliferation are still not clear. The present results showed that the serum level of progesterone and PAPPA was closely correlated by ELISA assay (p<0.01). PAPPA was detected in the villi of early embryo by RT-PCR, Western blot, immunohistochemistry and immunofluorescent staining. Moreover, PAPPA was significantly up-regulated by progesterone in trophoblastic (JAR) cells by Real-time PCR and ELISA assay (p<0.01); while the expression was decreased by the progesterone receptor inhibitor RU486. The down-regulation of PAPPA by siRNA transfection or up-regulation of PAPPA by progesterone treatment significantly decreased or increased the adhesion rate of trophoblastic cells to human uterine epithelial cell lines (RL95-2 and HEC-1A), respectively (p<0.01), as well as the proliferation of trophoblastic cells. In conclusion, PAPPA is up-regulated by progesterone, which promotes the adhesion and proliferation potential of trophoblastic cells. PMID:24817938

  4. Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

    SciTech Connect

    Zhang, Haimou; Qin, Gangjian; Liang, Gang; Li, Jinan; Chiu, Isaac; Barrington, Robert A.; Liu, Dongxu . E-mail: dxliu001@yahoo.com

    2007-07-13

    Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanism of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.

  5. Cleavage of Type I Collagen by Fibroblast Activation Protein-α Enhances Class A Scavenger Receptor Mediated Macrophage Adhesion

    PubMed Central

    Mazur, Anna; Holthoff, Emily; Vadali, Shanthi; Kelly, Thomas; Post, Steven R.

    2016-01-01

    Pathophysiological conditions such as fibrosis, inflammation, and tumor progression are associated with modification of the extracellular matrix (ECM). These modifications create ligands that differentially interact with cells to promote responses that drive pathological processes. Within the tumor stroma, fibroblasts are activated and increase the expression of type I collagen. In addition, activated fibroblasts specifically express fibroblast activation protein-α (FAP), a post-prolyl peptidase. Although FAP reportedly cleaves type I collagen and contributes to tumor progression, the specific pathophysiologic role of FAP is not clear. In this study, the possibility that FAP-mediated cleavage of type I collagen modulates macrophage interaction with collagen was examined using macrophage adhesion assays. Our results demonstrate that FAP selectively cleaves type I collagen resulting in increased macrophage adhesion. Increased macrophage adhesion to FAP-cleaved collagen was not affected by inhibiting integrin-mediated interactions, but was abolished in macrophages lacking the class A scavenger receptor (SR-A/CD204). Further, SR-A expressing macrophages localize with activated fibroblasts in breast tumors of MMTV-PyMT mice. Together, these results demonstrate that FAP-cleaved collagen is a substrate for SR-A-dependent macrophage adhesion, and suggest that by modifying the ECM, FAP plays a novel role in mediating communication between activated fibroblasts and macrophages. PMID:26934296

  6. Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.

    PubMed Central

    Haffner, C; Jarchau, T; Reinhard, M; Hoppe, J; Lohmann, S M; Walter, U

    1995-01-01

    The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks. Images PMID:7828592

  7. 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. PMID:25898165

  8. Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

    PubMed Central

    Aoh, Quyen L.; Graves, Lee M.; Duncan, Mara C.

    2011-01-01

    Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation. PMID:21832155

  9. Divalent cations and the protein surface co-ordinate the intensity of human platelet adhesion and P-selectin surface expression.

    PubMed

    Whiss, P A; Andersson, R G G

    2002-07-01

    At sites of blood vessel injury, platelets adhere to exposed vessel components, such as collagen, or immobilized fibrinogen derived from plasma or activated platelets. The divalent cations Mg(2+) and Ca(2+) are essential for platelet adhesion and activation, but Mg(2+) can also inhibit platelet activation. The present study evaluates, by an enzymatic method, the effects of various divalent cations on the adhesion of isolated human platelets to collagen, fibrinogen, albumin or plastic in vitro. By enzyme-linked immunosorbent assay, platelet surface expression of P-selectin was measured to estimate the state of activation on adherence. Mg(2+) increased platelet adhesion exclusively to collagen and fibrinogen at physiologically relevant concentrations. At higher concentrations, the adhesion declined. Ca(2+) induced a weak adhesion only to fibrinogen at physiological doses and a peak of increased adhesion to all protein-coated surfaces at 10 mmol/l. Mn(2+) elicited dose-dependent adhesion only to collagen and fibrinogen. Zn(2+), Ni(2+) and Cu(2+) increased the adhesion of platelets independently of the surface. Ca(2+) dose-dependently inhibited adhesion elicited by Mg(2+) to collagen and fibrinogen. No other combination of divalent cations elicited such an effect. Mg(2+)-dependent platelet adhesion to collagen and Ca(2+)-dependent adhesion to fibrinogen increased P-selectin expression. Thus, the present study shows that the outcome of the platelet adhesion depends on the surface and the access of divalent cations, which co-ordinate the intensity of platelet adhesion and P-selectin surface expression. PMID:12138368

  10. Role of Lactobacillus reuteri cell and mucus-binding protein A (CmbA) in adhesion to intestinal epithelial cells and mucus in vitro.

