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Sample records for 7tm receptor c-terminal

  1. Hitchhiking on the heptahelical highway: structure and function of 7TM receptor complexes

    PubMed Central

    Tesmer, John J. G.

    2017-01-01

    A revolution in the analysis of seven transmembrane (7TM) receptors has provided detailed information about how these physiologically important signalling proteins interact with extracellular cues. However, it has proven much more challenging to understand how they convey information to their principal intracellular targets: heterotrimeric G proteins, G protein-coupled receptor kinases, and arrestins. Recent structures now suggest a common mechanism that enables structurally diverse cytoplasmic proteins to hitch a ride on hundreds of different activated 7TM receptors in order to instigate physiological change. PMID:27093944

  2. Structure of the human smoothened receptor 7TM bound to an antitumor agent

    PubMed Central

    Wang, Chong; Wu, Huixian; Katritch, Vsevolod; Han, Gye Won; Huang, Xi-Ping; Liu, Wei; Siu, Fai Yiu; Roth, Bryan L.; Cherezov, Vadim; Stevens, Raymond C.

    2013-01-01

    The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. PMID:23636324

  3. Role of the C-terminal domain of PCSK9 in degradation of the LDL receptors.

    PubMed

    Holla, Øystein L; Cameron, Jamie; Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Laerdahl, Jon K; Leren, Trond P

    2011-10-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) at the cell surface and disrupts the normal recycling of the LDLR. In this study, we investigated the role of the C-terminal domain for the activity of PCSK9. Experiments in which conserved residues and histidines on the surface of the C-terminal domain were mutated indicated that no specific residues of the C-terminal domain, apart from those responsible for maintaining the overall structure, are required for the activity of PCSK9. Rather, the net charge of the C-terminal domain is important. The more positively charged the C-terminal domain, the higher the activity toward the LDLR. Moreover, replacement of the C-terminal domain with an unrelated protein of comparable size led to significant activity of the chimeric protein. We conclude that the role of the evolutionary, poorly conserved C-terminal domain for the activity of PCSK9 reflects its overall positive charge and size and not the presence of specific residues involved in protein-protein interactions.

  4. Differential subcellular localization of insulin receptor substrates depends on C-terminal regions and importin {beta}

    SciTech Connect

    Kabuta, Tomohiro; Take, Kazumi; Kabuta, Chihana; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2008-12-19

    Insulin receptor substrates (IRSs) play essential roles in signal transduction of insulin and insulin-like growth factors. Previously, we showed that IRS-3 is localized to the nucleus as well as the cytosol, while IRS-1 and 2 are mainly localized to the cytoplasm. In the present study, we found that importin {beta} directly interacts with IRS-3 and is able to mediate nuclear transport of IRS-3. Importin {beta} interacted with the pleckstrin homology domain, the phosphotyrosine binding domain and the C-terminal region of IRS-3; indeed all of these fragments exhibited predominant nuclear localization. By contrast, almost no interaction of importin {beta} with IRS-1 and -2 was observed, and their C-terminal regions displayed discrete spotty images in the cytosol. In addition, using chimeric proteins between IRS-1 and IRS-3, we revealed that the C-terminal regions are the main determinants of the differing subcellular localizations of IRS-1 and IRS-3.

  5. Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain.

    PubMed

    Lee, Nam Y; Hazlett, Theodore L; Koland, John G

    2006-05-01

    The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of alpha-helices and beta-sheets, with a marginal change in beta-sheet content occurring upon phosphorylation.

  6. Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain

    PubMed Central

    Lee, Nam Y.; Hazlett, Theodore L.; Koland, John G.

    2006-01-01

    The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the larger correlation time of EGFR-ICD was observed upon its autophosphorylation. This dynamic component was faster than predicted for the globular motion of the 62-kDa EGFR-ICD, suggesting an increase in the mobility of the C-terminal domain and a likely displacement of this domain from the kinase core. The interaction between the SH2 domain of c-Src and the phosphorylated EGFR C-terminal domain was shown to impede its mobility. Circular dichroism spectroscopy indicated that the EGFR C-terminal domain possessed a significant level of secondary structure in the form of α-helices and β-sheets, with a marginal change in β-sheet content occurring upon phosphorylation. PMID:16597832

  7. Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain

    PubMed Central

    Lee, Nam Y.; Koland, John G.

    2005-01-01

    The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2′(3′)-O-(2,4,6-trinitrophenyl)-adenosine 5′-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core. PMID:16199664

  8. Conformational changes accompany phosphorylation of the epidermal growth factor receptor C-terminal domain.

    PubMed

    Lee, Nam Y; Koland, John G

    2005-11-01

    The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C-terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C-terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C-terminal lengths were site-specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), binding at their active sites. This novel BFP/TNP-ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP-donor quenching by bound TNP-ATP. In particular, a marked reduction in energy transfer was observed for the full-length BFP-labeled EGFR-ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP-ATP-occupied active site for the full-length and C-terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP-ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation-dependent conformational changes in the C-terminal phosphorylation domain and its likely interaction with the kinase core.

  9. C-terminal dimerization of apo-cyclic AMP receptor protein validated in solution.

    PubMed

    Sim, Dae-Won; Choi, Jae Wan; Kim, Ji-Hun; Ryu, Kyoung-Seok; Kim, Myeongkyu; Yu, Hee-Wan; Jo, Ku-Sung; Kim, Eun-Hee; Seo, Min-Duk; Jeon, Young Ho; Lee, Bong-Jin; Kim, Young Pil; Won, Hyung-Sik

    2017-04-01

    Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP.

  10. C-terminal tail of FGF19 determines its specificity toward Klotho co-receptors.

    PubMed

    Wu, Xinle; Lemon, Bryan; Li, XiaoFan; Gupte, Jamila; Weiszmann, Jennifer; Stevens, Jennitte; Hawkins, Nessa; Shen, Wenyan; Lindberg, Richard; Chen, Jin-Long; Tian, Hui; Li, Yang

    2008-11-28

    FGF19 subfamily proteins (FGF19, FGF21, and FGF23) are unique members of fibroblast growth factors (FGFs) that regulate energy, bile acid, glucose, lipid, phosphate, and vitamin D homeostasis in an endocrine fashion. Their activities require the presence of alpha or betaKlotho, two related single-pass transmembrane proteins, as co-receptors in relevant target tissues. We previously showed that FGF19 can bind to both alpha and betaKlotho, whereas FGF21 and FGF23 can bind only to either betaKlotho or alphaKlotho, respectively in vitro. To determine the mechanism regulating the binding and specificity among FGF19 subfamily members to Klotho family proteins, chimeric proteins between FGF19 subfamily members or chimeric proteins between Klotho family members were constructed to probe the interaction between those two families. Our results showed that a chimera of FGF19 with the FGF21 C-terminal tail interacts only with betaKlotho and a chimera with the FGF23 C-terminal tail interacts only with alphaKlotho. FGF signaling assays also reflected the change of specificity we observed for the chimeras. These results identified the C-terminal tail of FGF19 as a region necessary for its recognition of Klotho family proteins. In addition, chimeras between alpha and betaKlotho were also generated to probe the regions in Klotho proteins that are important for signaling by this FGF subfamily. Both FGF23 and FGF21 require intact alpha or betaKlotho for signaling, respectively, whereas FGF19 can signal through a Klotho chimera consisting of the N terminus of alphaKlotho and the C terminus of betaKlotho. Our results provide the first glimpse of the regions that regulate the binding specificity between this unique family of FGFs and their co-receptors.

  11. Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.

    PubMed

    Baker, Sarah J; Morris, Judy L; Gibbins, Ian L

    2003-03-17

    In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.

  12. C-terminal motif of human neuropeptide Y4 receptor determines internalization and arrestin recruitment.

    PubMed

    Wanka, Lizzy; Babilon, Stefanie; Burkert, Kerstin; Mörl, Karin; Gurevich, Vsevolod V; Beck-Sickinger, Annette G

    2017-01-01

    The human neuropeptide Y4 receptor is a rhodopsin-like G protein-coupled receptor (GPCR), which contributes to anorexigenic signals. Thus, this receptor is a highly interesting target for metabolic diseases. As GPCR internalization and trafficking affect receptor signaling and vice versa, we aimed to investigate the molecular mechanism of hY4R desensitization and endocytosis. The role of distinct segments of the hY4R carboxyl terminus was investigated by fluorescence microscopy, binding assays, inositol turnover experiments and bioluminescence resonance energy transfer assays to examine the internalization behavior of hY4R and its interaction with arrestin-3. Based on results of C-terminal deletion mutants and substitution of single amino acids, the motif (7.78)EESEHLPLSTVHTEVSKGS(7.96) was identified, with glutamate, threonine and serine residues playing key roles, based on site-directed mutagenesis. Thus, we identified the internalization motif for the human neuropeptide Y4 receptor, which regulates arrestin-3 recruitment and receptor endocytosis.

  13. Nipah Virus Attachment Glycoprotein Stalk C-Terminal Region Links Receptor Binding to Fusion Triggering

    PubMed Central

    Liu, Qian; Bradel-Tretheway, Birgit; Monreal, Abrrey I.; Saludes, Jonel P.; Lu, Xiaonan; Nicola, Anthony V.

    2014-01-01

    ABSTRACT Membrane fusion is essential for paramyxovirus entry into target cells and for the cell-cell fusion (syncytia) that results from many paramyxoviral infections. The concerted efforts of two membrane-integral viral proteins, the attachment (HN, H, or G) and fusion (F) glycoproteins, mediate membrane fusion. The emergent Nipah virus (NiV) is a highly pathogenic and deadly zoonotic paramyxovirus. We recently reported that upon cell receptor ephrinB2 or ephrinB3 binding, at least two conformational changes occur in the NiV-G head, followed by one in the NiV-G stalk, that subsequently result in F triggering and F execution of membrane fusion. However, the domains and residues in NiV-G that trigger F and the specific events that link receptor binding to F triggering are unknown. In the present study, we identified a NiV-G stalk C-terminal region (amino acids 159 to 163) that is important for multiple G functions, including G tetramerization, conformational integrity, G-F interactions, receptor-induced conformational changes in G, and F triggering. On the basis of these results, we propose that this NiV-G region serves as an important structural and functional linker between the NiV-G head and the rest of the stalk and is critical in propagating the F-triggering signal via specific conformational changes that open a concealed F-triggering domain(s) in the G stalk. These findings broaden our understanding of the mechanism(s) of receptor-induced paramyxovirus F triggering during viral entry and cell-cell fusion. IMPORTANCE The emergent deadly viruses Nipah virus (NiV) and Hendra virus belong to the Henipavirus genus in the Paramyxoviridae family. NiV infections target endothelial cells and neurons and, in humans, result in 40 to 75% mortality rates. The broad tropism of the henipaviruses and the unavailability of therapeutics threaten the health of humans and livestock. Viral entry into host cells is the first step of henipavirus infections, which ultimately cause

  14. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

    PubMed

    Ebersole, Brittany; Petko, Jessica; Woll, Matthew; Murakami, Shoko; Sokolina, Kate; Wong, Victoria; Stagljar, Igor; Lüscher, Bernhard; Levenson, Robert

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  15. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

    PubMed Central

    Ebersole, Brittany; Petko, Jessica; Woll, Matthew; Murakami, Shoko; Sokolina, Kate; Wong, Victoria; Stagljar, Igor; Lüscher, Bernhard; Levenson, Robert

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R. PMID:26535572

  16. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  17. Structure of metabotropic glutamate receptor C-terminal domains in contact with interacting proteins.

    PubMed

    Enz, Ralf

    2012-01-01

    Metabotropic glutamate receptors (mGluRs) regulate intracellular signal pathways that control several physiological tasks, including neuronal excitability, learning, and memory. This is achieved by the formation of synaptic signal complexes, in which mGluRs assemble with functionally related proteins such as enzymes, scaffolds, and cytoskeletal anchor proteins. Thus, mGluR associated proteins actively participate in the regulation of glutamatergic neurotransmission. Importantly, dysfunction of mGluRs and interacting proteins may lead to impaired signal transduction and finally result in neurological disorders, e.g., night blindness, addiction, epilepsy, schizophrenia, autism spectrum disorders and Parkinson's disease. In contrast to solved crystal structures of extracellular N-terminal domains of some mGluR types, only a few studies analyzed the conformation of intracellular receptor domains. Intracellular C-termini of most mGluR types are subject to alternative splicing and can be further modified by phosphorylation and SUMOylation. In this way, diverse interaction sites for intracellular proteins that bind to and regulate the glutamate receptors are generated. Indeed, most of the known mGluR binding partners interact with the receptors' C-terminal domains. Within the last years, different laboratories analyzed the structure of these domains and described the geometry of the contact surface between mGluR C-termini and interacting proteins. Here, I will review recent progress in the structure characterization of mGluR C-termini and provide an up-to-date summary of the geometry of these domains in contact with binding partners.

  18. Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors.

    PubMed

    Sato, Takaaki; Kawasaki, Takashi; Mine, Shouhei; Matsumura, Hiroyoshi

    2016-11-18

    G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1-2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies.

  19. Functional Role of the C-Terminal Amphipathic Helix 8 of Olfactory Receptors and Other G Protein-Coupled Receptors

    PubMed Central

    Sato, Takaaki; Kawasaki, Takashi; Mine, Shouhei; Matsumura, Hiroyoshi

    2016-01-01

    G protein-coupled receptors (GPCRs) transduce various extracellular signals, such as neurotransmitters, hormones, light, and odorous chemicals, into intracellular signals via G protein activation during neurological, cardiovascular, sensory and reproductive signaling. Common and unique features of interactions between GPCRs and specific G proteins are important for structure-based design of drugs in order to treat GPCR-related diseases. Atomic resolution structures of GPCR complexes with G proteins have revealed shared and extensive interactions between the conserved DRY motif and other residues in transmembrane domains 3 (TM3), 5 and 6, and the target G protein C-terminal region. However, the initial interactions formed between GPCRs and their specific G proteins remain unclear. Alanine scanning mutagenesis of the murine olfactory receptor S6 (mOR-S6) indicated that the N-terminal acidic residue of helix 8 of mOR-S6 is responsible for initial transient and specific interactions with chimeric Gα15_olf, resulting in a response that is 2.2-fold more rapid and 1.7-fold more robust than the interaction with Gα15. Our mutagenesis analysis indicates that the hydrophobic core buried between helix 8 and TM1–2 of mOR-S6 is important for the activation of both Gα15_olf and Gα15. This review focuses on the functional role of the C-terminal amphipathic helix 8 based on several recent GPCR studies. PMID:27869740

  20. PCSK9-mediated degradation of the LDL receptor generates a 17 kDa C-terminal LDL receptor fragment.

    PubMed

    Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Leren, Trond P

    2013-06-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR) at the cell surface and reroutes the internalized LDLR to intracellular degradation. In this study, we have shown that PCSK9-mediated degradation of the full-length 160 kDa LDLR generates a 17 kDa C-terminal LDLR fragment. This fragment was not generated from mutant LDLRs resistant to PCSK9-mediated degradation or when degradation was prevented by chemicals such as ammonium chloride or the cysteine cathepsin inhibitor E64d. The observation that the 17 kDa fragment was only detected when the cells were cultured in the presence of the γ-secretase inhibitor DAPT indicates that this 17 kDa fragment undergoes γ-secretase cleavage within the transmembrane domain. The failure to detect the complementary 143 kDa ectodomain fragment is likely to be due to its rapid degradation in the endosomal lumen. The 17 kDa C-terminal LDLR fragment was also generated from a Class 5 mutant LDLR undergoing intracellular degradation. Thus, one may speculate that an LDLR with bound PCSK9 and a Class 5 LDLR with bound LDL are degraded by a similar mechanism that could involve ectodomain cleavage in the endosome.

  1. New melanocortin 1 receptor binding motif based on the C-terminal sequence of alpha-melanocyte-stimulating hormone.

    PubMed

    Schiöth, Helgi B; Muceniece, Ruta; Mutule, Ilga; Wikberg, Jarl E S

    2006-10-01

    The C-terminal tripeptide of the alpha-melanocyte stimulating hormone (alpha-MSH11-13) possesses strong antiinflammatory activity without known cellular target. In order to better understand the structural requirements for function of such motif, we designed, synthesized and tested out Trp- and Tyr-containing analogues of the alpha-MSH11-13. Seven alpha-MSH11-13 analogues were synthesized and characterized for their binding to the melanocortin receptors recombinantly expressed in insect (Sf9) cells, infected with baculovirus carrying corresponding MC receptor DNA. We also tested these analogues on B16-F1 mouse melanoma cells endogenously expressing the MC1 receptor for binding and for ability to increase cAMP levels as well as on COS-7 cells transfected with the human MC receptors. The data indicate that HS401 (Ac-Tyr-Lys-Pro-Val-NH2) and HS402 (Ac-Lys-Pro-Val-Tyr-NH2) selectively bound to the MC1 receptor and stimulated cAMP generation in a concentration dependent way while the other Tyr- and Trp-containing alpha-MSH11-13 analogues neither bound to MC receptors nor stimulated cAMP. We have thus identified new MC receptor binding motif derived from the C-terminal sequence of alpha-MSH. The tetrapeptides have novel properties as the both act via MC-ergic pathways and also carry the anti-inflammatory alpha-MSH11-13 message sequence.

  2. Transmembrane signalling at the epidermal growth factor receptor. Positive regulation by the C-terminal phosphotyrosine residues.

    PubMed Central

    Magni, M; Pandiella, A; Helin, K; Meldolesi, J; Beguinot, L

    1991-01-01

    Mutant epidermal growth factor (EGF) receptors (obtained by substitution of one, two or three C-terminal autophosphorylable tyrosine residues with phenylalanine residues or by deletion of the C-terminal 19 amino acids, including the distal tyrosine) were expressed in mouse NIH-3T3 fibroblast clones at densities comparable (less than 25% difference) with those in control clones expressing the wild-type receptor. Total EGF-induced phosphorylation of the mutated receptors was not appreciably changed with respect to controls, whereas autophosphorylation at tyrosine residues was decreased, especially in the double and the triple mutants. In the latter mutant, expression of the EGF-receptor-activated lipolytic enzyme phospholipase C gamma was unchanged, whereas its tyrosine phosphorylation induced by the growth factor was lowered to approx. 25% of that in the controls. In all of the cell clones employed, the accumulation of inositol phosphates induced by treatment with fetal calf serum varied only slightly, whereas the same effect induced by EGF was consistently lowered in those lines expressing mutated receptors. This decrease was moderate for those receptors missing only the distal tyrosine (point and deletion mutants), intermediate in the dual mutants and almost complete in the triple mutants. Likewise, increases in intracellular Ca2+ concentrations [( Ca2+]i) induced by fibroblast growth factor were approximately the same in all of the clones, whereas those induced by EGF were decreased in the mutants, again in proportion to the loss of the phosphorylable C-terminal tyrosine residues. The same trend occurred with membrane hyperpolarization, an effect secondary to the increase in [Ca2+]i via the activation of Ca2(+)-dependent K+ channels. We conclude that C-terminal autophosphorylable tyrosine residues play a positive role in the regulation of transmembrane signalling at the EGF receptor. The stepwise decrease in signal generation observed in single, double and triple

  3. PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments.

    PubMed

    Delloye-Bourgeois, Céline; Jacquier, Arnaud; Charoy, Camille; Reynaud, Florie; Nawabi, Homaira; Thoinet, Karine; Kindbeiter, Karine; Yoshida, Yutaka; Zagar, Yvrick; Kong, Youxin; Jones, Yvonne E; Falk, Julien; Chédotal, Alain; Castellani, Valérie

    2015-01-01

    Robo-Slit and Plexin-Semaphorin signaling participate in various developmental and pathogenic processes. During commissural axon guidance in the spinal cord, chemorepulsion by Semaphorin3B and Slits controls midline crossing. Slit processing generates an N-terminal fragment (SlitN) that binds to Robo1 and Robo2 receptors and mediates Slit repulsive activity, as well as a C-terminal fragment (SlitC) with an unknown receptor and bioactivity. We identified PlexinA1 as a Slit receptor and found that it binds the C-terminal Slit fragment specifically and transduces a SlitC signal independently of the Robos and the Neuropilins. PlexinA1-SlitC complexes are detected in spinal cord extracts, and ex vivo, SlitC binding to PlexinA1 elicits a repulsive commissural response. Analysis of various ligand and receptor knockout mice shows that PlexinA1-Slit and Robo-Slit signaling have complementary roles during commissural axon guidance. Thus, PlexinA1 mediates both Semaphorin and Slit signaling, and Slit processing generates two active fragments, each exerting distinct effects through specific receptors.

  4. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  5. Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils*

    PubMed Central

    Maaty, Walid S.; Lord, Connie I.; Gripentrog, Jeannie M.; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A.; Bothner, Brian; Jesaitis, Algirdas J.

    2013-01-01

    Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu312–Arg322 and Arg323–Lys350) and extracellular FPR1 peptide (Ile191–Arg201) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala323–Lys350 only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr325, Ser328, Thr329, Thr331, Ser332, Thr334, and Thr339. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of

  6. Identification of C-terminal phosphorylation sites of N-formyl peptide receptor-1 (FPR1) in human blood neutrophils.

    PubMed

    Maaty, Walid S; Lord, Connie I; Gripentrog, Jeannie M; Riesselman, Marcia; Keren-Aviram, Gal; Liu, Ting; Dratz, Edward A; Bothner, Brian; Jesaitis, Algirdas J

    2013-09-20

    Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 Mr) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu(312)-Arg(322) and Arg(323)-Lys(350)) and extracellular FPR1 peptide (Ile(191)-Arg(201)) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala(323)-Lys(350) only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr(325), Ser(328), Thr(329), Thr(331), Ser(332), Thr(334), and Thr(339). No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC50 = 33 ± 8 to 74 ± 21 nM. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites

  7. Structure-Activity Relationship Studies of N- and C-Terminally Modified Secretin Analogs for the Human Secretin Receptor

    PubMed Central

    Arokiaraj, Aloysius Wilfred Raj; Leprince, Jérôme; Lefranc, Benjamin; Vaudry, David; Allam, Ahmed A.; Ajarem, Jamaan; Chow, Billy K. C.

    2016-01-01

    The pleiotropic role of human secretin (hSCT) validates its potential use as a therapeutic agent. Nevertheless, the structure of secretin in complex with its receptor is necessary to develop a suitable therapeutic agent. Therefore, in an effort to design a three-dimensional virtual homology model and identify a peptide agonist and/or antagonist for the human secretin receptor (hSR), the significance of the primary sequence of secretin peptides in allosteric binding and activation was elucidated using virtual docking, FRET competitive binding and assessment of the cAMP response. Secretin analogs containing various N- or C-terminal modifications were prepared based on previous findings of the role of these domains in receptor binding and activation. These analogs exhibited very low or no binding affinity in a virtual model, and were found to neither exhibit in vitro binding nor agonistic or antagonistic properties. A parallel analysis of the analogs in the virtual model and in vitro studies revealed instability of these peptide analogs to bind and activate the receptor. PMID:26930505

  8. Sequence analysis of the non-recurring C-terminal domains shows that insect lipoprotein receptors constitute a distinct group of LDL receptor family members.