    PubMed

    Jensen, Hanne; Roos, Stefan; Jonsson, Hans; Rud, Ida; Grimmer, Stine; van Pijkeren, Jan-Peter; Britton, Robert A; Axelsson, Lars

    2014-04-01

    Lactobacillus reuteri, a symbiotic inhabitant of the gastrointestinal tract in humans and animals, is marketed as a probiotic. The ability to adhere to intestinal epithelial cells and mucus is an interesting property with regard to probiotic features such as colonization of the gastrointestinal tract and interaction with the host. Here, we present a study performed to elucidate the role of sortase (SrtA), four putative sortase-dependent proteins (SDPs), and one C-terminal membrane-anchored cell surface protein of Lactobacillus reuteri ATCC PTA 6475 in adhesion to Caco-2 cells and mucus in vitro. This included mutagenesis of the genes encoding these proteins and complementation of mutants. A null mutation in hmpref0536_10255 encoding srtA resulted in significantly reduced adhesion to Caco-2 cells and mucus, indicating involvement of SDPs in adhesion. Evaluation of the bacterial adhesion revealed that of the five putative surface protein mutants tested, only a null mutation in the hmpref0536_10633 gene, encoding a putative SDP with an LPxTG motif, resulted in a significant loss of adhesion to both Caco-2 cells and mucus. Complementation with the functional gene on a plasmid restored adhesion to Caco-2 cells. However, complete restoration of adhesion to mucus was not achieved. Overexpression of hmpref0536_10633 in strain ATCC PTA 6475 resulted in an increased adhesion to Caco-2 cells and mucus compared with the WT strain. We conclude from these results that, among the putative surface proteins tested, the protein encoded by hmpref0536_10633 plays a critical role in binding of Lactobacillus reuteri ATCC PTA 6475 to Caco-2 cells and mucus. Based on this, we propose that this LPxTG motif containing protein should be referred to as cell and mucus binding protein A (CmbA). PMID:24473252

  11. Myosin VI and its cargo adaptors – linking endocytosis and autophagy

    PubMed Central

    Tumbarello, David A.; Kendrick-Jones, John; Buss, Folma

    2013-01-01

    Summary The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway – both of which are involved in cargo trafficking and sorting – through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein–protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy. PMID:23781020

  12. TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium

    PubMed Central

    Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

    2012-01-01

    TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface. PMID:22219373

  13. Physiological Osmotic Induction of Leptospira interrogans Adhesion: LigA and LigB Bind Extracellular Matrix Proteins and Fibrinogen▿

    PubMed Central

    Choy, Henry A.; Kelley, Melissa M.; Chen, Tammy L.; Møller, Annette K.; Matsunaga, James; Haake, David A.

    2007-01-01

    Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests. PMID:17296754

  14. Supporting data for characterization of non-coding RNAs associated with the Neuronal growth regulator 1 (NEGR1) adhesion protein.

    PubMed

    Kaur, Prameet; Tan, Jun Rong; Karolina, Dwi Setyowati; Sepramaniam, Sugunavathi; Armugam, Arunmozhiarasi; Peter Wong, Tsun-Hon; Jeyaseelan, Kandiah

    2016-06-01

    Long non-coding RNAs and microRNAs control gene expression to determine central nervous system development and function. Neuronal growth regulator 1 (NEGR1) is a cell adhesion molecule that plays an important role in neurite outgrowth during neuronal development and its precise expression is crucial for correct brain development. The data described here is related to the research article titled "A long non-coding RNA, BC048612 and a microRNA, miR-203 coordinate the gene expression of Neuronal growth regulator 1 (NEGR1) adhesion protein" [1]. This data article contains detailed bioinformatics analysis of genetic signatures at the Negr1 gene locus retrieved from the UCSC genome browser. This approach could be adopted to identify putative regulatory non-coding RNAs in other tissues and diseases. PMID:26977442