    PubMed

    Rodenburg, Kees W; Smolenaars, Marcel M W; Van Hoof, Dennis; Van der Horst, Dick J

    2006-04-01

    Lipoprotein-mediated delivery of lipids in mammals involves endocytic receptors of the low density lipoprotein (LDL) receptor (LDLR) family. In contrast, in insects, the lipoprotein, lipophorin (Lp), functions as a reusable lipid shuttle in lipid delivery, and these animals, therefore, were not supposed to use endocytic receptors. However, recent data indicate additional endocytic uptake of Lp, mediated by a Lp receptor (LpR) of the LDLR family. The two N-terminal domains of LDLR family members are involved in ligand binding and dissociation, respectively, and are composed of a mosaic of multiple repeats. The three C-terminal domains, viz., the optional O-linked glycosylation domain, the transmembrane domain, and the intracellular domain, are of a non-repetitive sequence. The present classification of newly discovered LDLR family members, including the LpRs, bears no relevance to physiological function. Therefore, as a novel approach, the C-terminal domains of LDLR family members across the entire animal kingdom were used to perform a sequence comparison analysis in combination with a phylogenetic tree analysis. The LpRs appeared to segregate into a specific group distinct from the groups encompassing the other family members, and each of the three C-terminal domains of the insect receptors is composed of unique set of sequence motifs. Based on conservation of sequence motifs and organization of these motifs in the domains, LpR resembles most the groups of the LDLRs, very low density lipoprotein (VLDL) receptors, and vitellogenin receptors. However, in sequence aspects in which LpR deviates from these three receptor groups, it most notably resembles LDLR-related protein-2, or megalin. These features might explain the functional differences disclosed between insect and mammalian lipoprotein receptors.

  9. Unwinding of the C-Terminal Residues of Neuropeptide Y is critical for Y₂ Receptor Binding and Activation.

    PubMed

    Kaiser, Anette; Müller, Paul; Zellmann, Tristan; Scheidt, Holger A; Thomas, Lars; Bosse, Mathias; Meier, Rene; Meiler, Jens; Huster, Daniel; Beck-Sickinger, Annette G; Schmidt, Peter

    2015-06-15

    Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

  10. Selective suppression of the human aryl hydrocarbon receptor function can be mediated through binding interference at the C-terminal half of the receptor.

    PubMed

    Ren, Lina; Thompson, John D; Cheung, Michael; Ngo, Katherine; Sung, Sarah; Leong, Scott; Chan, William K

    2016-05-01

    The human aryl hydrocarbon receptor is a cytosolic signaling molecule which affects immune response and aberrant cell growth. Canonical signaling of the receptor requires the recruitment of coactivators to the promoter region to remodel local chromatin structure. We predicted that interference of this recruitment would block the aryl hydrocarbon receptor function. To prove that, we employed phage display to identify nine peptides of twelve-amino-acid in length which target the C-terminal half of the human aryl hydrocarbon receptor, including the region where coactivators bind. Eight 12mer peptides, in the form of GFP fusion, suppressed the ligand-dependent transcription of six AHR target genes (cyp1a1, cyp1a2, cyp1b1, ugt1a1, nqo1, and ahrr) in different patterns in Hep3B cells, whereas the AHR antagonist CH-223191 suppressed all these target genes similarly. Three of the 12mer peptides (namely 11-3, 1-7, and 7-3) suppressed the 3MC-induced, CYP1A1-dependent EROD activity and the ROS production caused by benzo[a]pyrene. These 12mer peptides suppressed the AHR function synergistically with CH-223191. In conclusion, we provide evidence that targeting the C-terminal half of the human aryl hydrocarbon receptor is a viable, new approach to selectively block the receptor function.

  11. Human thrombospondin's (TSP-1) C-terminal domain opens to interact with the CD-47 receptor: a molecular modeling study.

    PubMed

    Floquet, Nicolas; Dedieu, Stéphane; Martiny, Laurent; Dauchez, Manuel; Perahia, David

    2008-10-01

    Thrombospondin-1 (TSP-1) interaction with the membranous receptor CD-47 involves the peptide RFYVVMWK (4N-1) located in its C-terminal domain. However, the available X-ray structure of TSP-1 describes this peptide as completely buried into a hydrophobic pocket, preventing any interaction. Where classical standard methods failed, an appropriate approach combining normal mode analysis and an adapted protocol of energy minimization identified the large amplitude motions responsible of the partial solvent exposure of 4N-1. In agreement, the obtained model of the open TSP-1 was further used for protein-protein docking experiments against a homology model generated for CD-47. Considering the multiple applications of the CD-47 receptor as a target, our results open new pharmacological perspectives for the design of TSP-1:CD-47 inhibitors and CD-47 antagonists. We also suggest a common opening mechanism for proteins sharing the same fold as TSP-1. This work also suggests the usefulness of our approach in other topics in which predictions of protein-protein interactions are of importance.

  12. Functional Characterization of C-terminal Ryanodine Receptor 1 Variants Associated with Central Core Disease or Malignant Hyperthermia.

    PubMed

    Parker, Remai; Schiemann, Anja H; Langton, Elaine; Bulger, Terasa; Pollock, Neil; Bjorksten, Andrew; Gillies, Robyn; Hutchinson, David; Roxburgh, Richard; Stowell, Kathryn M

    2017-01-01

    Central core disease and malignant hyperthermia are human disorders of skeletal muscle resulting from aberrant Ca2+ handling. Most malignant hyperthermia and central core disease cases are associated with amino acid changes in the type 1 ryanodine receptor (RyR1), the skeletal muscle Ca2+-release channel. Malignant hyperthermia exhibits a gain-of-function phenotype, and central core disease results from loss of channel function. For a variant to be classified as pathogenic, functional studies must demonstrate a correlation with the pathophysiology of malignant hyperthermia or central core disease. We assessed the pathogenicity of four C-terminal variants of the ryanodine receptor using functional analysis. The variants were identified in families affected by either malignant hyperthermia or central core disease. Four variants were introduced separately into human cDNA encoding the skeletal muscle ryanodine receptor. Following transient expression in HEK-293T cells, functional studies were carried out using calcium release assays in response to an agonist. Two previously characterized variants and wild-type skeletal muscle ryanodine receptor were used as controls. The p.Met4640Ile variant associated with central core disease showed no difference in calcium release compared to wild-type. The p.Val4849Ile variant associated with malignant hyperthermia was more sensitive to agonist than wild-type but did not reach statistical significance and two variants (p.Phe4857Ser and p.Asp4918Asn) associated with central core disease were completely inactive. The p.Val4849Ile variant should be considered a risk factor for malignant hyperthermia, while the p.Phe4857Ser and p.Asp4918Asn variants should be classified as pathogenic for central core disease.

  13. Biased agonism and allosteric modulation of G protein-coupled receptor 183 - a 7TM receptor also known as Epstein-Barr virus-induced gene 2.

    PubMed

    Daugvilaite, Viktorija; Madsen, Christian Medom; Lückmann, Michael; Echeverria, Clara Castello; Sailer, Andreas Walter; Frimurer, Thomas Michael; Rosenkilde, Mette Marie; Benned-Jensen, Tau

    2017-07-01

    The GPCR Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored. We investigated the effects of the EBI2 endogenous agonist 7α,25-dihydroxycholesterol (7α,25-OHC) on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of the EBI2 receptor to investigate the structural basis of the allosteric modulation by sodium. Residue N114, located in the middle of transmembrane-III at position III:11/3.35, was found to function as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolished heterotrimeric G protein subunit Gi α activation by 7α,25-OHC even though the specific binding of [(3) H]-7α,25-OHC increased. In contrast, the N114A mutant was still able to recruit β-arrestin and even had an enhanced potency (18.7-fold) compared with EBI2 wild type. Sodium had a negative allosteric effect on oxysterol binding that was mediated via N114, verifying the key role of N114. This was further supported by molecular modelling of the ion binding site based on a EBI2 receptor homology model. Collectively, our data point to N114 as a key residue for EBI2 signalling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding. © 2017 The British Pharmacological Society.

  14. An overview of the sequence features of N- and C-terminal segments of the human chemokine receptors.

    PubMed

    Raucci, Raffaele; Costantini, Susan; Castello, Giuseppe; Colonna, Giovanni

    2014-12-01

    Chemokine receptors play a crucial role in the cellular signaling enrolling extracellular ligands chemotactic proteins which recruit immune cells. They possess seven trans-membrane helices, an extracellular N-terminal region with three extracellular hydrophilic loops being important for search and recognition of specific ligand(s), and an intracellular C-terminal region with three intracellular loops that couple G-proteins. Although the functional aspects of the terminal segments of the extra-and intra-cellular G proteins are universally identified, the molecular basis on which they rest are still unclear because they are not definable by means of X-rays due to their high mobility and are not easy to study in the membrane. The purpose of this work is to define which physical-chemical properties of the terminal segments of the human chemokine receptors are at the basis of their functional mechanisms. Therefore, we have evaluated their physical-chemical properties in terms of amino acid composition, local flexibility, disorder propensity, net charge distribution and putative sites of post-translational modifications. Our results support the conclusion that all 19 C-terminal and N-terminal segments of human chemokine receptors are very flexible due to the systematic presence of intrinsic disorder. Although, the purpose of this plasticity clearly appears that of controlling and modulating the binding of ligands, we provide evidence that the overlap of linearly charged stretches, intrinsic disorder and post-translational modification sites, consistently found in these motives, is a necessary feature to exert the function. The role of the intrinsic disorder has been discussed considering the structural information coming from intrinsically disordered model compounds which support the view that the chemokine terminals have to be considered as strong polyampholytes or polyelectrolytes where conformational ensembles and structural transitions between them are modulated by

  15. C-terminal threonines and serines play distinct roles in the desensitization of rhodopsin, a G protein-coupled receptor

    PubMed Central

    Azevedo, Anthony W; Doan, Thuy; Moaven, Hormoz; Sokal, Iza; Baameur, Faiza; Vishnivetskiy, Sergey A; Homan, Kristoff T; Tesmer, John JG; Gurevich, Vsevolod V; Chen, Jeannie; Rieke, Fred

    2015-01-01

    Rod photoreceptors generate measurable responses to single-photon activation of individual molecules of the G protein-coupled receptor (GPCR), rhodopsin. Timely rhodopsin desensitization depends on phosphorylation and arrestin binding, which quenches G protein activation. Rhodopsin phosphorylation has been measured biochemically at C-terminal serine residues, suggesting that these residues are critical for producing fast, low-noise responses. The role of native threonine residues is unclear. We compared single-photon responses from rhodopsin lacking native serine or threonine phosphorylation sites. Contrary to expectation, serine-only rhodopsin generated prolonged step-like single-photon responses that terminated abruptly and randomly, whereas threonine-only rhodopsin generated responses that were only modestly slower than normal. We show that the step-like responses of serine-only rhodopsin reflect slow and stochastic arrestin binding. Thus, threonine sites play a privileged role in promoting timely arrestin binding and rhodopsin desensitization. Similar coordination of phosphorylation and arrestin binding may more generally permit tight control of the duration of GPCR activity. DOI: http://dx.doi.org/10.7554/eLife.05981.001 PMID:25910054

  16. Analysis of the Role of the C-Terminal Tail in the Regulation of the Epidermal Growth Factor Receptor

    PubMed Central

    Kovacs, Erika; Das, Rahul; Wang, Qi; Collier, Timothy S.; Cantor, Aaron; Huang, Yongjian; Wong, Kathryn; Mirza, Amar; Barros, Tiago; Grob, Patricia; Jura, Natalia; Bose, Ron

    2015-01-01

    The ∼230-residue C-terminal tail of the epidermal growth factor receptor (EGFR) is phosphorylated upon activation. We examined whether this phosphorylation is affected by deletions within the tail and whether the two tails in the asymmetric active EGFR dimer are phosphorylated differently. We monitored autophosphorylation in cells using flow cytometry and found that the first ∼80 residues of the tail are inhibitory, as demonstrated previously. The entire ∼80-residue span is important for autoinhibition and needs to be released from both kinases that form the dimer. These results are interpreted in terms of crystal structures of the inactive kinase domain, including two new ones presented here. Deletions in the remaining portion of the tail do not affect autophosphorylation, except for a six-residue segment spanning Tyr 1086 that is critical for activation loop phosphorylation. Phosphorylation of the two tails in the dimer is asymmetric, with the activator tail being phosphorylated somewhat more strongly. Unexpectedly, we found that reconstitution of the transmembrane and cytoplasmic domains of EGFR in vesicles leads to a peculiar phenomenon in which kinase domains appear to be trapped between stacks of lipid bilayers. This artifactual trapping of kinases between membranes enhances an intrinsic functional asymmetry in the two tails in a dimer. PMID:26124280

  17. Differential DNA binding by the androgen and glucocorticoid receptors involves the second Zn-finger and a C-terminal extension of the DNA-binding domains.

    PubMed Central

    Schoenmakers, E; Alen, P; Verrijdt, G; Peeters, B; Verhoeven, G; Rombauts, W; Claessens, F

    1999-01-01

    The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat. PMID:10417312

  18. Annexin A2 is a C-terminal PCSK9-binding protein that regulates endogenous low density lipoprotein receptor levels.

    PubMed

    Mayer, Gaétan; Poirier, Steve; Seidah, Nabil G

    2008-11-14

    The proprotein convertase subtilisin/kexin-type 9 (PCSK9), which promotes degradation of the hepatic low density lipoprotein receptor (LDLR), is now recognized as a major player in plasma cholesterol metabolism. Several gain-of-function mutations in PCSK9 cause hypercholesterolemia and premature atherosclerosis, and thus, inhibition of PCSK9-induced degradation of the LDLR may be used to treat this deadly disease. Herein, we discovered an endogenous PCSK9 binding partner by Far Western blotting, co-immunoprecipitation, and pull-down assays. Following two-dimensional gel electrophoresis and mass spectrometry analysis, we demonstrated that PCSK9 binds to a approximately 33-kDa protein identified as annexin A2 (AnxA2) but not to the closely related annexin A1. Furthermore, our functional LDLR assays and small hairpin RNA studies show that AnxA2 and the AnxA2.p11 complex could prevent PCSK9-directed LDLR degradation in HuH7, HepG2, and Chinese hamster ovary cells. Immunocytochemistry revealed that PCSK9 and AnxA2 co-localize at the cell surface, indicating a possible competition with the LDLR. Structure-function analyses demonstrated that the C-terminal cysteine-histidine-rich domain of PCSK9 interacts specifically with the N-terminal repeat R1 of AnxA2. Mutational analysis of this 70-amino acid-long repeat indicated that the RRTKK81 sequence of AnxA2 is implicated in this binding because its mutation to AATAA81 prevents its interaction with PCSK9. To our knowledge, this work constitutes the first to show that PCSK9 activity on LDLR can be regulated by an endogenous inhibitor. The identification of the minimal inhibitory sequence of AnxA2 should pave the way toward the development of PCSK9 inhibitory lead molecules for the treatment of hypercholesterolemia.

  19. *The autoinhibitory C-terminal SH2 domain of phospholipase C–γ2 stabilizes B cell receptor signalosome assembly

    PubMed Central

    Wang, Jing; Sohn, Haewon; Sun, Guangping; Milner, Joshua D.; Pierce, Susan K.

    2014-01-01

    The binding of antigen to the B cell receptor (BCR) stimulates the assembly of a signaling complex (signalosome) composed initially of the kinases Lyn, spleen tyrosine kinase (Syk), and Bruton’s tyrosine kinase (Btk), as well as the adaptor protein B cell linker (BLNK). Together, these proteins recruit and activate phospholipase C–γ2 (PLC-γ2), a critical effector that stimulates increases in intracellular Ca2+ and activates various signaling pathways downstream of the BCR. Individuals with one copy of a mutant PLCG2 gene, which encodes a variant PLC-γ2 that lacks the autoinhibitory C-terminal Src homology 2 (cSH2) domain, exhibit PLC-γ2– associated antibody deficiencies and immune dysregulation (PLAID). Paradoxically, although COS-7 cells expressing the variant PLC-γ2 show enhanced basal and stimulated PLC-γ2 activity, B cells from PLAID patients show defective intracellular Ca2+ responses upon crosslinking of the BCR. We found that the cSH2 domain of PLC-γ2 played a critical role in stabilizing the early signaling complex that is stimulated by BCR crosslinking. In the presence of the variant PLC-γ2, Syk, Btk, and BLNK were only weakly phosphorylated and failed to stably associate with the BCR. Thus, BCRs could not form stable clusters, resulting in dysregulation of downstream signaling and trafficking of the BCR. Thus, the cSH2 domain functions not only to inhibit the active site of PLC-γ2, but also to directly or indirectly stabilize the early BCR signaling complex. PMID:25227611

  20. C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

    PubMed

    Hishinuma, Shigeru; Nozawa, Hiroki; Akatsu, Chizuru; Shoji, Masaru

    2016-11-01

    It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that Gq/11 -protein-coupled human histamine H1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [(3) H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [(3) H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively.

  1. Direct influence of C-terminally substituted amino acids in the Dmt-Tic pharmacophore on delta-opioid receptor selectivity and antagonism.

    PubMed

    Balboni, Gianfranco; Salvadori, Severo; Guerrini, Remo; Negri, Lucia; Giannini, Elisa; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H

    2004-07-29

    A series of 17 analogues were developed on the basis of the general formula H-Dmt-Tic-NH-CH(R)-R' (denotes chirality; R = charged, neutral, or aromatic functional group; R' = -OH or -NH(2)). These compounds were designed to test the following hypothesis: the physicochemical properties of third-residue substitutions C-terminal to Tic in the Dmt-Tic pharmacophore modify delta-opioid receptor selectivity and delta-opioid receptor antagonism through enhanced interactions with the mu-opioid receptor. The data substantiate the following conclusions: (i) all compounds had high receptor affinity [K(i)(delta) = 0.034-1.1 nM], while that for the mu-opioid receptor fluctuated by orders of magnitude [K(i)(mu) = 15.1-3966 nM]; (ii) delta-opioid receptor selectivity [K(i)(mu)/K(i)(delta)] declined 1000-fold from 22,600 to 21; (iii) a C-terminal carboxyl group enhanced selectivity but only as a consequence of the specific residue; (iv) amidated, positive charged residues [Lys-NH(2) (6), Arg-NH(2) (7)], and a negatively charged aromatic residue [Trp-OH (11)] enhanced mu-opioid affinity [K(i)(mu) = 17.0, 15.1, and 15.7 nM, respectively], while Gly-NH(2) (8), Ser-NH(2) (10), and His-OH (12) were nearly one-tenth as active; and (v) D-isomers exhibited mixed effects on mu-opioid receptor affinity (2' < 3' < 4' < 1' < 5') and decreased delta-selectivity in D-Asp-NH(2) (1') and D-Lys(Ac)-OH (5'). The analogues exhibited delta-opioid receptor antagonism (pA(2) = 6.9-10.07) and weak mu-opioid receptor agonism (IC(50) > 1 microM) except H-Dmt-Tic-Glu-NH(2) (3), which was a partial delta-opioid receptor agonist (IC(50) = 2.5 nM). Thus, these C-terminally extended analogues indicated that an amino acid residue containing a single charge, amino or guanidino functionality, or aromatic group substantially altered the delta-opioid receptor activity profile (selectivity and antagonism) of the Dmt-Tic pharmacophore, which suggests that the C-terminal constituent plays a major role in determining

  2. By Interacting with the C-terminal Phe of Apelin, Phe255 and Trp259 in Helix VI of the Apelin Receptor Are Critical for Internalization*

    PubMed Central

    Iturrioz, Xavier; Gerbier, Romain; Leroux, Vincent; Alvear-Perez, Rodrigo; Maigret, Bernard; Llorens-Cortes, Catherine

    2010-01-01

    Apelin is the endogenous ligand of the orphan seven-transmembrane domain (TM) G protein-coupled receptor APJ. Apelin is involved in the regulation of body fluid homeostasis and cardiovascular functions. We previously showed the importance of the C-terminal Phe of apelin 17 (K17F) in the hypotensive activity of this peptide. Here, we show either by deleting the Phe residue (K16P) or by substituting it by an Ala (K17A), that it plays a crucial role in apelin receptor internalization but not in apelin binding or in Gαi-protein coupling. Then we built a homology three-dimensional model of the human apelin receptor using the cholecystokinin receptor-1 model as a template, and we subsequently docked K17F into the binding site. We visualized a hydrophobic cavity at the bottom of the binding pocket in which the C-terminal Phe of K17F was embedded by Trp152 in TMIV and Trp259 and Phe255 in TMVI. Using molecular modeling and site-directed mutagenesis studies, we further showed that Phe255 and Trp259 are key residues in triggering receptor internalization without playing a role in apelin binding or in Gαi-protein coupling. These findings bring new insights into apelin receptor activation and show that Phe255 and Trp259, by interacting with the C-terminal Phe of the pyroglutamyl form of apelin 13 (pE13F) or K17F, are crucial for apelin receptor internalization. PMID:20675385

  3. Solution Structure and Sugar-Binding Mechanism of Mouse Latrophilin-1 RBL: a 7TM Receptor-Attached Lectin-Like Domain

    PubMed Central

    Vakonakis, Ioannis; Langenhan, Tobias; Prömel, Simone; Russ, Andreas; Campbell, Iain D.

    2008-01-01

    Summary Latrophilin-1 (Lat-1), a target receptor for α-Latrotoxin, is a putative G protein-coupled receptor implicated in synaptic function. The extracellular portion of Lat-1 contains a rhamnose binding lectin (RBL)-like domain of unknown structure. RBL domains, first isolated from the eggs of marine species, are also found in the ectodomains of other metazoan transmembrane proteins, including a recently discovered coreceptor of the neuronal axon guidance molecule SLT-1/Slit. Here, we describe a structure of this domain from the mouse Lat-1. RBL adopts a unique α/β fold with long structured loops important for monosaccharide recognition, as shown in the structure of a complex with L-rhamnose. Sequence alignments and mutagenesis show that residues important for carbohydrate binding are often absent in other receptor-attached examples of RBL, including the SLT-1/Slit coreceptor. We postulate that this domain class facilitates direct protein-protein interactions in many transmembrane receptors. PMID:18547526

  4. Neonatal Fc receptor stimulation induces ubiquitin c-terminal hydrolase-1 overexpression in podocytes through activation of p38 mitogen-activated protein kinase.

    PubMed

    Gan, Hualei; Feng, Songtao; Wu, Huijuan; Sun, Yu; Hu, Ruimin; Zhao, Zhonghua; Zhang, Zhigang

    2012-09-01

    Ubiquitin c-terminal hydrolase-1 is overexpressed in renal podocytes in some immune complex-mediated glomerulonephritides, an effect closely related to extensive podocyte injury. Neonatal Fc receptor is newly recognized to be present on human renal podocytes. It is presumed that neonatal Fc receptor serves as a sensor for immune stimulation transduction and is involved in the pathogenesis of podocyte injury. In our current study, we found that neonatal Fc receptor was constitutively expressed in normal podocytes and up-regulated by immune stimulation induced by antithymocyte serum. An increase in neonatal Fc receptor expression was observed in human podocytes within diseased glomeruli in 97 cases of various glomerulonephritides. The expression percentage was significantly higher in immune-mediated disease, including membranous nephropathy (46.7%), immunoglobin A nephropathy (66.7%), lupus nephritis (87.5%), and acute proliferative glomerulonephritis (100%), than in normal kidney samples (16.7%) (P < .05), whereas there was no significant difference between minimal-change disease and normal kidney. Further study showed that neonatal Fc receptor up-regulated the expression of ubiquitin c-terminal hydrolase-1 via activation of p38 in podocytes subjected to immune stimulation in vitro. These data suggest that neonatal Fc receptor acts as an immune sensor that evokes an inflammatory response, which may lead to functional and morphological changes in podocytes in glomerulonephritides.

  5. DEVELOPMENT OF THE SIGMA-1 RECEPTOR IN C-TERMINALS OF MOTONEURONS AND COLOCALIZATION WITH THE N,N’-DIMETHYLTRYPTAMINE FORMING ENZYME, INDOLE-N-METHYL TRANSFERASE

    PubMed Central

    Mavlyutov, Timur A.; Epstein, Miles L.; Liu, Patricia; Verbny, Yakov I.; Ziskind-Conhaim, Lea; Ruoho, Arnold E.

    2012-01-01

    The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein coupled receptors (GPCR). In the CNS the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that Indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and SIRs suggest that DMT is synthesized locally to effectively activate S1R in MN. PMID:22265729

  6. Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

    PubMed

    Mavlyutov, T A; Epstein, M L; Liu, P; Verbny, Y I; Ziskind-Conhaim, L; Ruoho, A E

    2012-03-29

    The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN.

  7. Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

    PubMed

    Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

    2017-09-01

    Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. NMR Structure of the C-Terminal Transmembrane Domain of the HDL Receptor, SR-BI, and a Functionally Relevant Leucine Zipper Motif.

    PubMed

    Chadwick, Alexandra C; Jensen, Davin R; Hanson, Paul J; Lange, Philip T; Proudfoot, Sarah C; Peterson, Francis C; Volkman, Brian F; Sahoo, Daisy

    2017-03-07

    The interaction of high-density lipoprotein (HDL) with its receptor, scavenger receptor BI (SR-BI), is critical for lowering plasma cholesterol levels and reducing the risk for cardiovascular disease. The HDL/SR-BI complex facilitates delivery of cholesterol into cells and is likely mediated by receptor dimerization. This work describes the use of nuclear magnetic resonance (NMR) spectroscopy to generate the first high-resolution structure of the C-terminal transmembrane domain of SR-BI. This region of SR-BI harbors a leucine zipper dimerization motif, which when mutated impairs the ability of the receptor to bind HDL and mediate cholesterol delivery. These losses in function correlate with the inability of SR-BI to form dimers. We also identify juxtamembrane regions of the extracellular domain of SR-BI that may interact with the lipid surface to facilitate cholesterol transport functions of the receptor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Intracellular localization of the M1 muscarinic acetylcholine receptor through clathrin-dependent constitutive internalization is mediated by a C-terminal tryptophan-based motif.

    PubMed

    Uwada, Junsuke; Yoshiki, Hatsumi; Masuoka, Takayoshi; Nishio, Matomo; Muramatsu, Ikunobu

    2014-07-15

    The M1 muscarinic acetylcholine receptor (M1-mAChR, encoded by CHRM1) is a G-protein-coupled membrane receptor that is activated by extracellular cholinergic stimuli. Recent investigations have revealed the intracellular localization of M1-mAChR. In this study, we observed constitutive internalization of M1-mAChR in mouse neuroblastoma N1E-115 cells without agonist stimulation. Constitutive internalization depended on dynamin, clathrin and the adaptor protein-2 (AP-2) complex. A WxxI motif in the M1-mAChR C-terminus is essential for its constitutive internalization, given that replacement of W(442) or I(445) with alanine residues abolished constitutive internalization. This WxxI motif resembles YxxΦ, which is the canonical binding motif for the μ2 subunit of the AP-2 complex. The M1-mAChR C-terminal WxxI motif interacted with AP-2 μ2. W442A and I445A mutants of the M1-mAChR C-terminal sequence lost AP-2-μ2-binding activity, whereas the W442Y mutant bound more effectively than wild type. Consistent with these results, W442A and I445A M1-mAChR mutants selectively localized to the cell surface. By contrast, the W442Y receptor mutant was found only at intracellular sites. Our data indicate that the cellular distribution of M1-mAChR is governed by the C-terminal tryptophan-based motif, which mediates constitutive internalization.

  10. Molecular and functional characterization of proteins interacting with the C-terminal domains of 5-HT2 receptors: emergence of 5-HT2 "receptosomes".

    PubMed

    Gavarini, Sophie; Bécamel, Carine; Chanrion, Benjamin; Bockaert, Joël; Marin, Philippe

    2004-06-01

    Many cellular functions are carried out by multiprotein complexes. The last five years of research have revealed that many G-protein coupled receptor (GPCR) functions that are not mediated by G proteins involve protein networks, which interact with their intracellular domains. This review focuses on one family of GPCRs activated by serotonin, the 5-HT(2) receptor family, which comprises three closely related subtypes, the 5-HT(2A), the 5-HT(2B) and the 5-HT(2c) receptors. These receptors still raise particular interest, because a large number of psychoactive drugs including hallucinogens, anti-psychotics, anxiolytics and anti-depressants, mediate their action, at least in part, through activation of 5-HT(2) receptors. Recent studies based on two-hybrid screens, proteomic, biochemical and cell biology approaches, have shown that the C-terminal domains of 5-HT(2) receptors interact with intracellular proteins. To date, the protein network associated with the C-terminus of the 5-HT(2C) receptor has been the most extensively characterized, using a proteomic approach combining affinity chromatography, mass spectrometry and immunoblotting. It includes scaffolding proteins containing one or several PDZ domains, signalling proteins and proteins of the cytoskeleton. Data indicating that the protein complexes interacting with 5-HT(2) receptor C-termini tightly control receptor trafficking and receptor-mediated signalling will also be reviewed.

  11. The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C-terminal juxtamembrane residues

    PubMed Central

    Peng, Weng Chuan; Lin, Xin; Torres, Jaume

    2009-01-01

    The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of α and β integrin TM interactions. However, we show herein that in FGFR3-TM, four C-terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3-TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C-terminal residues were present. In the absence of these four residues, A391E was dimer-destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3-TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR-TM. PMID:19165726

  12. The strong dimerization of the transmembrane domain of the fibroblast growth factor receptor (FGFR) is modulated by C-terminal juxtamembrane residues.

    PubMed

    Peng, Weng Chuan; Lin, Xin; Torres, Jaume

    2009-02-01

    The fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR subfamily of the receptor tyrosine kinases (RTKs) involved in signaling across the plasma membrane. Generally, ligand binding leads to receptor dimerization and activation. Dimerization involves the transmembrane (TM) domain, where mutations can lead to constitutive activation in certain cancer types and also in skeletal malformations. Thus, it has been postulated that FGFR homodimerization must be inherently weak to allow regulation, a feature reminiscent of alpha and beta integrin TM interactions. However, we show herein that in FGFR3-TM, four C-terminal residues, CRLR, have a profound destabilizing effect in an otherwise strongly dimerizing TM peptide. In the absence of these four residues, the dimerizing propensity of FGFR3-TM is comparable to glycophorin, as shown using various detergents. In addition, the expected enhanced dimerization induced by the mutation associated to the Crouzon syndrome A391E, was observed only when these four C-terminal residues were present. In the absence of these four residues, A391E was dimer-destabilizing. Finally, using site specific infrared dichroism and convergence with evolutionary conservation data, we have determined the backbone model of the FGFR3-TM homodimer in model lipid bilayers. This model is consistent with, and correlates with the effects of, most known pathological mutations found in FGFR-TM.

  13. β Adrenergic Receptor Kinase C-Terminal Peptide Gene-Therapy Improves β2-Adrenergic Receptor-Dependent Neoangiogenesis after Hindlimb Ischemia.

    PubMed

    Cannavo, Alessandro; Liccardo, Daniela; Lymperopoulos, Anastasios; Gambino, Giuseppina; D'Amico, Maria Loreta; Rengo, Franco; Koch, Walter J; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe

    2016-02-01

    After hindlimb ischemia (HI), increased catecholamine levels within the ischemic muscle can cause dysregulation of β2-adrenergic receptor (β2AR) signaling, leading to reduced revascularization. Indeed, in vivo β2AR overexpression via gene therapy enhances angiogenesis in a rat model of HI. G protein-coupled receptor kinase 2 (GRK2) is a key regulator of βAR signaling, and β adrenergic receptor kinase C-terminal peptide (βARKct), a peptide inhibitor of GRK2, has been shown to prevent βAR down-regulation and to protect cardiac myocytes and stem cells from ischemic injury through restoration of β2AR protective signaling (i.e., protein kinase B/endothelial nitric oxide synthase). Herein, we tested the potential therapeutic effects of adenoviral-mediated βARKct gene transfer in an experimental model of HI and its effects on βAR signaling and on endothelial cell (EC) function in vitro. Accordingly, in this study, we surgically induced HI in rats by femoral artery resection (FAR). Fifteen days of ischemia resulted in significant βAR down-regulation that was paralleled by an approximately 2-fold increase in GRK2 levels in the ischemic muscle. Importantly, in vivo gene transfer of the βARKct in the hindlimb of rats at the time of FAR resulted in a marked improvement of hindlimb perfusion, with increased capillary and βAR density in the ischemic muscle, compared with control groups. The effect of βARKct expression was also assessed in vitro in cultured ECs. Interestingly, ECs expressing the βARKct fenoterol, a β2AR-agonist, induced enhanced β2AR proangiogenic signaling and increased EC function. Our results suggest that βARKct gene therapy and subsequent GRK2 inhibition promotes angiogenesis in a model of HI by preventing ischemia-induced β2AR down-regulation.

  14. Proximal C-terminal domain of sulphonylurea receptor 2A interacts with pore-forming Kir6 subunits in KATP channels.

    PubMed Central

    Rainbow, Richard D; James, Marian; Hudman, Diane; Al Johi, Mohammed; Singh, Harprit; Watson, Peter J; Ashmole, Ian; Davies, Noel W; Lodwick, David; Norman, Robert I

    2004-01-01

    Functional KATP (ATP-sensitive potassium) channels are hetero-octamers of four Kir6 (inwardly rectifying potassium) channel subunits and four SUR (sulphonylurea receptor) subunits. Possible interactions between the C-terminal domain of SUR2A and Kir6.2 were investigated by co-immunoprecipitation of rat SUR2A C-terminal fragments with full-length Kir6.2 and by analysis of cloned KATP channel function and distribution in HEK-293 cells (human embryonic kidney 293 cells) in the presence of competing rSUR2A fragments. Three maltose-binding protein-SUR2A fusions, rSUR2A-CTA (rSUR2A residues 1254-1545), rSUR2A-CTB (residues 1254-1403) and rSUR2A-CTC (residues 1294-1403), were co-immunoprecipitated with full-length Kir6.2 using a polyclonal anti-Kir6.2 antiserum. A fourth C-terminal domain fragment, rSUR2A-CTD (residues 1358-1545) did not co-immunoprecipitate with Kir6.2 under the same conditions, indicating a direct interaction between Kir6.2 and a 65-amino-acid section of the cytoplasmic C-terminal region of rSUR2A between residues 1294 and 1358. ATP- and glibenclamide-sensitive K+ currents were decreased in HEK-293 cells expressing full-length Kir6 and SUR2 subunits that were transiently transfected with fragments rSUR2A-CTA, rSUR2A-CTC and rSUR2A-CTE (residues 1294-1359) compared with fragment rSUR2A-CTD or mock-transfected cells, suggesting either channel inhibition or a reduction in the number of functional KATP channels at the cell surface. Anti-KATP channel subunit-associated fluorescence in the cell membrane was substantially lower and intracellular fluorescence increased in rSUR2A-CTE expressing cells; thus, SUR2A fragments containing residues 1294-1358 reduce current by decreasing the number of channel subunits in the cell membrane. These results identify a site in the C-terminal domain of rSUR2A, between residues 1294 and 1358, whose direct interaction with full-length Kir6.2 is crucial for the assembly of functional KATP channels. PMID:14672537

  15. Agonist Binding and Desensitization of the μ-Opioid Receptor Is Modulated by Phosphorylation of the C-Terminal Tail Domain

    PubMed Central

    Arttamangkul, Seksiri; Bunzow, James R.; Williams, John T.

    2015-01-01

    Sustained activation of G protein–coupled receptors can lead to a rapid decline in signaling through acute receptor desensitization. In the case of the μ-opioid receptor (MOPr), this desensitization may play a role in the development of analgesic tolerance. It is understood that phosphorylation of MOPr promotes association with β-arrestin proteins, which then facilitates desensitization and receptor internalization. Agonists that induce acute desensitization have been shown to induce a noncanonical high-affinity agonist binding state in MOPr, conferring a persistent memory of prior receptor activation. In the current study, live-cell confocal imaging was used to investigate the role of receptor phosphorylation in agonist binding to MOPr. A phosphorylation cluster in the C-terminal tail of MOPr was identified as a mediator of agonist-induced affinity changes in MOPr. This site is unique from the primary phosphorylation cluster responsible for β-arrestin binding and internalization. Electrophysiologic measurements of receptor function suggest that both phosphorylation clusters may play a parallel role during acute receptor desensitization. Desensitization was unaffected by alanine mutation of either phosphorylation cluster, but was largely eliminated when both clusters were mutated. Overall, this work suggests that there are multiple effects of MOPr phosphorylation that appear to regulate MOPr function: one affecting β-arrestin binding and a second affecting agonist binding. PMID:25934731

  16. Downregulation of 5-HT7 Serotonin Receptors by the Atypical Antipsychotics Clozapine and Olanzapine. Role of Motifs in the C-Terminal Domain and Interaction with GASP-1.

    PubMed

    Manfra, Ornella; Van Craenenbroeck, Kathleen; Skieterska, Kamila; Frimurer, Thomas; Schwartz, Thue W; Levy, Finn Olav; Andressen, Kjetil Wessel

    2015-07-15

    The human 5-HT7 serotonin receptor, a G-protein-coupled receptor (GPCR), activates adenylyl cyclase constitutively and upon agonist activation. Biased ligands differentially activate 5-HT7 serotonin receptor desensitization, internalization and degradation in addition to G protein activation. We have previously found that the atypical antipsychotics clozapine and olanzapine inhibited G protein activation and, surprisingly, induced both internalization and lysosomal degradation of 5-HT7 receptors. Here, we aimed to determine the mechanism of clozapine- and olanzapine-mediated degradation of 5-HT7 receptors. In the C-terminus of the 5-HT7 receptor, we identified two YXXΦ motifs, LR residues, and a palmitoylated cysteine anchor as potential sites involved in receptor trafficking to lysosomes followed by receptor degradation. Mutating either of these sites inhibited clozapine- and olanzapine-mediated degradation of 5-HT7 receptors and also interfered with G protein activation. In addition, we tested whether receptor degradation was mediated by the GPCR-associated sorting protein-1 (GASP-1). We show that GASP-1 binds the 5-HT7 receptor and regulates the clozapine-mediated degradation. Mutations of the identified motifs and residues, located in or close to Helix-VIII of the 5-HT7 receptor, modified antipsychotic-stimulated binding of proteins (such as GASP-1), possibly by altering the flexibility of Helix-VIII, and also interfered with G protein activation. Taken together, our data demonstrate that binding of clozapine or olanzapine to the 5-HT7 receptor leads to antagonist-mediated lysosomal degradation by exposing key residues in the C-terminal tail that interact with GASP-1.

  17. C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity.

    PubMed

    Chillakuri, Chandramouli Reddy; Reinhart, Christoph; Michel, Hartmut

    2007-12-01

    We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.

  18. Identification of a Novel TGF-β-Binding Site in the Zona Pellucida C-terminal (ZP-C) Domain of TGF-β-Receptor-3 (TGFR-3)

    PubMed Central

    Diestel, Uschi; Resch, Marcus; Meinhardt, Kathrin; Weiler, Sigrid; Hellmann, Tina V.; Mueller, Thomas D.; Nickel, Joachim; Eichler, Jutta; Muller, Yves A.

    2013-01-01

    The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor β (TGF-β) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-β-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 Å crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3. PMID:23826237

  19. The role of the C-terminal extension (CTE) of the estrogen receptor alpha and beta DNA binding domain in DNA binding and interaction with HMGB.

    PubMed

    Melvin, Vida Senkus; Harrell, Chuck; Adelman, James S; Kraus, W Lee; Churchill, Mair; Edwards, Dean P

    2004-04-09

    HMGB-1/-2 are coregulatory proteins that facilitate the DNA binding and transcriptional activity of steroid receptor members of the nuclear receptor family of transcription factors. We investigated the influence and mechanism of action of HMGB-1/-2 (formerly known as HMG-1/-2) on estrogen receptor alpha (ERalpha) and ERbeta. Both ER subtypes were responsive to HMGB-1/-2 with respect to enhancement of receptor DNA binding affinity and transcriptional activity in cells. Responsiveness to HMGB-1/-2 was dependent on the C-terminal extension (CTE) region of the ER DNA binding domain (DBD) and correlated with a direct protein interaction between HMGB-1/-2 and the CTE. Thus the previously reported higher DNA binding affinity and transcription activity of ERalpha as compared with ERbeta is not due to a lack of ERbeta interaction with HMGB-1/-2. Using chimeric receptor DBDs, the higher intrinsic DNA binding affinity of ERalpha than ERbeta was shown to be due to a unique property of the ERalpha CTE, independent of HMGB-1/-2. The CTE of both ER subtypes was also shown to be required for interaction with ERE half-sites. These studies reveal the importance of the CTE and HMGB-1/-2 for ERalpha and ERbeta interaction with their cognate target DNAs.

  20. Preliminary crystallographic analysis of mouse Elf3 C-terminal DNA-binding domain in complex with type II TGF-[beta] receptor promoter DNA

    SciTech Connect

    Agarkar, Vinod B.; Babayeva, Nigar D.; Rizzino, Angie; Tahirov, Tahir H.

    2010-10-08

    Ets proteins are transcription factors that activate or repress the expression of genes that are involved in various biological processes, including cellular proliferation, differentiation, development, transformation and apoptosis. Like other Ets-family members, Elf3 functions as a sequence-specific DNA-binding transcriptional factor. A mouse Elf3 C-terminal fragment (amino-acid residues 269-371) containing the DNA-binding domain has been crystallized in complex with mouse type II TGF-{beta} receptor promoter (TR-II) DNA. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 42.66, b = 52, c = 99.78 {angstrom}, and diffracted to a resolution of 2.2 {angstrom}.

  1. Crystal Structure of Mouse Elf3 C-terminal DNA-binding Domain in Complex with Type II TGF-[beta] Receptor Promoter DNA

    SciTech Connect

    Agarkar, Vinod B.; Babayeva, Nigar D.; Wilder, Phillip J.; Rizzino, Angie; Tahirov, Tahir H.

    2010-08-18

    The Ets family of transcription factors is composed of more than 30 members. One of its members, Elf3, is expressed in virtually all epithelial cells as well as in many tumors, including breast tumors. Several studies observed that the promoter of the type II TGF-{beta} receptor gene (T{beta}R-II) is strongly stimulated by Elf3 via two adjacent Elf3 binding sites, the A-site and the B-site. Here, we report the 2.2 {angstrom} resolution crystal structure of a mouse Elf3 C-terminal fragment, containing the DNA-binding Ets domain, in complex with the B-site of mouse type II TGF-{beta} receptor promoter DNA (mT{beta}R-II{sub DNA}). Elf3 contacts the core GGAA motif of the B-site from a major groove similar to that of known Ets proteins. However, unlike other Ets proteins, Elf3 also contacts sequences of the A-site from the minor groove of the DNA. DNA binding experiments and cell-based transcription studies indicate that minor groove interaction by Arg349 located in the Ets domain is important for Elf3 function. Equally interesting, previous studies have shown that the C-terminal region of Elf3, which flanks the Ets domain, is required for Elf3 binding to DNA. In this study, we determined that Elf3 amino acid residues within this flanking region, including Trp361, are important for the structural integrity of the protein as well as for the Efl3 DNA binding and transactivation activity.

  2. A C-terminal segment of the V{sub 1}R vasopressin receptor is unstructured in the crystal structure of its chimera with the maltose-binding protein

    SciTech Connect

    Adikesavan, Nallini Vijayarangan; Mahmood, Syed Saad; Stanley, Nithianantham; Xu, Zhen; Wu, Nan; Thibonnier, Marc; Shoham, Menachem

    2005-04-01

    The 1.8 Å crystal structure of an MBP-fusion protein with the C-terminal cytoplasmic segment of the V1 vasopressin receptor reveals that the receptor segment is unstructured. The V{sub 1} vascular vasopressin receptor (V{sub 1}R) is a G-protein-coupled receptor (GPCR) involved in the regulation of body-fluid osmolality, blood volume and blood pressure. Signal transduction is mediated by the third intracellular loop of this seven-transmembrane protein as well as by the C-terminal cytoplasmic segment. A chimera of the maltose-binding protein (MBP) and the C-terminal segment of V{sub 1}R has been cloned, expressed, purified and crystallized. The crystals belong to space group P2{sub 1}, with unit-cell parameters a = 51.10, b = 66.56, c = 115.72 Å, β = 95.99°. The 1.8 Å crystal structure reveals the conformation of MBP and part of the linker region of this chimera, with the C-terminal segment being unstructured. This may reflect a conformational plasticity in the C-terminal segment that may be necessary for proper function of V{sub 1}R.

  3. Unlocking Doors without Keys: Activation of Src by Truncated C-terminal Intracellular Receptor Tyrosine Kinases Lacking Tyrosine Kinase Activity

    PubMed Central

    Mezquita, Belén; Mezquita, Pau; Pau, Montserrat; Mezquita, Jovita; Mezquita, Cristóbal

    2014-01-01

    One of the best examples of the renaissance of Src as an open door to cancer has been the demonstration that just five min of Src activation is sufficient for transformation and also for induction and maintenance of cancer stem cells [1]. Many tyrosine kinase receptors, through the binding of their ligands, become the keys that unlock the structure of Src and activate its oncogenic transduction pathways. Furthermore, intracellular isoforms of these receptors, devoid of any tyrosine kinase activity, still retain the ability to unlock Src. This has been shown with a truncated isoform of KIT (tr-KIT) and a truncated isoform of VEGFR-1 (i21-VEGFR-1), which are intracellular and require no ligand binding, but are nonetheless able to activate Src and induce cell migration and invasion of cancer cells. Expression of the i21-VEGFR-1 is upregulated by the Notch signaling pathway and repressed by miR-200c and retinoic acid in breast cancer cells. Both Notch inhibitors and retinoic acid have been proposed as potential therapies for invasive breast cancer. PMID:24709904

  4. C-Terminal Di-leucine Motif of Dopamine D1 Receptor Plays an Important Role in Its Plasma Membrane Trafficking

    PubMed Central

    Guo, Yan; Jose, Pedro A.

    2011-01-01

    The dopamine D1 receptor (D1R), a G protein-coupled receptor, plays a critical role in regulating blood pressure through its actions on renal hemodynamics and epithelial ion transport, which are highly linked to its intracellular trafficking. In this study, we generated a series of C-terminal mutants of D1R that were tagged with or without enhanced yellow fluorescent protein, and analyzed the consequences of these mutants on the plasma membrane trafficking of D1R and cyclic AMP response to D1R stimulation. D1R with mutations within the endocytic recycling signal (amino acid residues 360–382) continued to be functional, albeit decreased relative to wild-type D1R. Mutation of the palmitoylation site (347C>S) of D1R did not impair its trafficking to the plasma membrane, but abolished its ability to increase cyclic AMP accumulation. In contrast, replacement of di-leucines (344–345L>A) by alanines resulted in the retention of D1R in the early endosome, decreased its glycosylation, and prevented its targeting to the plasma membrane. Our studies suggest that di-L motif at the C-terminus of D1R is critical for the glycosylation and cell surface targeting of D1R. PMID:22206002

  5. Cleavage of the Interleukin-11 receptor induces processing of its C-terminal fragments by the gamma-secretase and the proteasome.

    PubMed

    Lokau, Juliane; Flynn, Charlotte M; Garbers, Christoph

    2017-09-16

    The cytokine Interleukin-11 (IL-11) signals through the membrane-bound IL-11 receptor (IL-11R), which is expressed in a cell-type specific manner. We have recently shown that the metalloprotease ADAM10 can cleave the IL-11R. The liberated soluble IL-11R (sIL-11R) ectodomain can bind its ligand, and the resulting IL-11/sIL-11R complex can activate cells that do not express the IL-11R (trans-signaling). In this study, we show that the remaining C-terminal fragment (CTF1) after ADAM10-mediated cleavage is subsequently cleaved within the membrane by the gamma-secretase complex, and that the resulting shorter CTF2 is further degraded by the proteasome. In contrast to other transmembrane receptors, e.g. Notch, we find no evidence that the IL-11R CTF can translocate into the nucleus to act as a transcription factor, suggesting that regulated intramembrane proteolysis of the IL-11R CTF acts as a mechanism to clear the plasma membrane from remaining protein fragments after cleavage of its ectodomain. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. A membrane-proximal, C-terminal α-helix is required for plasma membrane localization and function of the G Protein-coupled receptor (GPCR) TGR5.