  15. In vivo modification of tyrosine residues in recombinant mussel adhesive protein by tyrosinase co-expression in Escherichia coli

    PubMed Central

    2012-01-01

    Background In nature, mussel adhesive proteins (MAPs) show remarkable adhesive properties, biocompatibility, and biodegradability. Thus, they have been considered promising adhesive biomaterials for various biomedical and industrial applications. However, limited production of natural MAPs has hampered their practical applications. Recombinant production in bacterial cells could be one alternative to obtain useable amounts of MAPs, although additional post-translational modification of tyrosine residues into 3,4-dihydroxyphenyl-alanine (Dopa) and Dopaquinone is required. The superior properties of MAPs are mainly attributed to the introduction of quinone-derived intermolecular cross-links. To solve this problem, we utilized a co-expression strategy of recombinant MAP and tyrosinase in Escherichia coli to successfully modify tyrosine residues in vivo. Results A recombinant hybrid MAP, fp-151, was used as a target for in vivo modification, and a dual vector system of pET and pACYC-Duet provided co-expression of fp-151 and tyrosinase. As a result, fp-151 was over-expressed and mainly obtained from the soluble fraction in the co-expression system. Without tyrosinase co-expression, fp-151 was over-expressed in an insoluble form in inclusion bodies. The modification of tyrosine residues in the soluble-expressed fp-151 was clearly observed from nitroblue tetrazolium staining and liquid-chromatography-mass/mass spectrometry analyses. The purified, in vivo modified, fp-151 from the co-expression system showed approximately 4-fold higher bulk-scale adhesive strength compared to in vitro tyrosinase-treated fp-151. Conclusion Here, we reported a co-expression system to obtain in vivo modified MAP; additional in vitro tyrosinase modification was not needed to obtain adhesive properties and the in vivo modified MAP showed superior adhesive strength compared to in vitro modified protein. It is expected that this co-expression strategy will accelerate the use of functional MAPs in

  16. Expression and organization of basement membranes and focal adhesion proteins in pregnant myometrium is regulated by uterine stretch.

    PubMed

    Shynlova, Oksana; Chow, Michelle; Lye, Stephen J

    2009-10-01

    The mechanisms underlying the preparation of the uterus for labor are not fully understood. We have previously found a significant increase in the expression of messenger RNA (mRNAs) encoding extracellular basement membrane (BM) proteins of the smooth muscle cells (SMCs) in late pregnant rat myometrium. At term, the myometrium is stretched by growing fetuses and these mechanical signals are transmitted from extracellular matrix into SMCs through focal adhesions (FA). The aim of this study was to investigate the effect of gravidity on the expression and spatiotemporal distribution of major BM proteins, laminin-gamma2 and collagen IV, as well as typical FA constituents, vinculin and paxillin, in the myometrium during gestation and parturition, using a unilaterally pregnant rat model. We found that the expression of laminin-gamma2 and collagen IV proteins increased significantly with gestational age (P < .05) and was dependent on gravidity whereas vinculin and paxillin proteins were not affected. Near term, BM proteins from gravid horn myometrium demonstrated increased extracellular immunostaining and major rearrangement from sporadic protein distribution to organized, continuous, and regular structures surrounding the plasma membrane of each myocyte. Examination of FA proteins revealed that paxillin was translocated from the cytoplasm to the cell periphery, while vinculin was sequestered specifically to FAs. At labor, BM and FA proteins, organized in similar bead-like structures, were localized on opposing sides of SMC plasma membrane into 2 different compartments. We suggest that these stretch-induced changes facilitate formation of stable cell-matrix adhesions and provide the molecular basis for optimal force transduction during labor contractions. PMID:19602722

  17. Identification, characterization, and expression levels of putative adhesive proteins from the tube-dwelling polychaete Sabellaria alveolata.

    PubMed

    Becker, Pierre T; Lambert, Aurélie; Lejeune, Annabelle; Lanterbecq, Déborah; Flammang, Patrick

    2012-10-01

    The shelter of the tube-dwelling polychaete Sabellaria alveolata is composed of mineral particles assembled with spots of a proteinaceous cement. The adhesive proteins constituting the cement were identified on the basis of their sequence similarity with proteins of a phylogenetically related species, Phragmatopoma californica. Two positively charged proteins, Sa-1 and Sa-2, share common features: they both have a mass of 22 kDa; are rich in glycine, tyrosine and basic residues; and show repeated peptide motifs. The consensus repeat of Sa-1 is KGAYGAKGLGYGNKAGYGAYG (occurring 6-8 times), while Sa-2 displays the consensus heptapeptide VHKAAWG (5 times) and undecapeptide VHKAAGYGGYG (8 times). Two variants of a serine-rich protein, Sa-3A (22 kDa) and Sa-3B (21 kDa), were also identified. Their serine residues account for 75 mol% and are probably phosphorylated, meaning that Sa-3 is very acidic and negatively charged. Moreover, tyrosine residues of all adhesive proteins are presumably modified into DOPA. Although protein sequences are not well-conserved between S. alveolata and P. californica, their main characteristics (including amino acid composition, post-translational modifications, repeated patterns, isoelectric point, and mass) are shared by both species. This suggests that these features are more important for their function than the primary structure of the proteins. The mRNA abundance for each protein was estimated by quantitative real-time PCR, revealing relative expression levels of about 5, 11, 1.5, and 1 for Sa-1, -2, -3A, and -3B, respectively. These levels could be indicative of charge neutralization phenomena or could reflect their function (interface vs. bulk) in the cement. PMID:23111133

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2013-10-10

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

  20. Adhesions of extracellular surface-layer associated proteins in Lactobacillus M5-L and Q8-L.

    PubMed

    Zhang, Yingchun; Xiang, Xinling; Lu, Qianhui; Zhang, Lanwei; Ma, Fang; Wang, Linlin

    2016-02-01

    Surface-layer associated proteins (SLAP) that envelop Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L cell surfaces are involved in the adherence of these strain to the human intestinal cell line HT-29. To further elucidate some of the properties of these proteins, we assessed the yields and expressions of SLAP under different incubation conditions. An efficient and selective extraction of SLAP was obtained when cells of Lactobacillus were treated with 5 M LiCl at 37°C in aerobic conditions. The SLAP of Lactobacillus M5-L and Q8-L in cell extracts were visualized by SDS-PAGE and identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin-labeled HT-29 cells as adhesion proteins. Atomic force microscopy contact imaging revealed that Lactobacillus strains M5-L and Q8-L normally display a smooth, homogeneous surface, whereas the surfaces of M5-L and Q8-L treated with 5 M LiCl were rough and more heterogeneous. Analysis of adhesion forces revealed that the initial adhesion forces of 1.41 and 1.28 nN obtained for normal Lactobacillus M5-L and Q8-L strains, respectively, decreased to 0.70 and 0.48 nN, respectively, following 5 M LiCl treatment. Finally, the dominant 45-kDa protein bands of Lactobacillus Q8-L and Lactobacillus M5-L were identified as elongation factor Tu and surface antigen, respectively, by liquid chromatography-tandem mass spectrometry. PMID:26709174

  1. A human salivary protein which promotes adhesion of Streptococcus mutans serotype c strains to hydroxyapatite.

    PubMed Central

    Kishimoto, E; Hay, D I; Gibbons, R J

    1989-01-01

    The aim of this study was to investigate the nature of one of the factors in human submandibular-sublingual (SMSL) saliva which promotes the adhesion of Streptococcus mutans serotype c strains to hydroxyapatite (HA) surfaces. Gel filtration chromatography of SMSL saliva on Trisacryl GF2000 gave a void volume peak which contained the major fraction of adhesion-promoting activity for S. mutans JBP to HA. Maximum adhesion-promoting activity, however, eluted slightly later than the maximum 220-nm absorbance of the void volume peak. Gel filtration of the void volume material after treatment with sodium dodecyl sulfate (SDS) gave an early-eluting larger peak followed by a smaller peak with which the adhesion-promoting activity was associated. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed the presence of relatively slowly migrating material associated with the larger inactive peak, presumably mucin, and a faster-migrating band(s) associated with the smaller active peak. SDS-PAGE indicated molecular weights in the range of 300,000 to 350,000 by extrapolation from size standards. Comparison of SMSL from five individuals showed the presence of single bands or double bands associated with adhesion-promoting activity, indicating genetic polymorphism. The active material did not resemble either secretory immunoglobulin A, based on SDS-PAGE and immunoassay, or fibronectin, based on SDS-PAGE, and also differed in molecular weight from salivary mucins and salivary constituents previously reported to promote aggregation of certain oral bacteria, but a relationship to these materials cannot be excluded. This adhesion-promoting material may play a significant role in the initial colonization of tooth surfaces by S. mutans strains. Images PMID:2807544