    PubMed

    Spomer, Lina; Gertzen, Christoph G W; Schmitz, Birte; Häussinger, Dieter; Gohlke, Holger; Keitel, Verena

    2014-02-07

    The C terminus of G protein-coupled receptors (GPCRs) is important for G protein-coupling and activation; in addition, sorting motifs have been identified in the C termini of several GPCRs that facilitate correct trafficking from the endoplasmic reticulum to the plasma membrane. The C terminus of the GPCR TGR5 lacks any known sorting motif such that other factors must determine its trafficking. Here, we investigate deletion and substitution variants of the membrane-proximal C terminus of TGR5 with respect to plasma membrane localization and function using immunofluorescence staining, flow cytometry, and luciferase assays. Peptides of the membrane-proximal C-terminal variants are subjected to molecular dynamics simulations and analyzed with respect to their secondary structure. Our results reveal that TGR5 plasma membrane localization and responsiveness to extracellular ligands is fostered by a long (≥ 9 residues) α-helical stretch at the C terminus, whereas the presence of β-strands or only a short α-helical stretch leads to retention in the endoplasmic reticulum and a loss of function. As a proof-of-principle, chimeras of TGR5 containing the membrane-proximal amino acids of the β2 adrenergic receptor (β2AR), the sphingosine 1-phosphate receptor-1 (S1P1), or the κ-type opioid receptor (κOR) were generated. These TGR5β2AR, TGR5S1P1, or TGR5κOR chimeras were correctly sorted to the plasma membrane. As the exchanged amino acids of the β2AR, the S1P1, or the κOR form α-helices in crystal structures but lack significant sequence identity to the respective TGR5 sequence, we conclude that the secondary structure of the TGR5 membrane-proximal C terminus is the determining factor for plasma membrane localization and responsiveness towards extracellular ligands.

  7. Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts

    PubMed Central

    Masin, Marianela; Young, Christopher; Lim, KoiNi; Barnes, Sara J; Xu, Xing Jian; Marschall, Viola; Brutkowski, Wojciech; Mooney, Elizabeth R; Gorecki, Dariusz C; Murrell-Lagnado, Ruth

    2012-01-01

    BACKGROUND AND PURPOSE Splice variants of P2X7 receptor transcripts contribute to the diversity of receptor-mediated responses. Here, we investigated expression and function of C-terminal truncated (ΔC) variants of the mP2X7 receptor, which are predicted to escape inactivation in one strain of P2X7−/− mice (Pfizer KO). EXPERIMENTAL APPROACH Expression in wild-type (WT) and Pfizer KO tissue was investigated by reverse transcription (RT)-PCR and Western blot analysis. ΔC variants were also cloned and expressed in HEK293 cells to investigate their assembly, trafficking and function. KEY RESULTS RT-PCR indicates expression of a ΔC splice variant in brain, salivary gland (SG) and spleen from WT and Pfizer KO mice. An additional ΔC hybrid transcript, containing sequences of P2X7 upstream of exon 12, part of exon 13 followed in-frame by the sequence of the vector used to disrupt the P2X7 gene, was also identified in the KO mice. By blue native (BN) PAGE analysis and the use of cross linking reagents followed by SDS-PAGE, P2X7 trimers, dimers and monomers were detected in the spleen and SG of Pfizer KO mice. The molecular mass was reduced compared with P2X7 in WT mice tissue, consistent with a ΔC variant. When expressed in HEK293 cells the ΔC variants were inefficiently trafficked to the cell surface and agonist-evoked whole cell currents were small. Co-expressed with P2X7A, the ΔC splice variant acted in a dominant negative fashion to inhibit function. CONCLUSIONS AND IMPLICATIONS Pfizer KO mice are not null for P2X7 receptor expression but express ΔC variants with reduced function. PMID:21838754

  8. Expression, purification and reconstitution of the C-terminal transmembrane domain of scavenger receptor BI into detergent micelles for NMR analysis.

    PubMed

    Chadwick, Alexandra C; Jensen, Davin R; Peterson, Francis C; Volkman, Brian F; Sahoo, Daisy

    2015-03-01

    Scavenger receptor class B type I (SR-BI), the high density lipoprotein (HDL) receptor, is important for the delivery of HDL-cholesteryl esters to the liver for excretion via bile formation. The focus on therapeutic strategies aimed at reducing cholesterol levels highlights the critical need to understand the structural features of SR-BI that drive cholesterol removal. Yet, in the absence of a high-resolution structure of SR-BI, our understanding of how SR-BI interacts with HDL is limited. In this study, we have optimized the NMR solution conditions for the structural analysis of the C-terminal transmembrane domain of SR-BI that harbors putative domains required for receptor oligomerization. An isotopically-labeled SR-BI peptide encompassing residues 405-475 was bacterially-expressed and purified. [U-(15)N]-SR-BI(405-475) was incorporated into different detergent micelles and assessed by (1)H-(15)N-HSQC in order to determine which detergent micelle best maintained SR-BI(405-475) in a folded, native conformation for subsequent NMR analyses. We also determined the optimal detergent concentration used in micelles, as well as temperature, solution buffer and pH conditions. Based on (1)H-(15)N-HSQC peak dispersion, intensity, and uniformity, we determined that [U-(15)N]-SR-BI(405-475) should be incorporated into 5% detergent micelles consisting of 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-[1'-rac-glycerol] (LPPG) and data collected at 40°C in a non-buffered solution at pH 6.8. Furthermore, we demonstrate the ability of SR-BI(405-475) to form dimers upon chemical crosslinking. These studies represent the first steps in obtaining high-resolution structural information by NMR for the HDL receptor that plays a critical role in regulating whole body cholesterol removal.

  9. Importance of the C-terminal histidine residues of Helicobacter pylori GroES for Toll-like receptor 4 binding and interleukin-8 cytokine production

    PubMed Central

    Lee, Haur; Su, Yu-Lin; Huang, Bo-Shih; Hsieh, Feng-Tse; Chang, Ya-Hui; Tzeng, Shiou-Ru; Hsu, Chun-Hua; Huang, Po-Tsang; Lou, Kuo-Long; Wang, Yeng-Tseng; Chow, Lu-Ping

    2016-01-01

    Helicobacter pylori infection is associated with the development of gastric and duodenal ulcers as well as gastric cancer. GroES of H. pylori (HpGroES) was previously identified as a gastric cancer-associated virulence factor. Our group showed that HpGroES induces interleukin-8 (IL-8) cytokine release via a Toll-like receptor 4 (TLR4)-dependent mechanism and domain B of the protein is crucial for interactions with TLR4. In the present study, we investigated the importance of the histidine residues in domain B. To this end, a series of point mutants were expressed in Escherichia coli, and the corresponding proteins purified. Interestingly, H96, H104 and H115 were not essential, whereas H100, H102, H108, H113 and H118 were crucial for IL-8 production and TLR4 interactions in KATO-III cells. These residues were involved in nickel binding. Four of five residues, H102, H108, H113 and H118 induced certain conformation changes in extended domain B structure, which is essential for interactions with TLR4 and consequent IL-8 production. We conclude that interactions of nickel ions with histidine residues in domain B help to maintain the conformation of the C-terminal region to conserve the integrity of the HpGroES structure and modulate IL-8 release. PMID:27869178

  10. Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth*

    PubMed Central

    Kelly, Geraldine M.; Buckley, Deirdre A.; Kiely, Patrick A.; Adams, David R.; O'Connor, Rosemary

    2012-01-01

    Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the 1248SFYYS1252 motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling. PMID:22685298

  11. The sustainability of interactions between the orexin-1 receptor and β-arrestin-2 is defined by a single C-terminal cluster of hydroxy amino acids and modulates the kinetics of ERK MAPK regulation

    PubMed Central

    2005-01-01

    The orexin-1 receptor interacts with β-arrestin-2 in an agonist-dependent manner. In HEK-293T cells, these two proteins became co-internalized into acidic endosomes. Truncations from the C-terminal tail did not prevent agonist-induced internalization of the orexin-1 receptor or alter the pathway of internalization, although such mutants failed to interact with β-arrestin-2 in a sustained manner or produce its co-internalization. Mutation of a cluster of three threonine and one serine residue at the extreme C-terminus of the receptor greatly reduced interaction and abolished co-internalization of β-arrestin-2–GFP (green fluorescent protein). Despite the weak interactions of this C-terminally mutated form of the receptor with β-arrestin-2, studies in wild-type and β-arrestin-deficient mouse embryo fibroblasts confirmed that agonist-induced internalization of this mutant required expression of a β-arrestin. Although without effect on agonist-mediated elevation of intracellular Ca2+ levels, the C-terminally mutated form of the orexin-1 receptor was unable to sustain phosphorylation of the MAPKs (mitogen-activated protein kinases) ERK1 and ERK2 (extracellular-signal-regulated kinases 1 and 2) to the same extent as the wild-type receptor. These studies indicate that a single cluster of hydroxy amino acids within the C-terminal seven amino acids of the orexin-1 receptor determine the sustainability of interaction with β-arrestin-2, and indicate an important role of β-arrestin scaffolding in defining the kinetics of orexin-1 receptor-mediated ERK MAPK activation. PMID:15683363

  12. Development of 7TM receptor-ligand complex models using ligand-biased, semi-empirical helix-bundle repacking in torsion space: application to the agonist interaction of the human dopamine D2 receptor

    NASA Astrophysics Data System (ADS)

    Malo, Marcus; Persson, Ronnie; Svensson, Peder; Luthman, Kristina; Brive, Lars

    2013-03-01

    Prediction of 3D structures of membrane proteins, and of G-protein coupled receptors (GPCRs) in particular, is motivated by their importance in biological systems and the difficulties associated with experimental structure determination. In the present study, a novel method for the prediction of 3D structures of the membrane-embedded region of helical membrane proteins is presented. A large pool of candidate models are produced by repacking of the helices of a homology model using Monte Carlo sampling in torsion space, followed by ranking based on their geometric and ligand-binding properties. The trajectory is directed by weak initial restraints to orient helices towards the original model to improve computation efficiency, and by a ligand to guide the receptor towards a chosen conformational state. The method was validated by construction of the β1 adrenergic receptor model in complex with ( S)-cyanopindolol using bovine rhodopsin as template. In addition, models of the dopamine D2 receptor were produced with the selective and rigid agonist ( R)- N-propylapomorphine (( R)-NPA) present. A second quality assessment was implemented by evaluating the results from docking of a library of 29 ligands with known activity, which further discriminated between receptor models. Agonist binding and recognition by the dopamine D2 receptor is interpreted using the 3D structure model resulting from the approach. This method has a potential for modeling of all types of helical transmembrane proteins for which a structural template with sequence homology sufficient for homology modeling is not available or is in an incorrect conformational state, but for which sufficient empirical information is accessible.

  13. Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail.

    PubMed Central

    Liu, B; Nakashima, S; Adachi, T; Ito, Y; Takano, T; Shimizu, T; Nozawa, Y

    1997-01-01

    The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Calpha (PKCalpha) to membrane was similar to that of DG formation. In WT-H cells, PKCalpha was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCalpha was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCalpha. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that

  14. A concise synthesis of 1,4-dihydro-[1,4]diazepine-5,7-dione, a novel 7-TM receptor ligand core structure with melanocortin receptor agonist activity.

    PubMed

    Szewczyk, Jerzy R; Laudeman, Chris P; Sammond, Doug M; Villeneuve, Manon; Minick, Douglas J; Grizzle, Mary K; Daniels, Alejandro J; Andrews, John L; Ignar, Diane M

    2010-03-01

    Finding small non-peptide molecules for G protein-coupled receptors (GPCR) whose endogenous ligands are peptides, is a very important task for medicinal chemists. Over the years, compounds mimicking peptide structures have been discovered, and scaffolds emulating peptide backbones have been designed. In our work on GPCR ligands, including cholecystokinin receptor-1 (CCKR-1) agonists, we have employed benzodiazepines as a core structure. Looking for ways to reduce molecular weight and possibly improve physical properties of GPCR ligands, we embarked on the search for molecules providing similar scaffolds to the benzodiazepine with lower molecular weight. One of our target core structures was 1,4-dihydro-[1,4]diazepine-5,7-dione. There was not, however, a known synthetic route to such molecules. Here we report the discovery of a simple and concise method for synthesis of 2-[6-(1H-indazol-3-ylmethyl)-5,7-dioxo-4-phenyl-4,5,6,7-tetrahydro-[1,4]diazepin-1-yl]-N-isopropyl-N-phenyl-acetamide as an example of a compound containing the tetrahydrodiazepine-5,7-dione core. Compounds from this series were tested in numerous GPCR assays and demonstrated activity at melanocortin 1 and 4 receptors (MC1R and MC4R). Selected compounds from this series were tested in vivo in Peptide YY (PYY)-induced food intake. Compounds dosed by intracerebroventricular and oral routes reduced PYY-induced food intake and this effect was reversed by the cyclic peptide MC4R antagonist SHU9119.

  15. Unveiling the Membrane-Binding Properties of N-Terminal and C-Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

    PubMed Central

    2015-01-01

    G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2–31 (GRK52–31) was in random coil whereas GRK5546–565 was partially helical. When the subphase for the GRK52–31 peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK52–31 became partially helical. By inspecting the membrane behavior of two different segments of GRK52–31, namely, GRK52–24 and GRK525–31, we found that residues 25–31 are responsible for membrane binding, whereas the helical character is imparted by residues 2–24. With SFG, we deduced that the orientation angle of the helical segment of GRK52–31 is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca2+·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells. PMID:24401145

  16. Residues in the GluN1 C-terminal domain control kinetics and pharmacology of GluN1/GluN3A N-methyl-d-aspartate receptors.

    PubMed

    Cummings, Kirstie A; Belin, Sophie; Popescu, Gabriela K

    2017-03-29

    N-methyl-d-aspartate (NMDA) receptors assembled from GluN1 and GluN3 subunits are unique in that they form glycine-gated excitatory channels that are insensitive to glutamate and NMDA. Alternative splicing of the GluN1 subunit mRNA results in eight variants with regulated expression patterns and post-translational modifications. Here we investigate the role of residues in the GluN1 C-terminal alternatively spliced cassettes in receptor gating and modulation. We measured whole-cell currents from recombinant GluN1/GluN3A receptors expressed in HEK293 cells that differed in the sequence of their GluN1 C-terminal tail. We found that these residues controlled the level of steady-state activity and the degree to which activity was facilitated by zinc and protons. Further, we found that the phosphorylation status of sites specific to certain variants can also modulate channel activity. Based on these results we suggest that GluN1 C-terminal domain splicing may confer cell-specific and activity-dependent regulation onto the level and pharmacologic sensitivity of GluN1/GluN3A currents.

  17. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    ERIC Educational Resources Information Center

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  18. [Beta]-Adrenergic Receptor Activation Rescues Theta Frequency Stimulation-Induced LTP Deficits in Mice Expressing C-Terminally Truncated NMDA Receptor GluN2A Subunits

    ERIC Educational Resources Information Center

    Moody, Teena D.; Watabe, Ayako M.; Indersmitten, Tim; Komiyama, Noboru H.; Grant, Seth G. N.; O'Dell, Thomas J.

    2011-01-01

    Through protein interactions mediated by their cytoplasmic C termini the GluN2A and GluN2B subunits of NMDA receptors (NMDARs) have a key role in the formation of NMDAR signaling complexes at excitatory synapses. Although these signaling complexes are thought to have a crucial role in NMDAR-dependent forms of synaptic plasticity such as long-term…

  19. G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of Beta-Arrestin.

    PubMed

    Luo, Jiansong; Busillo, John M; Stumm, Ralf; Benovic, Jeffrey L

    2017-03-22

    Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gi-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared to CXCL12. Knockdown of GRK2, GRK3 or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional PKC isoforms reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of β-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a non-specific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of the process of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.

  20. Interaction between the ligand-binding domain of the LDL receptor and the C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDL receptor during endosomal acidification.

    PubMed

    Tveten, Kristian; Holla, Øystein L; Cameron, Jamie; Strøm, Thea Bismo; Berge, Knut Erik; Laerdahl, Jon K; Leren, Trond P

    2012-03-15

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the epidermal growth factor homology domain repeat A of the low-density lipoprotein receptor (LDLR) at the cell surface and disrupts recycling of the internalized LDLR. As a consequence, the LDLR is rerouted to the lysosomes for degradation. Although PCSK9 may bind to an LDLR lacking the ligand-binding domain, at least three ligand-binding repeats of the ligand-binding domain are required for PCSK9 to reroute the LDLR to the lysosomes. In this study, we have studied the binding of PCSK9 to an LDLR with or without the ligand-binding domain at increasingly acidic conditions in order to mimic the milieu of the LDLR:PCSK9 complex as it translocates from the cell membrane to the sorting endosomes. These studies have shown that PCSK9 is rapidly released from an LDLR lacking the ligand-binding domain at pH in the range of 6.9-6.1. A similar pattern of release at acidic pH was also observed for the binding to the normal LDLR of mutant PCSK9 lacking the C-terminal domain. Together these data indicate that an interaction between the negatively charged ligand-binding domain of the LDLR and the positively charged C-terminal domain of PCSK9 is required for PCSK9 to remain bound to the LDLR during the early phase of endosomal acidification as the LDLR translocates from the cell membrane to the sorting endosome.

  1. The C-terminal extension (CTE) of the nuclear hormone receptor DNA binding domain determines interactions and functional response to the HMGB-1/-2 co-regulatory proteins.

    PubMed

    Melvin, Vida Senkus; Roemer, Sarah C; Churchill, Mair E A; Edwards, Dean P

    2002-07-12

    Previously, we and others reported that the high mobility group proteins, HMGB-1/-2, enhance DNA binding in vitro and transactivation in situ by the steroid hormone subgroup of nuclear receptors but did not influence these functions of class II receptors. We show here that the DNA binding domain (DBD) is sufficient to account for the selective influence of HMGB-1/-2 on the steroid class of receptors. Furthermore, the use of chimeric DBDs reveals that this selectivity is dependent on the C-terminal extension (CTE), amino acid sequences adjacent to the zinc finger core DBD. HMGB-1/-2 interact directly with the DBDs of steroid but not class II receptors, and this interaction requires the CTE. This in vitro interaction correlates with a requirement of the CTE for maximal HMGB-1/-2 enhancement of DNA binding in vitro and transcriptional activation in cells. Finally, class II receptor DBDs have a much higher intrinsic affinity for DNA than steroid receptor DBDs, and this affinity difference is also dependent on the CTE. These results reveal the importance of the steroid receptor CTE for DNA binding affinity and functional response to HMGB-1/-2.

  2. An α-helical C-terminal tail segment of the skeletal L-type Ca2+ channel β1a subunit activates ryanodine receptor type 1 via a hydrophobic surface.

    PubMed

    Karunasekara, Yamuna; Rebbeck, Robyn T; Weaver, Llara M; Board, Philip G; Dulhunty, Angela F; Casarotto, Marco G

    2012-12-01

    Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the β(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide β(1a)V490-M524, corresponding to the C-terminal 35 residues of β(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in β(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type β(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue β(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.

  3. Dichotomy in the anxiolytic versus antidepressant effect of C-terminal truncation of the GluN2A subunit of NMDA receptors.

    PubMed

    Inta, Dragos; Vogt, Miriam A; Pfeiffer, Natascha; Köhr, Georg; Gass, Peter

    2013-06-15

    The glutamate system is thought to play an important role in modulating mood and anxiety. Ionotropic NMDA receptors critically influence neuronal circuits regulating emotional behaviour. Their pharmacological blockade triggers fast antidepressant and anxiolytic effects. In line with this concept, ablation of the GluN2A subunit of NMDA receptors induces antidepressant and anxiolytic effects. However, it is not known if absence of the GluN2A-containing NMDA channel or of the GluN2A-mediated intracellular signalling is responsible for these effects. To further investigate the contribution of the GluN2A-containing NMDA receptors in mood disorders we analysed mice lacking the intracellular C-terminus of the GluN2A subunit (GluN2AΔC/ΔC) in tests relevant for anxiety and depression. Interestingly, GluN2AΔC/ΔC mice showed decreased anxiety, but no anti-depressive-like phenotype, indicating a predominant role of the intracellular signalling of the GluN2A subunit in anxiety. These data suggest distinct roles of the GluN2A subunit as whole vs. its intracellular domain in modulating anxiety and depression-like symptoms and reveal differential molecular targets for the therapy of mood and anxiety disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Overproduction of the cyclic AMP receptor protein of Escherichia coli and expression of the engineered C-terminal DNA-binding domain.

    PubMed Central

    Gronenborn, A M; Clore, G M

    1986-01-01

    Overproduction of the cyclic AMP receptor protein (CRP) from Escherichia coli, up to 25% of the soluble cell protein, has been achieved in an inducible host-vector system under transcriptional control of the lambda promoter PL. This system is ideally suited for large scale production and purification of CRP. In addition, a structural gene for the DNA-binding domain of CRP has been constructed. To this end the nucleotide sequence coding for the C-terminus was fused to the sequence coding for the first 10 N-terminal amino acids and cloned into suitable vectors. Good expression was achieved using the lambda PL promoter. The gene product, beta CRP, is recognized by anti-CRP antibodies. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:3539103

  5. Physiological and pathophysiological control of synaptic GluN2B-NMDA receptors by the C-terminal domain of amyloid precursor protein.

    PubMed

    Pousinha, Paula A; Mouska, Xavier; Raymond, Elisabeth F; Gwizdek, Carole; Dhib, Gihen; Poupon, Gwenola; Zaragosi, Laure-Emmanuelle; Giudici, Camilla; Bethus, Ingrid; Pacary, Emilie; Willem, Michael; Marie, Hélène

    2017-07-06

    The amyloid precursor protein (APP) harbors physiological roles at synapses and is central to Alzheimer's disease (AD) pathogenesis. Evidence suggests that APP intracellular domain (AICD) could regulate synapse function, but the underlying molecular mechanisms remain unknown. We addressed AICD actions at synapses, per se, combining in vivo AICD expression, ex vivo AICD delivery or APP knock-down by in utero electroporation of shRNAs with whole-cell electrophysiology. We report a critical physiological role of AICD in controlling GluN2B-containing NMDA receptors (NMDARs) at immature excitatory synapses, via a transcription-dependent mechanism. We further show that AICD increase in mature neurons, as reported in AD, alters synaptic NMDAR composition to an immature-like GluN2B-rich profile. This disrupts synaptic signal integration, via over-activation of SK channels, and synapse plasticity, phenotypes rescued by GluN2B antagonism. We provide a new physiological role for AICD, which becomes pathological upon AICD increase in mature neurons. Thus, AICD could contribute to AD synaptic failure.

  6. Rat pristanoyl-CoA oxidase. cDNA cloning and recognition of its C-terminal (SQL) by the peroxisomal-targeting signal 1 receptor.