  2. Circulating renalase, catecholamines, and vascular adhesion protein 1 in hypertensive patients.

    PubMed

    Maciorkowska, Dominika; Zbroch, Edyta; Malyszko, Jolanta

    2015-11-01

    The aim of the study was to estimate and correlate circulating levels of renalase, vascular adhesion protein-1 (VAP-1), catecholamines in patients with primary hypertension. The renalase, VAP-1, and catecholamines concentration was estimated in 121 hypertensive patients. The correlation between renalase, VAP-1 levels and catecholamine concentration in blood, blood pressure control, pharmacological therapy, and medical history were taken in to consideration. The median office blood pressure was 145.5/86 mm Hg and was significantly higher than the median home blood pressure measurement value, which was 135/80 mm Hg, P < .05. Circulating renalase and VAP-1 (Me 9.57 μg/mL and Me = 326.7 ng/mL) levels were significantly higher in patients with hypertension comparing to healthy individuals (3.83 μg/mL and 248.37 ng/mL, P < .05). The correlation between renalase and noradrenalin concentration in blood was observed (r = 0.549; P < .05), also the correlation between VAP-1 and noradrenaline was noticed (r = 0.21, P = .029). Renalase level was higher in patients with coronary artery disease and correlated with decreased ejection fraction. VAP-1 concentration correlated also with left ventricular ejection fraction (r = -0.23, P = .013). Hypertensive patients with diabetes mellitus had almost statistically significant higher VAP-1 concentration compared with hypertensive patients without diabetes mellitus (Me = 403.22 ng/mL vs. Me = 326,68 ng/mL, P = .064). In multiple regression analysis, renalase was predicted by plasma dopamine and norepinephrine as also diastolic office blood pressure and left ventricle ejection fraction. Circulating renalase and VAP-1 levels are elevated in patients with poor blood pressure control. Its correlation with noradrenalin concentration need further studies to find out the role of renalase as also VAP-1 in pathogenesis and treatment of hypertension. PMID:26403854

  3. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

    PubMed

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  4. Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells

    PubMed Central

    Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2015-01-01

    Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest. PMID:26370773

  5. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

    PubMed

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  6. The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy

    PubMed Central

    Sergé, Arnauld

    2016-01-01

    The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis. PMID:27200348

  7. Involvement of NF-κB-mediated expression of galectin-3-binding protein in TNF-α-induced breast cancer cell adhesion.

    PubMed

    Noma, Naruto; Simizu, Siro; Kambayashi, Yusuke; Kabe, Yasuaki; Suematsu, Makoto; Umezawa, Kazuo

    2012-06-01

    Galectin-3-binding protein (G3BP) is highly expressed in various types of cancer and is thought to be involved in cancer malignancy; however, the role of G3BP in breast cancer cells is not fully understood. In this study, we investigated the role of NF-κB in the adhesion of breast cancer cells to a substrate by using (-)-DHMEQ, a specific inhibitor of NF-κB. (-)-DHMEQ inhibited both TNF-α-induced G3BP expression and cell adhesion in human breast cancer cell lines. We also found that knockdown of G3BP suppressed the adhesion, while its overexpression increased the adhesion. These data reveal that (-)-DHMEQ suppresses breast cancer cell adhesion by inhibiting NF-κB-regulated G3BP expression. PMID:22447108

  8. In-situ coupling between kinase activities and protein dynamics within single focal adhesions

    PubMed Central

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  9. In-situ coupling between kinase activities and protein dynamics within single focal adhesions.

    PubMed

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  10. The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein.

    PubMed

    Burzio, L A; Saéz, C; Pardo, J; Waite, J H; Burzio, L O

    2000-06-15

    The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure. PMID:11004549

  11. A role for the retinoblastoma protein as a regulator of mouse osteoblast cell adhesion: implications for osteogenesis and osteosarcoma formation.

    PubMed

    Sosa-García, Bernadette; Gunduz, Volkan; Vázquez-Rivera, Viviana; Cress, W Douglas; Wright, Gabriela; Bian, Haikuo; Hinds, Philip W; Santiago-Cardona, Pedro G