    PubMed

    Vanhooren, J C; Fransen, M; de Béthune, B; Baumgart, E; Baes, M; Torrekens, S; Van Leuven, F; Mannaerts, G P; Van Veldhoven, P P

    1996-07-15

    The composite pristanoyl-CoA oxidase cDNA sequence, derived from two overlapping clones from a rat liver cDNA library and a 5'-RACE (rapid amplification of cDNA ends) PCR fragment, consisted of 2600 bases and contained an open reading frame of 2100 bases, encoding a protein of 700 amino acids with a calculated molecular mass of 78445 Da. This value is somewhat larger than the reported molecular mass of 70 kDa as determined earlier by SDS-gel electrophoresis. The amino acid identity with rat palmitoyl-CoA oxidase was rather low (28%) and barely higher than that with the yeast acyl-CoA oxidases (20%), suggesting that the palmitoyl-CoA oxidase/pristanoyl-CoA oxidase duplication occurred early in evolution. The carboxy-terminal tripeptide of pristanoyl-CoA oxidase was SQL. In vitro studies with the bacterially expressed human peroxisomal-targeting signal-1 import receptor indicated that SQL functions as a peroxisome-targeting signal. Northern analysis of tissues from control and clofibrate treated rats demonstrated that the pristanoyl-CoA oxidase gene is transcribed in liver and extrahepatic tissues and that transcription is not enhanced by treatment of rats with peroxisome proliferators. No mRNA could be detected by northern analysis of human tissues, suggesting that the human pristanoyl-CoA oxidase gene, if present, is only poorly or not transcribed.

  7. CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor.

    PubMed

    Lander, Noelia; Chiurillo, Miguel A; Storey, Melissa; Vercesi, Anibal E; Docampo, Roberto

    2016-12-02

    Methods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP3R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was co-localized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP3R, endogenously tagged TcIP3R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Impact of the coxsackie and adenovirus receptor (CAR) on glioma cell growth and invasion: requirement for the C-terminal domain.

    PubMed

    Huang, Kuo-Cheng; Altinoz, Meric; Wosik, Karolina; Larochelle, Nancy; Koty, Zafiro; Zhu, Lixia; Holland, Paul C; Nalbantoglu, Josephine

    2005-02-20

    Expression of the coxsackie and adenovirus receptor (CAR) is downregulated in malignant glioma cell lines and is barely detectable in high-grade primary astrocytoma (glioblastoma multiforme). We determined the effect of forced CAR expression on the invasion and growth of the human glioma cell line U87-MG, which does not express any CAR. Although retrovirally mediated expression of full-length CAR in U87-MG cells did not affect monolayer growth in vitro, it did reduce glioma cell invasion in a 3-dimensional spheroid model. Furthermore, in xenograft experiments, intracerebral implantation of glioma cells expressing full-length CAR resulted in tumors with a significantly reduced volume compared to tumors generated by control vector-transduced U87-MG cells. In contrast, U87-MG cells expressing transmembrane CAR with a deletion of the entire cytoplasmic domain (except for the first 2 intracellular juxtamembrane cysteine amino acids) had rates of invasion and tumor growth that were similar to those of the control cells. This difference in behavior between the 2 forms of CAR was not due to improper cell surface localization of the cytoplasmically deleted CAR as determined by comparable immunostaining of unpermeabilized cells, equivalent adenoviral transduction of the cells and similar extent of fractionation into lipid-rich domains. Taken together, these results suggest that the decrease or loss of CAR expression in malignant glioma may confer a selective advantage in growth and invasion to these tumors.

  9. Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain.

    PubMed

    Song, Dan; Nishiyama, Mariko; Kimura, Sadao

    2016-10-01

    R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT1 receptor signaling. Intracellular Ca(2+) responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.

  10. Effect of semax and its C-terminal fragment Pro-Gly-Pro on the expression of VEGF family genes and their receptors in experimental focal ischemia of the rat brain.

    PubMed

    Medvedeva, Ekaterina V; Dmitrieva, Veronika G; Povarova, Oksana V; Limborska, Svetlana A; Skvortsova, Veronika I; Myasoedov, Nikolay F; Dergunova, Lyudmila V

    2013-02-01

    The synthetic peptide Semax (Met-Glu-His-Phe-Pro-Gly-Pro) is used successfully in acute stroke therapy. In spite of numerous studies on the subject, many aspects of the neuroprotective effects of the peptide remain unknown. We studied the action of Semax and its C-terminal tripeptide Pro-Gly-Pro on the expression of the VEGF gene family (Vegf-a, Vegf-b, Vegf-c, Vegf-d, and Plgf) and their receptors (Vegfr-1, Vegfr-2, and Vegfr-3) in the frontoparietal cortex region of the rat brain at 3, 24, and 72 h after permanent left middle cerebral artery occlusion (pMCAO). The relative mRNA level of the genes studied was assessed using real-time reverse transcription-PCR. The Vegf-b and Vegf-d genes were most affected by the peptides, which resulted in their most noticeable activation at 3 h after pMCAO. The level of Vegf-d transcripts decreased considerably, whereas the mRNA level of the Vegf-b gene was significantly increased after 72 h of treatment with each of the peptides. In addition, the effects of the peptides on the expression of the Vegf-b and Vegf-d genes were the opposite of the action of ischemia. It is suggested that the identified effects of the peptides diminish the effects of ischemia, thus participating in the positive therapeutic effect of Semax on ischemic stroke.

  11. Alternative Splicing of Toll-Like Receptor 9 Transcript in Teleost Fish Grouper Is Regulated by NF-κB Signaling via Phosphorylation of the C-Terminal Domain of the RPB1 Subunit of RNA Polymerase II

    PubMed Central

    Lee, Frank Fang-Yao; Hui, Cho-Fat; Chang, Tien-Hsien; Chiou, Pinwen Peter

    2016-01-01

    Similar to its mammalian counterparts, teleost Toll-like receptor 9 (TLR9) recognizes unmethylated CpG DNA presented in the genome of bacteria or DNA viruses and initiates signaling pathway(s) for immune responses. We have previously shown that the TLR9 pathway in grouper, an economically important teleost, can be debilitated by an inhibitory gTLR9B isoform, whose production is mediated by RNA alternative splicing. However, how does grouper TLR9 (gTLR9) signaling impinge on the RNA splicing machinery to produce gTlr9B is unknown. Here we show that the gTlr9 alternative splicing is regulated through ligand-induced phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II). We first observed that ligand-activated NF- κB pathway biased the production of the gTlr9B isoform. Because NF- κB is known to recruit p-TEFb kinase, which phosphorylates the Pol II CTD at Ser2 residues, we examined p-TEFb’s role in alternative splicing. We found that promoting p-TEFb kinase activity significantly favored the production of the gTlr9B isoform, whereas inhibiting p-TEFb yielded an opposite result. We further showed that p-TEFb-mediated production of the gTlr9B isoform down-regulates its own immune responses, suggesting a self-limiting mechanism. Taken together, our data indicate a feedback mechanism of the gTLR9 signaling pathway to regulate the alternative splicing machinery, which in turn produces an inhibitor to the pathway. PMID:27658294

  12. A novel C-terminal truncating NR5A1 mutation in dizygotic twins

    PubMed Central

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction. PMID:28326187

  13. A novel C-terminal truncating NR5A1 mutation in dizygotic twins.

    PubMed

    Hattori, Atsushi; Zukeran, Hiroaki; Igarashi, Maki; Toguchi, Suzuka; Toubaru, Yuji; Inoue, Takanobu; Katoh-Fukui, Yuko; Fukami, Maki

    2017-01-01

    Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is a nuclear receptor involved in gonadal and adrenal development. We identified a novel C-terminally truncating NR5A1 mutation, p.Leu423Trpfs*7, in dizygotic twins with 46,XY disorders of sex development. Our results highlight the functional importance of C-terminal region of NR5A1 and indicate that NR5A1 mutations can be associated with intrafamilial phenotypic variations, progressive testicular dysfunction, hypogonadotropic hypogonadism, and borderline adrenal dysfunction.

  14. C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

    PubMed Central

    Gong, Jihong; Lai, Ying; Li, Xiaohong; Wang, Mengxian; Leitz, Jeremy; Hu, Yachong; Zhang, Yunxiang; Choi, Ucheor B.; Cipriano, Daniel; Pfuetzner, Richard A.; Südhof, Thomas C.; Yang, Xiaofei; Brunger, Axel T.

    2016-01-01

    In presynaptic nerve terminals, complexin regulates spontaneous “mini” neurotransmitter release and activates Ca2+-triggered synchronized neurotransmitter release. We studied the role of the C-terminal domain of mammalian complexin in these processes using single-particle optical imaging and electrophysiology. The C-terminal domain is important for regulating spontaneous release in neuronal cultures and suppressing Ca2+-independent fusion in vitro, but it is not essential for evoked release in neuronal cultures and in vitro. This domain interacts with membranes in a curvature-dependent fashion similar to a previous study with worm complexin [Snead D, Wragg RT, Dittman JS, Eliezer D (2014) Membrane curvature sensing by the C-terminal domain of complexin. Nat Commun 5:4955]. The curvature-sensing value of the C-terminal domain is comparable to that of α-synuclein. Upon replacement of the C-terminal domain with membrane-localizing elements, preferential localization to the synaptic vesicle membrane, but not to the plasma membrane, results in suppression of spontaneous release in neurons. Membrane localization had no measurable effect on evoked postsynaptic currents of AMPA-type glutamate receptors, but mislocalization to the plasma membrane increases both the variability and the mean of the synchronous decay time constant of NMDA-type glutamate receptor evoked postsynaptic currents. PMID:27821736

  15. High Affinity Agonists of the Neuropeptide Y (NPY) Y4 Receptor Derived from the C-Terminal Pentapeptide of Human Pancreatic Polypeptide (hPP): Synthesis, Stereochemical Discrimination, and Radiolabeling.

    PubMed

    Kuhn, Kilian K; Ertl, Thomas; Dukorn, Stefanie; Keller, Max; Bernhardt, Günther; Reiser, Oliver; Buschauer, Armin

    2016-07-14

    The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH2) (BVD-74D, 2) was described in the literature as a high affinity Y4 receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N(ω)-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [(3)H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y4R radioligands ([(3)H]-(2R,7R)-10, [(3)H]18) with subnanomolar Kd values.

  16. The C-terminal domain of the long form of cellular FLICE-inhibitory protein (c-FLIPL) inhibits the interaction of the caspase 8 prodomain with the receptor-interacting protein 1 (RIP1) death domain and regulates caspase 8-dependent nuclear factor κB (NF-κB) activation.

    PubMed

    Matsuda, Iyo; Matsuo, Kentaro; Matsushita, Yuka; Haruna, Yasushi; Niwa, Masamitsu; Kataoka, Takao

    2014-02-14

    Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation.

  17. The C-terminal Domain of the Long Form of Cellular FLICE-inhibitory Protein (c-FLIPL) Inhibits the Interaction of the Caspase 8 Prodomain with the Receptor-interacting Protein 1 (RIP1) Death Domain and Regulates Caspase 8-dependent Nuclear Factor κB (NF-κB) Activation*

    PubMed Central

    Matsuda, Iyo; Matsuo, Kentaro; Matsushita, Yuka; Haruna, Yasushi; Niwa, Masamitsu; Kataoka, Takao

    2014-01-01

    Caspase 8 plays an essential role in the regulation of apoptotic and non-apoptotic signaling pathways. The long form of cellular FLICE-inhibitory protein (c-FLIPL) has been shown previously to regulate caspase 8-dependent nuclear factor κB (NF-κB) activation by receptor-interacting protein 1 (RIP1) and TNF receptor-associated factor 2 (TRAF2). In this study, the molecular mechanism by which c-FLIPL regulates caspase 8-dependent NF-κB activation was further explored in the human embryonic kidney cell line HEK 293 and variant cells barely expressing caspase 8. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone greatly diminished caspase 8-dependent NF-κB activation induced by Fas ligand (FasL) when c-FLIPL, but not its N-terminal fragment c-FLIP(p43), was expressed. The prodomain of caspase 8 was found to interact with the RIP1 death domain and to be sufficient to mediate NF-κB activation induced by FasL or c-FLIP(p43). The interaction of the RIP1 death domain with caspase 8 was inhibited by c-FLIPL but not c-FLIP(p43). Thus, these results reveal that the C-terminal domain of c-FLIPL specifically inhibits the interaction of the caspase 8 prodomain with the RIP1 death domain and, thereby, regulates caspase 8-dependent NF-κB activation. PMID:24398693

  18. Kinetic and stability properties of Penicillium chrysogenum ATP sulfurylase missing the C-terminal regulatory domain.

    PubMed

    Hanna, Eissa; Ng, Kit Fai; MacRae, Ian J; Bley, Christopher J; Fisher, Andrew J; Segel, Irwin H

    2004-02-06

    ATP sulfurylase from Penicillium chrysogenum is a homohexameric enzyme that is subject to allosteric inhibition by 3'-phosphoadenosine 5'-phosphosulfate. In contrast to the wild type enzyme, recombinant ATP sulfurylase lacking the C-terminal allosteric domain was monomeric and noncooperative. All kcat values were decreased (the adenosine 5'-phosphosulfate (adenylylsulfate) (APS) synthesis reaction to 17% of the wild type value). Additionally, the Michaelis constants for MgATP and sulfate (or molybdate), the dissociation constant of E.APS, and the monovalent oxyanion dissociation constants of dead end E.MgATP.oxyanion complexes were all increased. APS release (the k6 step) was rate-limiting in the wild type enzyme. Without the C-terminal domain, the composite k5 step (isomerization of the central complex and MgPPi release) became rate-limiting. The cumulative results indicate that besides (a) serving as a receptor for the allosteric inhibitor, the C-terminal domain (b) stabilizes the hexameric structure and indirectly, individual subunits. Additionally, (c) the domain interacts with and perfects the catalytic site such that one or more steps following the formation of the binary E.MgATP and E.SO4(2-) complexes and preceding the release of MgPPi are optimized. The more negative entropy of activation of the truncated enzyme for APS synthesis is consistent with a role of the C-terminal domain in promoting the effective orientation of MgATP and sulfate at the active site.

  19. Modules for C-terminal epitope tagging of Tetrahymena genes

    PubMed Central

    Kataoka, Kensuke; Schoeberl, Ursula E.; Mochizuki, Kazufumi

    2010-01-01

    Although epitope tagging has been widely used for analyzing protein function in many organisms, there are few genetic tools for epitope tagging in Tetrahymena. In this study, we describe several C-terminal epitope tagging modules that can be used to express tagged proteins in Tetrahymena cells by both plasmid- and PCR-based strategies. PMID:20624430

  20. Resonance assignments and secondary structure of apolipoprotein E C-terminal domain in DHPC micelles.

    PubMed

    Lo, Chi-Jen; Chyan, Chia-Lin; Chen, Yi-Chen; Chang, Chi-Fon; Huang, Hsien-Bin; Lin, Ta-Hsien

    2015-04-01

    Human apolipoprotein E (apoE) has been known to play a key role in the transport of plasma cholesterol and lipoprotein metabolism. It is an apolipoprotein of 299 amino acids with a molecular mass, ~34 kDa. ApoE has three major isoforms, apoE2, apoE3, and apoE4 which differ only at residue 112 or 158. ApoE consists of two independently folded domains (N-terminal and C-terminal domain) separated by a hinge region. The N-terminal domain and C-terminal domain of apoE are responsible for the binding to receptor and to lipid, respectively. Since the high resolution structures of apoE in lipids are still unavailable to date, we therefore aim to resolve the structures in lipids by NMR. Here, we reported the resonance assignments and secondary structure distribution of the C-terminal domain of wild-type human apoE (residue 195-299) in the micelles formed by dihexanoylphosphatidylcholine. Our results may provide a novel structural model of apoE in micelles and may shed new light on the molecular mechanisms underlying the apoE related biological processes.

  1. Structural and functional comparisons of retroviral envelope protein C-terminal domains: still much to learn.

    PubMed

    Steckbeck, Jonathan D; Kuhlmann, Anne-Sophie; Montelaro, Ronald C

    2014-01-16

    Retroviruses are a family of viruses that cause a broad range of pathologies in animals and humans, from the apparently harmless, long-term genomic insertion of endogenous retroviruses, to tumors induced by the oncogenic retroviruses and acquired immunodeficiency syndrome (AIDS) resulting from human immunodeficiency virus infection. Disease can be the result of diverse mechanisms, including tumorigenesis induced by viral oncogenes or immune destruction, leading to the gradual loss of CD4 T-cells. Of the virally encoded proteins common to all retroviruses, the envelope (Env) displays perhaps the most diverse functionality. Env is primarily responsible for binding the cellular receptor and for effecting the fusion process, with these functions mediated by protein domains localized to the exterior of the virus. The remaining C-terminal domain may have the most variable functionality of all retroviral proteins. The C-terminal domains from three prototypical retroviruses are discussed, focusing on the different structures and functions, which include fusion activation, tumorigenesis and viral assembly and lifecycle influences. Despite these genetic and functional differences, however, the C-terminal domains of these viruses share a common feature in the modulation of Env ectodomain conformation. Despite their differences, perhaps each system still has information to share with the others.

  2. Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

    PubMed Central

    Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

    2015-01-01

    The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

  3. C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

    PubMed Central

    Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

    2014-01-01

    A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

  4. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

    PubMed

    Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

    2013-10-01

    The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

  5. C-terminal engineering of CXCL12 and CCL5 chemokines: functional characterization by electrophysiological recordings.

    PubMed

    Picciocchi, Antoine; Siaučiūnaiteė-Gaubard, Lina; Petit-Hartlein, Isabelle; Sadir, Rabia; Revilloud, Jean; Caro, Lydia; Vivaudou, Michel; Fieschi, Franck; Moreau, Christophe; Vivès, Corinne

    2014-01-01

    Chemokines are chemotactic cytokines comprised of 70-100 amino acids. The chemokines CXCL12 and CCL5 are the endogenous ligands of the CXCR4 and CCR5 G protein-coupled receptors that are also HIV co-receptors. Biochemical, structural and functional studies of receptors are ligand-consuming and the cost of commercial chemokines hinders their use in such studies. Here, we describe methods for the expression, refolding, purification, and functional characterization of CXCL12 and CCL5 constructs incorporating C-terminal epitope tags. The model tags used were hexahistidines and Strep-Tag for affinity purification, and the double lanthanoid binding tag for fluorescence imaging and crystal structure resolution. The ability of modified and purified chemokines to bind and activate CXCR4 and CCR5 receptors was tested in Xenopus oocytes expressing the receptors, together with a Kir3 G-protein activated K(+) channel that served as a reporter of receptor activation. Results demonstrate that tags greatly influence the biochemical properties of the recombinant chemokines. Besides, despite the absence of any evidence for CXCL12 or CCL5 C-terminus involvement in receptor binding and activation, we demonstrated unpredictable effects of tag insertion on the ligand apparent affinity and efficacy or on the ligand dissociation. These tagged chemokines should constitute useful tools for the selective purification of properly-folded chemokines receptors and the study of their native quaternary structures.

  6. Effect of surface coating of KYb2F7:Tm3+ on optical properties and biomedical applications

    NASA Astrophysics Data System (ADS)

    Pedraza, Francisco J.; Avalos, Julio C.; Yust, Brian G.; Tsin, Andrew; Sardar, Dhiraj K.

    2016-09-01

    This project aims to provide an insight on the effects of biocompatible polymers on the optical properties and the nanoparticle-cell interaction of KYb2F7:Tm3+ nanocrystals that exhibit strong near infrared (NIR) fluorescence. KYb2F7:Tm3+ nanocrystals were synthesized with a diameter of 20-30 nm and surface modified with poly(ethylene glycol), Pluronic® F-127, and poly(N-vinylpyrrolidone), due to the associated advantages. Some of these include biocompatibility and biodistribution in the instance of agglomeration and hydrophobicity as well as the addition of a targeting agent and drug loading by further functionalization. Despite the decrease in fluorescence intensity induced by the surface modification, thulium’s emission fingerprint was easily detected. Moreover, surface modified KYb2F7:Tm3+ nanocrystals failed to induce a toxic response on endothelial cells following a 24 h uptake period up to concentrations of 100 μg ml-1. In vitro toxicity and confocal imaging have demonstrated the versatility of these NIR fluorescence nanocrystals in biomedical imaging, drug delivery, and photodynamic therapy.

  7. Effect of surface coating of KYb2F7:Tm3+ on optical properties and biomedical applications

    PubMed Central

    Pedraza, Francisco J; Avalos, Julio C; Yust, Brian G; Tsin, Andrew; Sardar, Dhiraj K

    2016-01-01

    This project aims to provide an insight on the effects of biocompatible polymers on the optical properties and the nanoparticle-cell interaction of KYb2F7:Tm3+ nanocrystals that exhibit strong near infrared (NIR) fluorescence. KYb2F7:Tm3+ nanocrystals were synthesized with a diameter of 20–30 nm and surface modified with poly(ethylene glycol), Pluronic® F-127, and poly(N-vinylpyrrolidone), due to the associated advantages. Some of these include biocompatibility and biodistribution in the instance of agglomeration and hydrophobicity as well as the addition of a targeting agent and drug loading by further functionalization. Despite the decrease in fluorescence intensity induced by the surface modification, thulium’s emission fingerprint was easily detected. Moreover, surface modified KYb2F7:Tm3+ nanocrystals failed to induce a toxic response on endothelial cells following a 24 h uptake period up to concentrations of 100 μg ml−1. In vitro toxicity and confocal imaging have demonstrated the versatility of these NIR fluorescence nanocrystals in biomedical imaging, drug delivery, and photodynamic therapy. PMID:27518385

  8. Crystal structure of the C-terminal domain of mouse TLR9

    PubMed Central

    Collins, Bernard; Wilson, Ian A.

    2014-01-01

    Toll-like receptors (TLRs) are important pattern recognition receptors that function in innate immunity. Elucidating the structure and signaling mechanisms of TLR9, a sensor of foreign and endogenous DNA, is essential for understanding its critical roles in immunity and autoimmunity. Abundant evidence suggests that the TLR9-CTD (C-terminal domain) by itself is capable of DNA-binding and signaling. We present the crystal structure of unliganded mouse TLR9-CTD. TLR9-CTD exhibits one unique feature, a cluster of stacked aromatic and arginine side chains on its concave face. Overall, its structure is most related to the TLR8-CTD, suggesting a similar mode of ligand binding and signaling. PMID:24888966

  9. Crystallization of the C-terminal globular domain of avian reovirus fibre

    SciTech Connect

    Raaij, Mark J. van; Hermo Parrado, X. Lois; Guardado Calvo, Pablo; Fox, Gavin C.; Llamas-Saiz, Antonio L.; Costas, Celina; Martínez-Costas, José; Benavente, Javier

    2005-07-01

    Partial proteolysis of the avian reovirus cell-attachment protein σC yields a major homotrimeric C-terminal fragment that presumably contains the receptor-binding domain. This fragment has been crystallized in the presence and absence of zinc sulfate and cadmium sulfate. One of the crystal forms diffracts synchrotron X-rays to 2.2–2.3 Å. Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6{sub 3}22 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the Zn{sup II}- and Cd{sup II}-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine.

  10. Nonlinear dynamics of C-terminal tails in cellular microtubules

    NASA Astrophysics Data System (ADS)

    Sekulic, Dalibor L.; Sataric, Bogdan M.; Zdravkovic, Slobodan; Bugay, Aleksandr N.; Sataric, Miljko V.

    2016-07-01

    The mechanical and electrical properties, and information processing capabilities of microtubules are the permanent subject of interest for carrying out experiments in vitro and in silico, as well as for theoretical attempts to elucidate the underlying processes. In this paper, we developed a new model of the mechano-electrical waves elicited in the rows of very flexible C-terminal tails which decorate the outer surface of each microtubule. The fact that C-terminal tails play very diverse roles in many cellular functions, such as recruitment of motor proteins and microtubule-associated proteins, motivated us to consider their collective dynamics as the source of localized waves aimed for communication between microtubule and associated proteins. Our approach is based on the ferroelectric liquid crystal model and it leads to the effective asymmetric double-well potential which brings about the conditions for the appearance of kink-waves conducted by intrinsic electric fields embedded in microtubules. These kinks can serve as the signals for control and regulation of intracellular traffic along microtubules performed by processive motions of motor proteins, primarly from kinesin and dynein families. On the other hand, they can be precursors for initiation of dynamical instability of microtubules by recruiting the proper proteins responsible for the depolymerization process.

  11. Evolution of the RNA polymerase II C-terminal domain

    PubMed Central

    Stiller, John W.; Hall, Benjamin D.

    2002-01-01

    In recent years a great deal of biochemical and genetic research has focused on the C-terminal domain (CTD) of the largest subunit (RPB1) of DNA-dependent RNA polymerase II. This strongly conserved domain of tandemly repeated heptapeptides has been linked functionally to important steps in the initiation and processing of mRNA transcripts in both animals and fungi. Although they are absolutely required for viability in these organisms, C-terminal tandem repeats do not occur in RPB1 sequences from diverse eukaryotic taxa. Here we present phylogenetic analyses of RPB1 sequences showing that canonical CTD heptads are strongly conserved in only a subset of eukaryotic groups, all apparently descended from a single common ancestor. Moreover, eukaryotic groups in which the most complex patterns of ontogenetic development occur are descended from this CTD-containing ancestor. Consistent with the results of genetic and biochemical investigations of CTD function, these analyses suggest that the enhanced control over RNA polymerase II transcription conveyed by acquired CTD/protein interactions was an important step in the evolution of intricate patterns of gene expression that are a hallmark of large, developmentally complex eukaryotic organisms. PMID:11972039

  12. Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains.

    PubMed

    Carpenter, K A; Schmidt, R; von Mentzer, B; Haglund, U; Roberts, E; Walpole, C

    2001-07-27

    The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V

  13. A C-terminal membrane association domain of phototropin 2 is necessary for chloroplast movement.

    PubMed

    Kong, Sam-Geun; Kagawa, Takatoshi; Wada, Masamitsu; Nagatani, Akira

    2013-01-01

    Phototropins (phot1 and phot2), plant-specific blue light receptor kinases, mediate a range of physiological responses in Arabidopsis, including phototropism, chloroplast photorelocation movement, stomatal opening and leaf flattening. Phototropins consist of two photoreceptive domains at their N-terminus, LOV1 (light, oxygen or voltage 1) and LOV2, and a serine/threonine kinase domain at their C-terminus. Here, we determined the molecular moiety for the membrane association of phototropins using the yeast CytoTrap and Arabidopsis protoplast systems. We then examined the physiological significance of the membrane association of phototropins. This detailed study with serial deletions narrowed down the association domain to a relatively small part of the C-terminal domain of phototropin. The functional analysis of phot2 deletion mutants in the phot2-deficient Adiantum and Arabidopsis mutants revealed that the ability to mediate the chloroplast avoidance response correlated well with phot2's membrane association, especially with the Golgi apparatus. Taken together, our data suggest that a small part of the C-terminal domain of phototropins is necessary not only for membrane association but also for the physiological activities that elicit phototropin-specific responses.

  14. Intrinsic Disorder of the C-Terminal Domain of Drosophila Methoprene-Tolerant Protein

    PubMed Central

    Kolonko, Marta; Ożga, Katarzyna; Hołubowicz, Rafał; Taube, Michał; Kozak, Maciej; Ożyhar, Andrzej; Greb-Markiewicz, Beata

    2016-01-01

    Methoprene tolerant protein (Met) has recently been confirmed as the long-sought juvenile hormone (JH) receptor. This protein plays a significant role in the cross-talk of the 20-hydroxyecdysone (20E) and JH signalling pathways, which are important for control of insect development and maturation. Met belongs to the basic helix-loop-helix/Per-Arnt-Sim (bHLH-PAS) family of transcription factors. In these proteins, bHLH domains are typically responsible for DNA binding and dimerization, whereas the PAS domains are crucial for the choice of dimerization partner and the specificity of target gene activation. The C-terminal region is usually responsible for the regulation of protein complex activity. The sequence of the Met C-terminal region (MetC) is not homologous to any sequence deposited in the Protein Data Bank (PDB) and has not been structurally characterized to date. In this study, we show that the MetC exhibits properties typical for an intrinsically disordered protein (IDP). The final averaged structure obtained with small angle X-ray scattering (SAXS) experiments indicates that intrinsically disordered MetC exists in an extended conformation. This extended shape and the long unfolded regions characterise proteins with high flexibility and dynamics. Therefore, we suggest that the multiplicity of conformations adopted by the disordered MetC is crucial for its activity as a biological switch modulating the cross-talk of different signalling pathways in insects. PMID:27657508

  15. Autoinhibition of the Nuclease ARTEMIS Is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains.

    PubMed

    Niewolik, Doris; Peter, Ingrid; Butscher, Carmen; Schwarz, Klaus

    2017-02-24

    The nuclease ARTEMIS is essential for the development of B and T lymphocytes. It is required for opening DNA hairpins generated during antigen receptor gene assembly from variable (V), diversity (D), and joining (J) subgenic elements (V(D)J recombination). As a member of the non-homologous end-joining pathway, it is also involved in repairing a subset of pathological DNA double strand breaks. Loss of ARTEMIS function therefore results in radiosensitive severe combined immunodeficiency (RS-SCID). The hairpin opening activity is dependent on the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which can bind to and phosphorylate ARTEMIS. The ARTEMIS C terminus is dispensable for cellular V(D)J recombination and in vitro nuclease assays with C-terminally truncated ARTEMIS showing DNA-PKcs-independent hairpin opening activity. Therefore, it has been postulated that ARTEMIS is regulated via autoinhibition by its C terminus. To obtain evidence for the autoinhibition model, we performed co-immunoprecipitation experiments with combinations of ARTEMIS mutants. We show that an N-terminal fragment comprising the catalytic domain can interact both with itself and with a C-terminal fragment. Amino acid exchanges N456A+S457A+E458Q in the C terminus of full-length ARTEMIS resulted in unmasking of the N terminus and in increased ARTEMIS activity in cellular V(D)J recombination assays. Mutations in ARTEMIS-deficient patients impaired the interaction with the C terminus and also affected protein stability. The interaction between the N- and C-terminal domains was not DNA-PKcs-dependent, and phosphomimetic mutations in the C-terminal domain did not result in unmasking of the catalytic domain. Our experiments provide strong evidence that a physical interaction between the C-terminal and catalytic domains mediates ARTEMIS autoinhibition. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. C-Terminal Functionalization of Nylon-3 Polymers: Effects of C-Terminal Groups on Antibacterial and Hemolytic Activities

    PubMed Central

    Zhang, Jihua; Markiewicz, Matthew J.; Mowery, Brendan P.; Weisblum, Bernard

    2012-01-01

    Nylon-3 polymers contain β-amino acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as co-initiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete we introduce a new β-lactam, approximately one equivalent relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials. PMID:22168316

  17. C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

    PubMed

    Zhang, Jihua; Markiewicz, Matthew J; Mowery, Brendan P; Weisblum, Bernard; Stahl, Shannon S; Gellman, Samuel H

    2012-02-13

    Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain. Controlling the unit at the C-termini of nylon-3 polymer chains, however, has been problematic. Here we describe a strategy for specifying C-terminal functionality that is based on the polymerization mechanism. After the anionic ring-opening polymerization is complete, we introduce a new β-lactam, approximately 1 equiv relative to the expected number of polymer chains. Because the polymer chains bear a reactive imide group at their C-termini, this new β-lactam should become attached at this position. If the terminating β-lactam bears a distinctive functional group, that functionality should be affixed to most or all C-termini in the reaction mixture. We use the new technique to compare the impact of N- and C-terminal placement of a critical hydrophobic fragment on the biological activity profile of nylon-3 copolymers. The synthetic advance described here should prove to be generally useful for tailoring the properties of nylon-3 materials.

  18. Crystallization of the C-terminal domain of the bacteriophage T5 L-shaped fibre.

    PubMed

    Garcia-Doval, Carmela; Luque, Daniel; Castón, José R; Boulanger, Pascale; van Raaij, Mark J

    2013-12-01

    Tails of bacteriophage T5 (a member of the Siphoviridae family) were studied by electron microscopy. For the distal parts of the L-shaped tail fibres, which are involved in host cell receptor binding, a low-resolution volume was calculated. Several C-terminal fragments of the fibre were expressed and purified. Crystals of two of them were obtained that belonged to space groups P63 and R32 and diffracted synchrotron radiation to 2.3 and 2.9 Å resolution, respectively. A single-wavelength anomalous dispersion data set to 2.5 Å resolution was also collected from a selenomethionine-derivatized crystal of one of the fragments, which belonged to space group C2.

  19. Structure-activity relationships of C-terminal tri- and tetrapeptide fragments that inhibit gastrin activity.

    PubMed

    Martinez, J; Bali, J P; Magous, R; Laur, J; Lignon, M F; Briet, C; Nisato, D; Castro, B

    1985-03-01

    A series of tri- and tetrapeptide derivatives, analogues of the gastrin C-terminal region with no phenylalanine residue, were synthesized. These peptides were tested for their ability to inhibit gastrin-stimulated acid secretion in vivo as well as binding of [125I]-(Nle11)-HG-13 to gastric mucosal cell receptors in vitro. Most of the peptides tested exhibited gastrin antagonist activity in vivo and in vitro. Most active derivatives were 20-30 times more potent than the well-known gastrin antagonist derivatives proglumide and benzotript and had 20-200 times more binding affinity. The smallest fragment exhibiting antagonist activity was the tripeptide Boc-L-tryptophyl-L-methionyl-L-aspartic acid amide.

  20. Design and structure-activity relationships of C-terminal cyclic neurotensin fragment analogues.

    PubMed

    Sefler, A M; He, J X; Sawyer, T K; Holub, K E; Omecinsky, D O; Reily, M D; Thanabal, V; Akunne, H C; Cody, W L

    1995-01-20

    Neurotensin (NT) is a linear tridecapeptide with a broad range of central and peripheral pharmacological effects. The C-terminal hexapeptide of NT (NT8-13) has been shown to possess similar properties to NT itself, and in fact, an analogue of NT8-13 (N alpha MeArg8-Lys-Pro-Trp-Tle-Leu13, Tle = tert-leucine) has been reported to possess central activity after peripheral administration. Cyclic derivatives of this hexapeptide were synthesized by a combination of solution and solid-phase peptide synthetic methodologies, and several analogues had low nanomolar binding affinity for the NT receptor. In particular, cyclo[Arg-Lys-Pro-Trp-Glu]-Leu (cyclized between the alpha amine of Arg and the gamma carboxylate of Glu) possessed 16 nM NT receptor affinity and was determined to be an agonist in vitro. 1H-NMR and 13C-edited 1H-NMR spectroscopy were performed on this and related cyclic analogues to help identify structural properties which may be important for receptor recognition. These cyclic peptides represent novel molecular probes to further investigate NT receptor pharmacology, as well as to advance our understanding of the structure-conformation relationships of NT and to help establish a working basis for additional pharmacophore mapping studies.

  1. Insulin resistance uncoupled from dyslipidemia due to C-terminal PIK3R1 mutations

    PubMed Central

    Huang-Doran, Isabel; Tomlinson, Patsy; Payne, Felicity; Gast, Alexandra; Sleigh, Alison; Bottomley, William; Harris, Julie; Daly, Allan; Rocha, Nuno; Rudge, Simon; Clark, Jonathan; Kwok, Albert; Romeo, Stefano; McCann, Emma; Müksch, Barbara; Dattani, Mehul; Zucchini, Stefano; Wakelam, Michael; Foukas, Lazaros C.; Savage, David B.; Murphy, Rinki; O’Rahilly, Stephen; Semple, Robert K.

    2016-01-01

    Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in PIK3R1, encoding the p85α/p55α/p50α subunits of PI3K, which act between INSR and AKT in insulin signaling. Four of 5 patients had extreme insulin resistance without dyslipidemia or hepatic steatosis. In 3 of these 4, plasma adiponectin was preserved, as in insulin receptor dysfunction. The fourth patient and her healthy mother had low plasma adiponectin associated with a potentially novel mutation, p.Asp231Ala, in adiponectin itself. Cells studied from one patient with the p.Tyr657X PIK3R1 mutation expressed abundant truncated PIK3R1 products and showed severely reduced insulin-stimulated association of mutant but not WT p85α with IRS1, but normal downstream signaling. In 3T3-L1 preadipocytes, mutant p85α overexpression attenuated insulin-induced AKT phosphorylation and adipocyte differentiation. Thus, PIK3R1 C-terminal mutations impair insulin signaling only in some cellular contexts and produce a subphenotype of insulin resistance resembling INSR dysfunction but unlike AKT2 dysfunction, implicating PI3K in the pathogenesis of key components of the metabolic syndrome. PMID:27766312

  2. C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

    PubMed

    Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

    2017-01-01

    C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed.

  3. Biased signaling favoring gi over β-arrestin promoted by an apelin fragment lacking the C-terminal phenylalanine.

    PubMed

    Ceraudo, Emilie; Galanth, Cécile; Carpentier, Eric; Banegas-Font, Inmaculada; Schonegge, Anne-Marie; Alvear-Perez, Rodrigo; Iturrioz, Xavier; Bouvier, Michel; Llorens-Cortes, Catherine

    2014-08-29

    Apelin plays a prominent role in body fluid and cardiovascular homeostasis. We previously showed that the C-terminal Phe of apelin 17 (K17F) is crucial for triggering apelin receptor internalization and decreasing blood pressure (BP) but is not required for apelin binding or Gi protein coupling. Based on these findings, we hypothesized that the important role of the C-terminal Phe in BP decrease may be as a Gi-independent but β-arrestin-dependent signaling pathway that could involve MAPKs. For this purpose, we have used apelin fragments K17F and K16P (K17F with the C-terminal Phe deleted), which exhibit opposite profiles on apelin receptor internalization and BP. Using BRET-based biosensors, we showed that whereas K17F activates Gi and promotes β-arrestin recruitment to the receptor, K16P had a much reduced ability to promote β-arrestin recruitment while maintaining its Gi activating property, revealing the biased agonist character of K16P. We further show that both β-arrestin recruitment and apelin receptor internalization contribute to the K17F-stimulated ERK1/2 activity, whereas the K16P-promoted ERK1/2 activity is entirely Gi-dependent. In addition to providing new insights on the structural basis underlying the functional selectivity of apelin peptides, our study indicates that the β-arrestin-dependent ERK1/2 activation and not the Gi-dependent signaling may participate in K17F-induced BP decrease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Mechanistic studies of ubiquitin C-terminal hydrolase L1.

    PubMed

    Case, April; Stein, Ross L

    2006-02-21

    Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.

  5. A C-terminal HSP90 inhibitor restores glucocorticoid sensitivity and relieves a mouse allograft model of Cushing disease.

    PubMed

    Riebold, Mathias; Kozany, Christian; Freiburger, Lee; Sattler, Michael; Buchfelder, Michael; Hausch, Felix; Stalla, Günter K; Paez-Pereda, Marcelo

    2015-03-01

    One function of the glucocorticoid receptor (GR) in corticotroph cells is to suppress the transcription of the gene encoding proopiomelanocortin (POMC), the precursor of the stress hormone adrenocorticotropin (ACTH). Cushing disease is a neuroendocrine condition caused by partially glucocorticoid-resistant corticotroph adenomas that excessively secrete ACTH, which leads to hypercortisolism. Mutations that impair GR function explain glucocorticoid resistance only in sporadic cases. However, the proper folding of GR depends on direct interactions with the chaperone heat shock protein 90 (HSP90, refs. 7,8). We show here that corticotroph adenomas overexpress HSP90 compared to the normal pituitary. N- and C-terminal HSP90 inhibitors act at different steps of the HSP90 catalytic cycle to regulate corticotroph cell proliferation and GR transcriptional activity. C-terminal inhibitors cause the release of mature GR from HSP90, which promotes its exit from the chaperone cycle and potentiates its transcriptional activity in a corticotroph cell line and in primary cultures of human corticotroph adenomas. In an allograft mouse model, the C-terminal HSP90 inhibitor silibinin showed anti-tumorigenic effects, partially reverted hormonal alterations, and alleviated symptoms of Cushing disease. These results suggest that the pathogenesis of Cushing disease caused by overexpression of heat shock proteins and consequently misregulated GR sensitivity may be overcome pharmacologically with an appropriate HSP90 inhibitor.

  6. Protein splicing of inteins with atypical glutamine and aspartate C-terminal residues.

    PubMed

    Amitai, Gil; Dassa, Bareket; Pietrokovski, Shmuel

    2004-01-30

    Inteins are protein-splicing domains present in many proteins. They self-catalyze their excision from the host protein, ligating their former flanks by a peptide bond. The C-terminal residue of inteins is typically an asparagine (Asn). Cyclization of this residue to succinimide causes the final detachment of inteins from their hosts. We studied protein-splicing activity of two inteins with atypical C-terminal residues. One having a C-terminal glutamine (Gln), isolated from Chilo iridescent virus (CIV), and another unique intein, first reported here, with a C-terminal aspartate, isolated from Carboxydothermus hydrogenoformans (Chy). Protein-splicing activity was examined in the wild-type inteins and in several mutants with N- and C-terminal amino acid substitutions. We demonstrate that both wild-type inteins can protein splice, probably by new variations of the typical protein-splicing mechanism. Substituting the atypical C-terminal residue to the typical Asn retained protein-splicing only in the CIV intein. All diverse C-terminal substitutions in the Chy intein (Asp(345) to Asn, Gln, Glu, and Ala) abolished protein-splicing and generated N- and C-terminal cleavage. The observed C-terminal cleavage in the Chy intein ending with Ala cannot be explained by cyclization of this residue. We present and discuss several new models for reactions in the protein-splicing pathway.

  7. Oligomerization of the polycystin-2 C-terminal tail and effects on its Ca2+-binding properties.

    PubMed

    Yang, Yifei; Keeler, Camille; Kuo, Ivana Y; Lolis, Elias J; Ehrlich, Barbara E; Hodsdon, Michael E

    2015-04-17

    Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.

  8. The C-terminal tails of 4,4'-diphenylmethane-bis(methyl) carbamate are essential for binding to receptor for advanced glycation end products to attenuate advanced glycation end products-induced inflammation and apoptosis responses in human umbilical vein endothelial cells.

    PubMed

    Feng, Liang; Zhu, Mao-mao; Bu, Wei-quan; Wang, Chun-fei; Zheng, Zhao-guang; Wang, Ru-shang; Jia, Xiao-bin; Zhu, Quan

    2016-01-01

    A novel compound 4,4'-diphenylmethane-bis(methyl) carbamate (CM1) was shown to possess preventive activity on AGEs-induced human umbilical vein endothelial cells (HUVECs) damage via binding to RAGE. However, the underlying structural basis of CM1 on binding to RAGE was not fully understood. In the present study, CM1 analogues were designed and synthesized to compare the activity differences on inhibiting AGEs-induced inflammatory response including TGF-β1, RAGE protein expression in HUVECs, and macrophages migration and adhesion to HUVECs. In addition, the cell viability and anti-apoptosis activities of CM1 analogues were also examined. These results indicated that CM1 had higher activities on preventing AGEs-induced HUVECs damage (inflammation, cell viability and apoptosis) than other analogues. The bioaffinity assay was conducted by CMC and demonstrated that the IC50 and dissociation equilibrium constants (Kd) of CM1 were lower whereas the Bmax was higher than other analogues. The incubation of RAGE protein with CM1 analogues by equilibrium dialysis method showed CM1 had a stronger binding rate than other CM1 analogues. Our findings suggested that the C-terminal tails (methoxycarbonyl groups) of CM1 were the active groups for binding to RAGE and then led to the attenuation on RAGE-mediated endothelial dysfunction. © 2015 Royal Pharmaceutical Society.

  9. Physical association of GPR54 C-terminal with protein phosphatase 2A

    SciTech Connect

    Evans, Barry J.; Wang Zixuan; Mobley, La'Tonya; Khosravi, Davood; Fujii, Nobutaka; Navenot, Jean-Marc; Peiper, Stephen C.

    2008-12-26

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  10. Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers.

    PubMed

    Windisch, Dirk; Ziegler, Colin; Bürck, Jochen; Ulrich, Anne S

    2014-12-01

    E5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state (15)N- and (31)P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.

  11. An Autoinhibitory Helix in the C-Terminal Region of Phospholipase C-β Mediates Gαq Activation

    PubMed Central

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J. G.

    2011-01-01

    Phospholipase C-β (PLCβ) is a key regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to Gq. We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLCβ3 dramatically increase basal activity and diminish stimulation by Gαq. Gαq binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLCβ. PMID:21822282

  12. AMINO ACID COMPOSITION AND C-TERMINAL RESIDUES OF ALGAL BILIPROTEINS,

    DTIC Science & Technology

    R-phycoerythrin from Ceramium rubrum and C- phycocyanin from Nostoc nuscorum were obtained in purified form by fractional crystallization, followed by...as amino acids. Alanine was identified as the only C-terminal amino acid of R-phycoerythrin, each molecule of which contained about 12 terminal groups. Serine was identified as the only C-terminal group of C- phycocyanin . (Author)

  13. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach

    PubMed Central

    Tirupula, Kalyan C.; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S.

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A ‘cardiac-specific finger print’ of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a ‘MAS-signalosome’ model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of ‘signalosome’ components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor. PMID

  14. MAS C-Terminal Tail Interacting Proteins Identified by Mass Spectrometry- Based Proteomic Approach.

    PubMed

    Tirupula, Kalyan C; Zhang, Dongmei; Osbourne, Appledene; Chatterjee, Arunachal; Desnoyer, Russ; Willard, Belinda; Karnik, Sadashiva S

    2015-01-01

    Propagation of signals from G protein-coupled receptors (GPCRs) in cells is primarily mediated by protein-protein interactions. MAS is a GPCR that was initially discovered as an oncogene and is now known to play an important role in cardiovascular physiology. Current literature suggests that MAS interacts with common heterotrimeric G-proteins, but MAS interaction with proteins which might mediate G protein-independent or atypical signaling is unknown. In this study we hypothesized that MAS C-terminal tail (Ct) is a major determinant of receptor-scaffold protein interactions mediating MAS signaling. Mass-spectrometry based proteomic analysis was used to comprehensively identify the proteins that interact with MAS Ct comprising the PDZ-binding motif (PDZ-BM). We identified both PDZ and non-PDZ proteins from human embryonic kidney cell line, mouse atrial cardiomyocyte cell line and human heart tissue to interact specifically with MAS Ct. For the first time our study provides a panel of PDZ and other proteins that potentially interact with MAS with high significance. A 'cardiac-specific finger print' of MAS interacting PDZ proteins was identified which includes DLG1, MAGI1 and SNTA. Cell based experiments with wild-type and mutant MAS lacking the PDZ-BM validated MAS interaction with PDZ proteins DLG1 and TJP2. Bioinformatics analysis suggested well-known multi-protein scaffold complexes involved in nitric oxide signaling (NOS), cell-cell signaling of neuromuscular junctions, synapses and epithelial cells. Majority of these protein hits were predicted to be part of disease categories comprising cancers and malignant tumors. We propose a 'MAS-signalosome' model to stimulate further research in understanding the molecular mechanism of MAS function. Identifying hierarchy of interactions of 'signalosome' components with MAS will be a necessary step in future to fully understand the physiological and pathological functions of this enigmatic receptor.

  15. Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding.

    PubMed

    Xu, Wei; Xu, Xianzhong; Leal, Walter S; Ames, James B

    2011-01-07

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.

  16. Extrusion of the C-terminal Helix in Navel Orangeworm Moth Pheromone-Binding Protein (AtraPBP1) Controls Pheromone Binding†

    PubMed Central

    Xu, Wei; Xu, Xianzhong; Leal, Walter S.; Ames, James B.