    2010-01-01

    The retinoblastoma protein (pRb) is a cell cycle regulator inactivated in most human cancers. Loss of pRb function results from mutations in the gene coding for pRb or for any of its upstream regulators. Although pRb is predominantly known as a cell cycle repressor, our data point to additional pRb functions in cell adhesion. Our data show that pRb regulates the expression of a wide repertoire of cell adhesion genes and regulates the assembly of the adherens junctions required for cell adhesion. We conducted our studies in osteoblasts, which depend on both pRb and on cell-to-cell contacts for their differentiation and function. We generated knockout mice in which the RB gene was excised specifically in osteoblasts using the cre-lox P system and found that osteoblasts from pRb knockout mice did not assemble adherens junction at their membranes. pRb depletion in wild type osteoblasts using RNAi also disrupted adherens junctions. Microarrays comparing pRb-expressing and pRb-deficient osteoblasts showed that pRb controls the expression of a number of cell adhesion genes, including cadherins. Furthermore, pRb knockout mice showed bone abnormalities consistent with osteoblast adhesion defects. We also found that pRb controls the function of merlin, a well-known regulator of adherens junction assembly, by repressing Rac1 and its effector Pak1. Using qRT-PCR, immunoblots, co-immunoprecipitation assays, and immunofluorescent labeling, we observed that pRb loss resulted in Rac1 and Pak1 overexpression concomitant with merlin inactivation by Pak1, merlin detachment from the membrane, and adherens junction loss. Our data support a pRb function in cell adhesion while elucidating the mechanism for this function. Our work suggests that in some tumor types pRb inactivation results in both a loss of cell cycle control that promotes initial tumor growth as well as in a loss of cell-to-cell contacts, which contributes to later stages of metastasis. PMID:21085651

  12. A protein fragment of streptococcal cell surface antigen I/II which prevents adhesion of Streptococcus mutans.

    PubMed Central

    Munro, G H; Evans, P; Todryk, S; Buckett, P; Kelly, C G; Lehner, T

    1993-01-01

    Attachment of Streptococcus mutans to the tooth surface involves a cell surface protein with an M(r) of 185,000, termed streptococcal antigen (SA) I/II. Four overlapping fragments of the gene encoding SA I/II were amplified by polymerase chain reaction, cloned, and expressed in Escherichia coli. The recombinant polypeptides were assayed for adhesion-binding activity to salivary receptors and for recognition by a panel of monoclonal antibodies (MAbs) raised against SA I/II. Two of the MAbs which are known to prevent colonization of S. mutans in vivo bound the recombinant polypeptide comprising residues 816 to 1161. In vitro adhesion of S. mutans to saliva-coated hydroxyapatite beads was also inhibited specifically by a polypeptide (residues 816 to 1213) encompassing the same region. The evidence from the MAbs preventing colonization of S. mutans and the adherence inhibition assay suggests that an adhesion-binding activity resides within the portion of SA I/II comprising residues 816 to 1213, which is highly conserved among oral streptococcal species. Images PMID:7691754

  13. β Integrin-like protein-mediated adhesion and its disturbances during cell cultivation of the mussel Mytilus trossulus.

    PubMed

    Maiorova, Mariia A; Odintsova, Nelly A

    2015-08-01

    In this study, we focus on the specific contribution of β integrin-like protein to adhesion-mediated events in molluscan larval cells in culture that could not have been investigated within the whole animal. An analysis of disturbances to cell-substratum adhesion, caused by the integrin receptor inhibiting Arg-Gly-Asp-Ser (RGDS)-peptide, the Ca(2+)/Mg(2+)-chelators and the stress influence of freezing-thawing, reveals that all these factors resulted in the partial destruction of the integrin-extracellular matrix (ECM) interaction in culture and, in particular, changes in the distribution and relative abundance of β integrin-positive cells. The experiments, carried out on selected substrates, found that β integrin-positive cells demonstrate different affinities for the substrates. This finding further supports the assumption that epithelial differentiation in cultivated cells of larval Mytilus may be mediated by β integrin-like proteins via binding to laminin; direct binding to other components of the ECM could not be demonstrated. The mussel β integrin-positive cells are not involved in myogenic or neuronal differentiation on any of the substrates but part of them has tubulin-positive cilia, forming some epithelia-like structures. Our data indicate that β integrin-positive cells are able to proliferate in vitro which suggests that they could participate in renewing the digestive epithelium in larvae. The findings provide evidence that the distribution pattern of β integrin-like protein depends on the cell type and the factors influencing the adhesion. PMID:25673210

  14. Resistance to protein adsorption and adhesion of fibroblasts on nanocrystalline diamond films: the role of topography and boron doping.

    PubMed

    Alcaide, María; Papaioannou, Stavros; Taylor, Andrew; Fekete, Ladislav; Gurevich, Leonid; Zachar, Vladimir; Pennisi, Cristian Pablo

    2016-05-01

    Boron-doped nanocrystalline diamond (BNCD) fil