    2011-01-01

    The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from Amyelois transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129–142) causes more than 100-fold increase in pheromone binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ~2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding. PMID:21130734

  17. Mechanism for the Selective Interaction of C-terminal Eps15 Homology Domain Proteins with Specific Asn-Pro-Phe-containing Partners*

    PubMed Central

    Kieken, Fabien; Sharma, Mahak; Jović, Marko; Giridharan, Sai Srinivas Panapakkam; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

    2010-01-01

    Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues “fine tune” the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs. PMID:20106972

  18. Structural Aspects of N-Glycosylations and the C-terminal Region in Human Glypican-1*

    PubMed Central

    Awad, Wael; Adamczyk, Barbara; Örnros, Jessica; Karlsson, Niclas G.; Mani, Katrin; Logan, Derek T.

    2015-01-01

    Glypicans are multifunctional cell surface proteoglycans involved in several important cellular signaling pathways. Glypican-1 (Gpc1) is the predominant heparan sulfate proteoglycan in the developing and adult human brain. The two N-linked glycans and the C-terminal domain that attach the core protein to the cell membrane are not resolved in the Gpc1 crystal structure. Therefore, we have studied Gpc1 using crystallography, small angle x-ray scattering, and chromatographic approaches to elucidate the composition, structure, and function of the N-glycans and the C terminus and also the topology of Gpc1 with respect to the membrane. The C terminus is shown to be highly flexible in solution, but it orients the core protein transverse to the membrane, directing a surface evolutionarily conserved in Gpc1 orthologs toward the membrane, where it may interact with signaling molecules and/or membrane receptors on the cell surface, or even the enzymes involved in heparan sulfate substitution in the Golgi apparatus. Furthermore, the N-glycans are shown to extend the protein stability and lifetime by protection against proteolysis and aggregation. PMID:26203194

  19. Regulation of synaptic structure by ubiquitin C-terminal hydrolase L1.

    PubMed

    Cartier, Anna E; Djakovic, Stevan N; Salehi, Afshin; Wilson, Scott M; Masliah, Eliezer; Patrick, Gentry N

    2009-06-17

    Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a deubiquitinating enzyme that is selectively and abundantly expressed in the brain, and its activity is required for normal synaptic function. Here, we show that UCH-L1 functions in maintaining normal synaptic structure in hippocampal neurons. We found that UCH-L1 activity is rapidly upregulated by NMDA receptor activation, which leads to an increase in the levels of free monomeric ubiquitin. Conversely, pharmacological inhibition of UCH-L1 significantly reduces monomeric ubiquitin levels and causes dramatic alterations in synaptic protein distribution and spine morphology. Inhibition of UCH-L1 activity increases spine size while decreasing spine density. Furthermore, there is a concomitant increase in the size of presynaptic and postsynaptic protein clusters. Interestingly, however, ectopic expression of ubiquitin restores normal synaptic structure in UCH-L1-inhibited neurons. These findings point to a significant role of UCH-L1 in synaptic remodeling, most likely by modulating free monomeric ubiquitin levels in an activity-dependent manner.

  20. HGF signaling regulates Claudin-3 dynamics through its C-terminal tyrosine residues.

    PubMed

    Twiss, Floor; Oldenkamp, Michiel; Hiemstra, Annemieke; Zhou, Houjiang; Matheron, Lucrèce; Mohammed, Shabaz; de Rooij, Johan

    2013-10-01

    The hormone HGF regulates morphogenesis and regeneration of multiple organs and increased HGF signaling is strongly associated with metastatic cancer. At the cellular level, one of the distinct effects of HGF is the de-stabilization of cell-cell junctions. Several molecular mechanisms have been shown to be involved that mostly culminate at the E-cadherin adhesion complex. One of the key determinants in HGF-driven morphological changes is the actomyosin cytoskeleton whose organization and physical parameters changes upon stimulation. Here we have investigated how HGF affects the different actomyosin-associated cell-cell junction complexes, Nectin Junctions, Adherens Junctions and Tight Junctions in MDCK cells. We find that components of all complexes stay present at cell-cell contacts until their physical dissociation. We find that at cell-cell junctions, the mobility of Claudin-3, but not that of other cell-cell adhesion receptors, is affected by HGF. This depends on tyrosine residues that likely affect PDZ-domain interactions at the C-terminal tail of Claudin-3, although their phosphorylation is not directly regulated by HGF. Thus we uncovered Claudins as novel targets of HGF signaling at cell-cell junctions.

  1. Design, Synthesis and Biological Evaluation of Biphenylamide Derivatives as Hsp90 C-terminal Inhibitors

    PubMed Central

    Zhao, Huiping; Garg, Gaurav; Zhao, Jinbo; Moroni, Elisabetta; Girgis, Antwan; Franco, Lucas S.; Singh, Swapnil; Colombo, Giorgio; Blagg, Brian S. J.

    2015-01-01

    Modulation of Hsp90 C-terminal function represents a promising therapeutic approach for the treatment of cancer and neurodegenerative diseases. Current drug discovery efforts toward Hsp90 C-terminal inhibition focus on novobiocin, an antibiotic that was transformed into an Hsp90 inhibitor. Based on structural information obtained during the development of novobiocin derivatives and molecular docking studies, scaffolds containing a biphenyl moiety in lieu of the coumarin ring present in novobiocin were identified as new Hsp90 C-terminal inhibitors. Structure-activity relationship studies produced new derivatives that inhibit the proliferation of breast cancer cell lines at nanomolar concentrations, which corresponded directly with Hsp90 inhibition. PMID:25462258

  2. Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

    PubMed

    Striberny, Bernd; Melton, Anthony E; Schwacke, Rainer; Krause, Kirsten; Fischer, Karsten; Goertzen, Leslie R; Rashotte, Aaron M

    2017-02-01

    Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

  3. The C-terminal 42 residues of the Tula virus Gn protein regulate interferon induction.

    PubMed

    Matthys, Valery; Gorbunova, Elena E; Gavrilovskaya, Irina N; Pepini, Timothy; Mackow, Erich R

    2011-05-01

    Hantaviruses primarily infect the endothelial cell lining of capillaries and cause two vascular permeability-based diseases. The ability of pathogenic hantaviruses to regulate the early induction of interferon determines whether hantaviruses replicate in endothelial cells. Tula virus (TULV) and Prospect Hill virus (PHV) are hantaviruses which infect human endothelial cells but fail to cause human disease. PHV is unable to inhibit early interferon (IFN) responses and fails to replicate within human endothelial cells. However, TULV replicates successfully in human endothelial cells, suggesting that TULV is capable of regulating cellular IFN responses. We observed a >300-fold reduction in the IFN-stimulated genes (ISGs) MxA and ISG56 following TULV versus PHV infection of endothelial cells 1 day postinfection. Similar to results with pathogenic hantaviruses, expressing the TULV Gn protein cytoplasmic tail (Gn-T) blocked RIG-I- and TBK1-directed transcription from IFN-stimulated response elements (ISREs) and IFN-β promoters (>90%) but not transcription directed by constitutively active IFN regulatory factor-3 (IRF3). In contrast, expressing the PHV Gn-T had no effect on TBK1-induced transcriptional responses. Analysis of Gn-T truncations demonstrated that the C-terminal 42 residues of the Gn-T (Gn-T-C42) from TULV, but not PHV, inhibited IFN induction >70%. These findings demonstrate that the TULV Gn-T inhibits IFN- and ISRE-directed responses upstream of IRF3 at the level of the TBK1 complex and further define a 42-residue domain of the TULV Gn-T that inhibits IFN induction. In contrast to pathogenic hantavirus Gn-Ts, the TULV Gn-T lacks a C-terminal degron domain and failed to bind tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), a TBK1 complex component required for IRF3 activation. These findings indicate that the nonpathogenic TULV Gn-T regulates IFN induction but accomplishes this via unique interactions with cellular TBK1 complexes. These

  4. Solution structure and dynamics of C-terminal regulatory domain of Vibrio vulnificus extracellular metalloprotease

    SciTech Connect

    Yun, Ji-Hye; Kim, Heeyoun; Park, Jung Eun; Lee, Jung Sup; Lee, Weontae

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer We have determined solution structures of vEP C-terminal regulatory domain. Black-Right-Pointing-Pointer vEP C-ter100 has a compact {beta}-barrel structure with eight anti-parallel {beta}-strands. Black-Right-Pointing-Pointer Solution structure of vEP C-ter100 shares its molecular topology with that of the collagen-binding domain of collagenase. Black-Right-Pointing-Pointer Residues in the {beta}3 region of vEP C-ter100 might be important in putative ligand/receptor binding. Black-Right-Pointing-Pointer vEP C-ter100 interacts strongly with iron ion. -- Abstract: An extracellular metalloprotease (vEP) secreted by Vibrio vulnificus ATCC29307 is a 45-kDa proteolytic enzyme that has prothrombin activation and fibrinolytic activities during bacterial infection. The action of vEP could result in clotting that could serve to protect the bacteria from the host defense machinery. Very recently, we showed that the C-terminal propeptide (C-ter100), which is unique to vEP, is involved in regulation of vEP activity. To understand the structural basis of this function of vEP C-ter100, we have determined the solution structure and backbone dynamics using multidimensional nuclear magnetic resonance spectroscopy. The solution structure shows that vEP C-ter100 is composed of eight anti-parallel {beta}-strands with a unique fold that has a compact {beta}-barrel formation which stabilized by hydrophobic and hydrogen bonding networks. Protein dynamics shows that the overall structure, including loops, is very rigid and stabilized. By structural database analysis, we found that vEP C-ter100 shares its topology with that of the collagen-binding domain of collagenase, despite low sequence homology between the two domains. Fluorescence assay reveals that vEP C-ter100 interacts strongly with iron (Fe{sup 3+}). These findings suggest that vEP protease might recruit substrate molecules, such as collagen, by binding at C-ter100 and that vEP participates

  5. The β(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail.

    PubMed

    Rebbeck, Robyn T; Karunasekara, Yamuna; Gallant, Esther M; Board, Philip G; Beard, Nicole A; Casarotto, Marco G; Dulhunty, Angela F

    2011-02-16

    Although it has been suggested that the C-terminal tail of the β(1a) subunit of the skeletal dihyropyridine receptor (DHPR) may contribute to voltage-activated Ca(2+) release in skeletal muscle by interacting with the skeletal ryanodine receptor (RyR1), a direct functional interaction between the two proteins has not been demonstrated previously. Such an interaction is reported here. A peptide with the sequence of the C-terminal 35 residues of β(1a) bound to RyR1 in affinity chromatography. The full-length β(1a) subunit and the C-terminal peptide increased [(3)H]ryanodine binding and RyR1 channel activity with an AC(50) of 450-600 pM under optimal conditions. The effect of the peptide was dependent on cytoplasmic Ca(2+), ATP, and Mg(2+) concentrations. There was no effect of the peptide when channel activity was very low as a result of Mg(2+) inhibition or addition of 100 nM Ca(2+) (without ATP). Maximum increases were seen with 1-10 μM Ca(2+), in the absence of Mg(2+) inhibition. A control peptide with the C-terminal 35 residues in a scrambled sequence did not bind to RyR1 or alter [(3)H]ryanodine binding or channel activity. This high-affinity in vitro functional interaction between the C-terminal 35 residues of the DHPR β(1a) subunit and RyR1 may support an in vivo function of β(1a) during voltage-activated Ca(2+) release.

  6. Solid phase synthesis of a GHRP analog containing C-terminal thioamide group.

    PubMed

    Majer, Z; Zewdu, M; Hollósi, M; Sepródi, J; Vadász, Z; Teplán, I

    1988-02-15

    [Lyst6]GHRP, the C-terminally thionated analog of the highly potent growth hormone releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 was prepared by using solid support. The success of the synthesis showed that Lawesson's reagent can be used for selective thionation of an amide group not only in solution but also on the surface of a resin. The C-terminal thioamide group proved to be stable under the conditions of the solid phase synthesis.

  7. C-Terminal Modification of Fully Unprotected Peptide Hydrazides via in Situ Generation of Isocyanates.

    PubMed

    Vinogradov, Alexander A; Simon, Mark D; Pentelute, Bradley L

    2016-03-18

    A method for chemo- and regioselective conjugation of nucleophiles to fully unprotected peptides and proteins via in situ generation of C-terminal isocyanates is reported. Oxidation of C-terminal peptide hydrazides in aqueous media followed by Curtius rearrangement of acyl azides reliably generates isocyanates, which react with a variety of external nucleophiles, such as hydrazines, hydrazides, aromatic thiols, and hydroxylamines. Multiple peptides and a 53 kDa protein hydrazide were conjugated to different nucleophiles using this reaction.

  8. The activity of protein phosphatase 5 towards native clients is modulated by the middle- and C-terminal domains of Hsp90

    PubMed Central

    Haslbeck, Veronika; Eckl, Julia M.; Drazic, Adrian; Rutz, Daniel A.; Lorenz, Oliver R.; Zimmermann, Kerstin; Kriehuber, Thomas; Lindemann, Claudia; Madl, Tobias; Richter, Klaus

    2015-01-01

    Protein phosphatase 5 is involved in the regulation of kinases and transcription factors. The dephosphorylation activity is modulated by the molecular chaperone Hsp90, which binds to the TPR-domain of protein phosphatase 5. This interaction is dependent on the C-terminal MEEVD motif of Hsp90. We show that C-terminal Hsp90 fragments differ in their regulation of the phosphatase activity hinting to a more complex interaction. Also hydrodynamic parameters from analytical ultracentrifugation and small-angle X-ray scattering data suggest a compact structure for the Hsp90-protein phosphatase 5 complexes. Using crosslinking experiments coupled with mass spectrometric analysis and structural modelling we identify sites, which link the middle/C-terminal domain interface of C. elegans Hsp90 to the phosphatase domain of the corresponding kinase. Studying the relevance of the domains of Hsp90 for turnover of native substrates we find that ternary complexes with the glucocorticoid receptor (GR) are cooperatively formed by full-length Hsp90 and PPH-5. Our data suggest that the direct stimulation of the phosphatase activity by C-terminal Hsp90 fragments leads to increased dephosphorylation rates. These are further modulated by the binding of clients to the N-terminal and middle domain of Hsp90 and their presentation to the phosphatase within the phosphatase-Hsp90 complex. PMID:26593036

  9. Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

    PubMed Central

    Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

    2014-01-01

    The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

  10. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension.

    PubMed

    Kataoka, T; Holler, N; Micheau, O; Martinon, F; Tinel, A; Hofmann, K; Tschopp, J

    2001-06-01

    The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. No interaction of Bcl-rambo with either anti-apoptotic (Bcl-2, Bcl-x(L), Bcl-w, A1, MCL-1, E1B-19K, and BHRF1) or pro-apoptotic (Bax, Bak, Bik, Bid, Bim, and Bad) members of the Bcl-2 family was observed. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bcl-rambo constitutes a novel type of pro-apoptotic Bcl-2 member that triggers cell death independently of its BH motifs.

  11. Cloning and Characterization of a 7 Transmembrane Receptor from the Adherent Cells of Chicken Peripheral Blood Mononuclear Cells

    PubMed Central

    Chen, Yu San; Wu, Hsing Chieh; Shien, Jui Hung; Chiu, Hua Hsien; Lee, Long Huw

    2014-01-01

    A cDNA encoding a 7 transmembrane (7TM) receptor gene from the adherent cells of chicken peripheral blood mononuclear cells (PBMC) was cloned and characterized. The open reading frame of the chicken-7TM (Ch-7TM) receptor gene was 1008 nucleotides long, encoding a protein of 335 amino acid residues with a molecular mass of approximately 37.1 kDa. Hydrophobic stretches indicated the presence of 7 TM domains. Moreover, the complete nucleotide sequences encoding 7TM of duck (Du-7TM) and goose (Go-7TM), corresponding to the open reading frame of Ch-7TM, were determined. Each of the Du- and Go-7TM encoding regions comprised 990 nucleotides, representing an 18-nucleotide deletion in alignment with the Ch-7TM encoding region, resulting in a 6-amino-acid deletion at the 3′-end. No signal peptides were predicted. Six phosphorylation sites were predicted and conserved for all three 7TMs. The proteins of the three 7TMs were similar, with 11 conserved cysteine residues. No glycosylation sites could be predicted. The results of the pairwise comparisons indicated that the Ch-7TM encoding region and Ch-7TM protein were the least similar to those of Du- and Go-7TMs. These results were in accordance with those of the phylogenetic analysis, which indicated that the Du- and Go-7TM encoding regions clustered, but were separated from the Ch-7TM encoding region. Monoclonal antibody B28D5 was prepared from spleens of mice immunized with the bacterially expressed N-terminal (55 amino acid residues) region of the Ch-7TM protein for further use. Double staining with B28D5 and KUL01 suggested that Ch-7TM was expressed in subsets of the adherent cells, among which a subset that was recognized with both antibodies was likely of monocyte and macrophage lineage. However, the fluorescence intensities of B28D5 and, particularly, KUL01 decreased after the adherent cells were incubated for additional 48 h. PMID:24466279

  12. The Impact of the Human DNA Topoisomerase II C-Terminal Domain on Activity

    PubMed Central

    Meczes, Emma L.; Gilroy, Kathryn L.; West, Katherine L.; Austin, Caroline A.

    2008-01-01

    Background Type II DNA topoisomerases (topos) are essential enzymes needed for the resolution of topological problems that occur during DNA metabolic processes. Topos carry out an ATP-dependent strand passage reaction whereby one double helix is passed through a transient break in another. Humans have two topoII isoforms, α and β, which while enzymatically similar are differentially expressed and regulated, and are thought to have different cellular roles. The C-terminal domain (CTD) of the enzyme has the most diversity, and has been implicated in regulation. We sought to investigate the impact of the CTD domain on activity. Methodology/Principle Findings We have investigated the role of the human topoII C-terminal domain by creating constructs encoding C-terminally truncated recombinant topoIIα and β and topoIIα+β-tail and topoIIβ+α-tail chimeric proteins. We then investigated function in vivo in a yeast system, and in vitro in activity assays. We find that the C-terminal domain of human topoII isoforms is needed for in vivo function of the enzyme, but not needed for cleavage activity. C-terminally truncated enzymes had similar strand passage activity to full length enzymes, but the presence of the opposite C-terminal domain had a large effect, with the topoIIα-CTD increasing activity, and the topoIIβ-CTD decreasing activity. Conclusions/Significance In vivo complementation data show that the topoIIα C-terminal domain is needed for growth, but the topoIIβ isoform is able to support low levels of growth without a C-terminal domain. This may indicate that topoIIβ has an additional localisation signal. In vitro data suggest that, while the lack of any C-terminal domain has little effect on activity, the presence of either the topoIIα or β C-terminal domain can affect strand passage activity. Data indicates that the topoIIβ-CTD may be a negative regulator. This is the first report of in vitro data with chimeric human topoIIs. PMID:18335031

  13. C-terminal calcitonin gene-related peptide fragments and vasopressin but not somatostatin-28 induce miosis in monkeys.

    PubMed

    Almegård, B; Bill, A

    1993-11-30

    The miotic effects of C-terminal calcitonin gene-related peptide (CGRP) fragments, somatostatin-28 and vasopressin have been evaluated with special attention being paid to possible interactions with cholecystokinin (CCK)A receptors. The peptides were injected intracamerally to anesthetized monkeys pretreated with indomethacin and atropine. CGRP-(32-37) induced a miosis with a potency 1000 times lower than that previously found with sulphated CCK-8. Two other fragments, CGRP-(30-37) and CGRP-(31-37), also had miotic properties. The CGRP-(32-37)-induced miosis was antagonized by the CCKA receptor antagonist loxiglumide. No contractile effect was elicited by 67 pmol-7.4 nmol somatostatin-28. Vasopressin (360 pmol) caused a small reduction in pupil size. Loxiglumide pretreatment did not affect the reduction in pupil size but a vasopressin receptor antagonist partly inhibited the response. The results indicate that CGRP-(32-37) is a miotic with low potency but high efficacy in the monkey eye, probably interacting with CCKA receptors, and that vasopressin is a mitotic with low potency and efficacy, probably acting via vasopressin receptors.

  14. Detection of prosecretory mitogen lacritin in nonprimate tears primarily as a C-terminal-like fragment.

    PubMed

    Laurie, Diane E; Splan, Rebecca K; Green, Kari; Still, Katherine M; McKown, Robert L; Laurie, Gordon W

    2012-09-12

    Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal-reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair.

  15. Regulatory function of the C-terminal segment of guanylate cyclase-activating protein 2.

    PubMed

    Zernii, Evgeni Yu; Grigoriev, Ilya I; Nazipova, Aliya A; Scholten, Alexander; Kolpakova, Tatiana V; Zinchenko, Dmitry V; Kazakov, Alexey S; Senin, Ivan I; Permyakov, Sergei E; Dell'Orco, Daniele; Philippov, Pavel P; Koch, Karl-W

    2015-10-01

    Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases. Copyright © 2015 Elsevier B.V. All rights

  16. Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

    PubMed Central

    Volkmann, Gerrit; Liu, Xiang-Qin

    2009-01-01

    Background Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. Methodology A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. Conclusions We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups. PMID:20027230

  17. Structural characterization of two pore-forming peptides: consequences of introducing a C-terminal tryptophan.

    PubMed

    Herrera, Alvaro I; Al-Rawi, Ahlam; Cook, Gabriel A; Gao, Jian; Iwamoto, Takeo; Prakash, Om; Tomich, John M; Chen, Jianhan

    2010-08-01

    Synthetic channel-forming peptides that can restore chloride conductance across epithelial membranes could provide a novel treatment of channelopathies such as cystic fibrosis. Among a series of 22-residue peptides derived from the second transmembrane segment of the glycine receptor alpha(1)-subunit (M2GlyR), p22-S22W (KKKKP ARVGL GITTV LTMTT QW) is particularly promising with robust membrane insertion and assembly. The concentration to reach one-half maximal short circuit current is reduced to 45 +/- 6 microM from that of 210 +/- 70 microM of peptide p22 (KKKKP ARVGL GITTV LTMTT QS). However, this is accompanied with nearly 50% reduction in conductance. Toward obtaining a molecular level understanding of the channel activities, we combine information from solution NMR, existing biophysical data, and molecular modeling to construct atomistic models of the putative pentameric channels of p22 and p22-S22W. Simulations in membrane bilayers demonstrate that these structural models, even though highly flexible, are stable and remain adequately open for ion conductance. The membrane-anchoring tryptophan residues not only rigidify the whole channel, suggesting increased stability, but also lead to global changes in the pore profile. Specifically, the p22-S22W pore has a smaller opening on average, consistent with lower measured conductance. Direct observation of several incidences of chloride transport suggests several qualitative features of how these channels might selectively conduct anions. The current study thus helps to rationalize the functional consequences of introducing a single C-terminal tryptophan. Availability of these structural models also paves the way for future work to rationally modify and improve M2GlyR-derived peptides toward potential peptide-based channel replacement therapy.

  18. PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch.

    PubMed

    Espejo, Alexsandra B; Gao, Guozhen; Black, Karynne; Gayatri, Sitaram; Veland, Nicolas; Kim, Jeesun; Chen, Taiping; Sudol, Marius; Walker, Cheryl; Bedford, Mark T

    2017-02-10

    PRMT5 is the primary enzyme responsible for the deposition of the symmetric dimethylarginine in mammalian cells. In an effort to understand how PRMT5 is regulated, we identified a threonine phosphorylation site within a C-terminal tail motif, which is targeted by the Akt/serum- and glucocorticoid-inducible kinases. While investigating the function of this posttranslational modification, we serendipitously discovered that its free C-terminal tail binds PDZ domains (when unphosphorylated) and 14-3-3 proteins (when phosphorylated). In essence, a phosphorylation event within the last few residues of the C-terminal tail generates a posttranslational modification-dependent PDZ/14-3-3 interaction "switch." The C-terminal motif of PRMT5 is required for plasma membrane association, and loss of this switching capacity is not compatible with life. This signaling phenomenon was recently reported for the HPV E6 oncoprotein but has not yet been observed for mammalian proteins. To investigate the prevalence of PDZ/14-3-3 switching in signal transduction, we built a protein domain microarray that harbors PDZ domains and 14-3-3 proteins. We have used this microarray to interrogate the C-terminal tails of a small group of candidate proteins and identified ERBB4, PGHS2, and IRK1 (as well as E6 and PRMT5) as conforming to this signaling mode, suggesting that PDZ/14-3-3 switching may be a broad biological paradigm.

  19. Structural function of C-terminal amidation of endomorphin. Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid, studied by 1H-NMR spectroscopy, molecular calculation, and X-ray crystallography.

    PubMed

    In, Yasuko; Minoura, Katsuhiko; Tomoo, Koji; Sasaki, Yusuke; Lazarus, Lawrence H; Okada, Yoshio; Ishida, Toshimasa

    2005-10-01

    To investigate the structural function of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH(2)), an endogenous micro-opioid receptor ligand, the solution conformations of EM2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH) in TFE (trifluoroethanol), water (pH 2.7 and 5.2), and aqueous DPC (dodecylphosphocholine) micelles (pH 3.5 and 5.2) were investigated by the combination of 2D (1)H-NMR measurement and molecular modelling calculation. Both peptides were in equilibrium between the cis and trans rotamers around the Tyr--Pro w bond with population ratios of 1 : 1 to 1 : 2 in dimethyl sulfoxide, TFE and water, whereas they predominantly took the trans rotamer in DPC micelle, except in EM2OH at pH 5.2, which had a trans/cis rotamer ratio of 2 : 1. Fifty possible 3D conformers were generated for each peptide, taking different electronic states depending on the type of solvent and pH (neutral and monocationic forms for EM2, and zwitterionic and monocation forms for EM2OH) by the dynamical simulated annealing method, under the proton-proton distance constraints derived from the ROE cross-peak intensities. These conformers were then roughly classified into four groups of two open [reverse S (rS)- and numerical 7 (n7)-type] and two folded (F1- and F2-type) conformers according to the conformational pattern of the backbone structure. Most EM2 conformers in neutral (in TFE) and monocationic (in water and DPC micelles) forms adopted the open structure (mixture of major rS-type and minor n7-type conformers) despite the trans/cis rotamer form. On the other hand, the zwitterionic EM2OH in TFE, water and DPC micelles showed an increased population of F1- and F2-type folded conformers, the population of which varied depending on their electronic state and pH. Most of these folded conformers took an F1-type structure similar to that stabilized by an intramolecular hydrogen bond of (Tyr1)NH(3) (+)...COO(-)(Phe4), observed in its crystal structure

  20. The VSG C-terminal domain is inaccessible to antibodies on live trypanosomes.

    PubMed

    Schwede, Angela; Jones, Nicola; Engstler, Markus; Carrington, Mark

    2011-02-01

    In the mammalian host, the Trypanosoma brucei cell surface is covered with a densely packed protein coat of a single protein, the variant surface glycoprotein (VSG). The VSG is believed to shield invariant surface proteins from host antibodies but there is limited information on how far antibodies can penetrate into the VSG monolayer. Here, the VSG surface coat was probed to determine whether it acts as a barrier to binding of antibodies to the membrane proximal VSG C-terminal domain. The binding of C-terminal domain antibodies to VSG221 or VSG118 was compared with antibodies recognising the cognate whole VSGs. The C-terminal VSG domain was inaccessible to antibodies on live cells but not on fixed cells. This provides further evidence that the VSG coat acts as a barrier and protects the cell from antibodies that would otherwise bind to some of the other externally disposed proteins.

  1. Evolutionary bridges to new protein folds: design of C-terminal Cro protein chameleon sequences

    PubMed Central

    Anderson, William J.; Van Dorn, Laura O.; Ingram, Wendy M.; Cordes, Matthew H. J.

    2011-01-01

    Regions of amino-acid sequence that are compatible with multiple folds may facilitate evolutionary transitions in protein structure. In a previous study, we described a heuristically designed chameleon sequence (SASF1, structurally ambivalent sequence fragment 1) that could adopt either of two naturally occurring conformations (α-helical or β-sheet) when incorporated as part of the C-terminal dimerization subdomain of two structurally divergent transcription factors, P22 Cro and λ Cro. Here we describe longer chameleon designs (SASF2 and SASF3) that in the case of SASF3 correspond to the full C-terminal half of the ordered region of a P22 Cro/λ Cro sequence alignment (residues 34–57). P22-SASF2 and λWDD-SASF2 show moderate thermal stability in denaturation curves monitored by circular dichroism (Tm values of 46 and 55°C, respectively), while P22-SASF3 and λWDD-SASF3 have somewhat reduced stability (Tm values of 33 and 49°C, respectively). 13C and 1H NMR secondary chemical shift analysis confirms two C-terminal α-helices for P22-SASF2 (residues 36–45 and 54–57) and two C-terminal β-strands for λWDD-SASF2 (residues 40–45 and 50–52), corresponding to secondary structure locations in the two parent sequences. Backbone relaxation data show that both chameleon sequences have a relatively well-ordered structure. Comparisons of 15N-1H correlation spectra for SASF2 and SASF3-containing proteins strongly suggest that SASF3 retains the chameleonism of SASF2. Both Cro C-terminal conformations can be encoded in a single sequence, showing the plausibility of linking different Cro folds by smooth evolutionary transitions. The N-terminal subdomain, though largely conserved in structure, also exerts an important contextual influence on the structure of the C-terminal region. PMID:21676898

  2. Detection of Prosecretory Mitogen Lacritin in Nonprimate Tears Primarily as a C-Terminal-Like Fragment

    PubMed Central

    Laurie, Diane E.; Splan, Rebecca K.; Green, Kari; Still, Katherine M.; McKown, Robert L.; Laurie, Gordon W.

    2012-01-01

    Purpose. Lacritin is a human tear glycoprotein that promotes basal tear protein secretion in cultured rat lacrimal acinar cells and proliferation of subconfluent human corneal epithelial cells. When topically added to rabbit eyes, lacritin promotes basal tearing. Despite these activities on several species, lacritin's presence in nonprimate tears or other tissues has not been explored. Here we probed for lacritin in normal horse tears. Methods. Sequences were collected from the Ensembl genomic alignment of human LACRT gene with high-quality draft horse genome (EquCab2.0) and analyzed. Normal horse tears were collected and assayed by Western blotting, ELISA, and mass spectrometry. Newly generated rabbit antibodies, respectively, against N- and C-terminal regions of human lacritin were employed. Results. Identity was 75% and 45%, respectively, at nucleotide and protein levels. Structural features were conserved, including a C-terminal amphipathic α-helix. Anti-C-terminal antibodies strongly detected a ∼13 kDa band in horse tears that was validated by mass spectrometry. In human tears, the same antibody detected uncleaved lacritin (∼24 kDa) strongly and C-terminal fragments of ∼13 and ∼11 kDa weakly. Anti-N-terminal antibodies were slightly reactive with a ∼24 kDa horse antigen and showed no reaction with the anti-C-terminal–reactive ∼13 kDa species. Similar respective levels of horse C-terminal versus N-terminal immunoreactivity were apparent by ELISA. Conclusions. Lacritin is present in horse tears, largely as a C-terminal fragment homologous to the mitogenic and bactericidal region in human lacritin, suggesting potential benefit in corneal wound repair. PMID:22871838

  3. C-terminal constrained phenylalanine as a pharmacophoric unit in peptide-based proteasome inhibitors.

    PubMed

    Baldisserotto, Anna; Marastoni, Mauro; Lazzari, Ilaria; Trapella, Claudio; Gavioli, Riccardo; Tomatis, Roberto

    2008-07-01

    Here we report the synthesis and biological properties of peptide-based molecules bearing constrained analogues of phenylalanine at the C-terminal. Compounds were tested as proteasome subunits' inhibitors. Dehydro-peptides showed good inhibition, in particular against trypsin-like (T-L) proteasome activity while some C-terminal Tic-derivatives inhibit only caspase-like activity in enzymatic beta1 subunits with a certain degree of efficacy. The best analogues of the series demonstrated good resistance to proteolysis and a capacity to permeate the cell membrane.

  4. Specific inhibition of AGC protein kinases by antibodies against C-terminal epitopes.

    PubMed

    Traincard, François; Giacomoni, Véronique; Veron, Michel

    2004-08-13

    The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.

  5. Crystal Structure of the C-terminal Region of Streptococcus mutans Antigen I/II and Characterization of Salivary Agglutinin Adherence Domains

    SciTech Connect

    Larson, Matthew R.; Rajashankar, Kanagalaghatta R.; Crowley, Paula J.; Kelly, Charles; Mitchell, Tim J.; Brady, L. Jeannine; Deivanayagam, Champion

    2012-05-29

    The Streptococcus mutans antigen I/II (AgI/II) is a cell surface-localized protein that adheres to salivary components and extracellular matrix molecules. Here we report the 2.5 {angstrom} resolution crystal structure of the complete C-terminal region of AgI/II. The C-terminal region is comprised of three major domains: C{sub 1}, C{sub 2}, and C{sub 3}. Each domain adopts a DE-variant IgG fold, with two {beta}-sheets whose A and F strands are linked through an intramolecular isopeptide bond. The adherence of the C-terminal AgI/II fragments to the putative tooth surface receptor salivary agglutinin (SAG), as monitored by surface plasmon resonance, indicated that the minimal region of binding was contained within the first and second DE-variant-IgG domains (C{sub 1} and C{sub 2}) of the C terminus. The minimal C-terminal region that could inhibit S. mutans adherence to SAG was also confirmed to be within the C{sub 1} and C{sub 2} domains. Competition experiments demonstrated that the C- and N-terminal regions of AgI/II adhere to distinct sites on SAG. A cleft formed at the intersection between these C{sub 1} and C{sub 2} domains bound glucose molecules from the cryo-protectant solution, revealing a putative binding site for its highly glycosylated receptor SAG. Finally, electron microscopy images confirmed the elongated structure of AgI/II and enabled building a composite tertiary model that encompasses its two distinct binding regions.

  6. C-Terminal Region of MAP7 Domain Containing Protein 3 (MAP7D3) Promotes Microtubule Polymerization by Binding at the C-Terminal Tail of Tubulin

    PubMed Central

    Yadav, Saroj; Verma, Paul J.; Panda, Dulal

    2014-01-01

    MAP7 domain containing protein 3 (MAP7D3), a newly identified microtubule associated protein, has been shown to promote microtubule assembly and stability. Its microtubule binding region has been reported to consist of two coiled coil motifs located at the N-terminus. It possesses a MAP7 domain near the C-terminus and belongs to the microtubule associated protein 7 (MAP7) family. The MAP7 domain of MAP7 protein has been shown to bind to kinesin-1; however, the role of MAP7 domain in MAP7D3 remains unknown. Based on the bioinformatics analysis of MAP7D3, we hypothesized that the MAP7 domain of MAP7D3 may have microtubule binding activity. Indeed, we found that MAP7 domain of MAP7D3 bound to microtubules as well as enhanced the assembly of microtubules in vitro. Interestingly, a longer fragment MDCT that contained the MAP7 domain (MD) with the C-terminal tail (CT) of the protein promoted microtubule polymerization to a greater extent than MD and CT individually. MDCT stabilized microtubules against dilution induced disassembly. MDCT bound to reconstituted microtubules with an apparent dissociation constant of 3.0±0.5 µM. An immunostaining experiment showed that MDCT localized along the length of the preassembled microtubules. Competition experiments with tau indicated that MDCT shares its binding site on microtubules with tau. Further, we present evidence indicating that MDCT binds to the C-terminal tail of tubulin. In addition, MDCT could bind to tubulin in HeLa cell extract. Here, we report a microtubule binding region in the C-terminal region of MAP7D3 that may have a role in regulating microtubule assembly dynamics. PMID:24927501

  7. Distinct roles for the N- and C-terminal regions in the cytotoxicity of pierisin-1, a putative ADP-ribosylating toxin from cabbage butterfly, against mammalian cells

    PubMed Central

    Kanazawa, Takashi; Watanabe, Masahiko; Matsushima-Hibiya, Yuko; Kono, Takuo; Tanaka, Noriaki; Koyama, Kotaro; Sugimura, Takashi; Wakabayashi, Keiji

    2001-01-01

    Pierisin-1 is an 850-aa cytotoxic protein found in the cabbage butterfly, Pieris rapae, and has been suggested to consist of an N-terminal region with ADP-ribosyltransferase domain and of a C-terminal region that might have a receptor-binding domain. To elucidate the role of each region, we investigated the functions of various fragments of pierisin-1. In vitro expressed polypeptide consisting of amino acid residues 1–233 or 234–850 of pierisin-1 alone did not show cytotoxicity against human cervical carcinoma HeLa cells. However, the presence of both polypeptides in the culture medium showed some of the original cytotoxic activity. Introduction of the N-terminal polypeptide alone by electroporation also induced cell death in HeLa cells, and even in the mouse melanoma MEB4 cells insensitive to pierisin-1. Thus, the N-terminal region has a principal role in the cytotoxicity of pierisin-1 inside mammalian cells. Analyses of incorporated pierisin-1 indicated that the entire protein, regardless of whether it consisted of a single polypeptide or two separate N- and C-terminal polypeptides, was incorporated into HeLa cells. However, neither of the terminal polypeptides was incorporated when each polypeptide was present separately. These findings indicate that the C-terminal region is important for the incorporation of pierisin-1. Moreover, presence of receptor for pierisin-1 in the lipid fraction of cell membrane was suggested. The cytotoxic effects of pierisin-1 were enhanced by previous treatment with trypsin, producing “nicked” pierisin-1. Generation of the N-terminal fragment in HeLa cells was detected after application of intact entire molecule of pierisin-1. From the above observations, it is suggested that after incorporation of pierisin-1 into the cell by interaction of its C-terminal region with the receptor in the cell membrane, the entire protein is cleaved into the N- and C-terminal fragments with intracellular protease, and the N-terminal fragment

  8. Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.

    PubMed

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-11-26

    NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

  9. Alpha-A crystallin: quantitation of C-terminal modification during lens aging

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

  10. Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

    PubMed Central

    Reulen, Sanne WA; van Baal, Ingrid; Raats, Jos MH; Merkx, Maarten

    2009-01-01

    Background Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging. Results Targeting to the periplasm of E. coli allowed efficient production of correctly-folded single-domain antibody (sdAb)-intein fusions proteins. On column purification and 2-mercapthoethanesulfonic acid (MESNA)-induced cleavage yielded single-domain antibodies with a reactive C-terminal MESNA thioester in good yields. These thioester-functionalized single-domain antibodies allowed synthesis of immunomicelles via native chemical ligation in a single step. Conclusion A novel procedure was developed to obtain soluble, well-folded single-domain antibodies with reactive C-terminal thioesters in good yields. These proteins are promising building blocks for the chemoselective functionalization via NCL of a broad range of nanoparticle scaffolds, including micelles, liposomes and dendrimers. PMID:19619333

  11. Alpha-A crystallin: quantitation of C-terminal modification during lens aging

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Gopalakrishnan, S.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

  12. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    USDA-ARS?s Scientific Manuscript database

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  13. Mutational and Functional Analysis of the C-Terminal Region of the C3H Mouse Mammary Tumor Virus Superantigen

    PubMed Central

    Wrona, Thomas J.; Lozano, Mary; Binhazim, Awadh A.; Dudley, Jaquelin P.

    1998-01-01

    The mouse mammary tumor virus (MMTV) encodes within the U3 region of the long terminal repeat (LTR) a protein known as the superantigen (Sag). Sag is needed for the efficient transmission of milk-borne virus from the gut to target tissue in the mammary gland. MMTV-infected B cells in the gut express Sag as a type II transmembrane protein that is recognized by the variable region of particular beta chains (Vβ) of the T-cell receptor (TCR) on the surface of T cells. Recognition of Sag by particular TCRs results in T-cell stimulation, release of cytokines, and amplification of MMTV infection in lymphoid cells that are needed for infection of adolescent mammary tissue. Because the C-terminal 30 to 40 amino acids of Sag are variable and correlate with recognition of particular TCR Vβ chains, we prepared a series of C-terminal Sag mutations that were introduced into a cloned infectious MMTV provirus. Virus-producing XC rat cells were used for injection of susceptible BALB/c mice, and these mice were monitored for functional Sag activity by the deletion of C3H MMTV Sag-reactive (CD4+ Vβ14+) T cells. Injected mice also were analyzed for mutant infection and tumor formation in mammary glands as well as milk-borne transmission of MMTV to offspring. Most mutations abrogated Sag function, although one mutation (HPA242) that changed the negative charge of the extreme C terminus to a positive charge created a weaker Sag that slowed the kinetics of Sag-mediated T-cell deletion. Despite the lack of Sag activity, many of the sag mutant viruses were capable of sporadic infections of the mammary glands of injected mice but not of offspring mice, indicating that functional Sag increases the probability of milk-borne MMTV infection. Furthermore, although most viruses encoding nonfunctional Sags were unable to cause mammary tumors, tumors were induced by such viruses carrying mutations in a negative regulatory element that overlaps the sag gene within the LTR, suggesting that loss of

  14. Predictors of intact and C-terminal fibroblast growth factor 23 in Gambian children

    PubMed Central

    Braithwaite, Vickie; Jones, Kerry S; Assar, Shima; Schoenmakers, Inez; Prentice, Ann

    2013-01-01

    Elevated C-terminal fibroblast growth factor 23 (C-FGF23) concentrations have been reported in Gambian children with and without putative Ca-deficiency rickets. The aims of this study were to investigate whether i) elevated C-FGF23 concentrations in Gambian children persist long term; ii) they are associated with higher intact FGF23 concentrations (I-FGF23), poor iron status and shorter 25-hydroxyvitamin D half-life (25OHD-t1/2); and iii) the persistence and predictors of elevated FGF23 concentrations differ between children with and without a history of rickets. Children (8–16 years, n=64) with a history of rickets and a C-FGF23 concentration >125 RU/ml (bone deformity (BD), n=20) and local community children with a previously measured elevated C-FGF23 concentration (LC+, n=20) or a previously measured C-FGF23 concentration within the normal range (LC−, n=24) participated. BD children had no remaining signs of bone deformities. C-FGF23 concentration had normalised in BD children, but remained elevated in LC+ children. All the children had I-FGF23 concentration within the normal range, but I-FGF23 concentration was higher and iron status poorer in LC+ children. 1,25-dihydroxyvitamin D was the strongest negative predictor of I-FGF23 concentration (R2=18%; P=0.0006) and soluble transferrin receptor was the strongest positive predictor of C-FGF23 concentration (R2=33%; P≤0.0001). C-FGF23 and I-FGF23 concentrations were poorly correlated with each other (R2=5.3%; P=0.07). 25OHD-t1/2 was shorter in BD children than in LC− children (mean (s.d.): 24.5 (6.1) and 31.5 (11.5) days respectively; P=0.05). This study demonstrated that elevated C-FGF23 concentrations normalised over time in Gambian children with a history of rickets but not in local children, suggesting a different aetiology; that children with resolved rickets had a shorter 25OHD-t1/2, suggesting a long-standing increased expenditure of 25OHD, and that iron deficiency is a predictor of elevated C

  15. C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.

    PubMed

    Li, Wenyi; O'Brien-Simpson, Neil M; Yao, Shenggen; Tailhades, Julien; Reynolds, Eric C; Dawson, Raymond M; Otvos, Laszlo; Hossain, Mohammed Akhter; Separovic, Frances; Wade, John D

    2017-01-05

    Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL(-1) ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action.

  16. Comparative Analysis of the Biochemical and Functional Properties of C-Terminal Domains of Autotransporters ▿

    PubMed Central

    Marín, Elvira; Bodelón, Gustavo; Fernández, Luis Ángel

    2010-01-01

    Autotransporters (ATs) are the largest group of proteins secreted by Gram-negative bacteria and include many virulence factors from human pathogens. ATs are synthesized as large precursors with a C-terminal domain that is inserted in the outer membrane (OM) and is essential for the translocation of an N-terminal passenger domain to the extracellular milieu. Several mechanisms have been proposed for AT secretion. Self-translocation models suggest transport across a hydrophilic channel formed by an internal pore of the β-barrel or by the oligomerization of C-terminal domains. Alternatively, an assisted-translocation model suggests that transport employs a conserved machinery of the bacterial OM such as the Bam complex. In this work we have investigated AT secretion by carrying out a comparative study to analyze the conserved biochemical and functional features of different C-terminal domains selected from ATs of gammaproteobacteria, betaproteobacteria, alphaproteobacteria, and epsilonproteobacteria. Our results indicate that C-terminal domains having an N-terminal α-helix and a β-barrel constitute functional transport units for the translocation of peptides and immunoglobulin domains with disulfide bonds. In vivo and in vitro analyses show that multimerization is not a conserved feature in AT C-terminal domains. Furthermore, we demonstrate that the deletion of the conserved α-helix severely impairs β-barrel folding and OM insertion and thereby blocks passenger domain secretion. These observations suggest that the AT β-barrel without its α-helix cannot form a stable hydrophilic channel in the OM for protein translocation. The implications of our data for an understanding of AT secretion are discussed. PMID:20802036

  17. Chemokine-Like Factor 1-Derived C-Terminal Peptides Induce the Proliferation of Dermal Microvascular Endothelial Cells in Psoriasis

    PubMed Central

    Tan, Yaqi; Wang, Yixuan; Li, Li; Xia, Jinyu; Peng, Shiguang; He, Yanling

    2015-01-01

    Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation. PMID:25915746

  18. Evolutionary bridges to new protein folds: design of C-terminal Cro protein chameleon sequences.

    PubMed

    Anderson, William J; Van Dorn, Laura O; Ingram, Wendy M; Cordes, Matthew H J

    2011-09-01

    Regions of amino-acid sequence that are compatible with multiple folds may facilitate evolutionary transitions in protein structure. In a previous study, we described a heuristically designed chameleon sequence (SASF1, structurally ambivalent sequence fragment 1) that could adopt either of two naturally occurring conformations (α-helical or β-sheet) when incorporated as part of the C-terminal dimerization subdomain of two structurally divergent transcription factors, P22 Cro and λ Cro. Here we describe longer chameleon designs (SASF2 and SASF3) that in the case of SASF3 correspond to the full C-terminal half of the ordered region of a P22 Cro/λ Cro sequence alignment (residues 34-57). P22-SASF2 and λ(WDD)-SASF2 show moderate thermal stability in denaturation curves monitored by circular dichroism (T(m) values of 46 and 55°C, respectively), while P22-SASF3 and λ(WDD)-SASF3 have somewhat reduced stability (T(m) values of 33 and 49°C, respectively). (13)C and (1)H NMR secondary chemical shift analysis confirms two C-terminal α-helices for P22-SASF2 (residues 36-45 and 54-57) and two C-terminal β-strands for λ(WDD)-SASF2 (residues 40-45 and 50-52), corresponding to secondary structure locations in the two parent sequences. Backbone relaxation data show that both chameleon sequences have a relatively well-ordered structure. Comparisons of (15)N-(1)H correlation spectra for SASF2 and SASF3-containing proteins strongly suggest that SASF3 retains the chameleonism of SASF2. Both Cro C-terminal conformations can be encoded in a single sequence, showing the plausibility of linking different Cro folds by smooth evolutionary transitions. The N-terminal subdomain, though largely conserved in structure, also exerts an important contextual influence on the structure of the C-terminal region.

  19. GBNV encoded movement protein (NSm) remodels ER network via C-terminal coiled coil domain

    SciTech Connect

    Singh, Pratibha; Savithri, H.S.

    2015-08-15

    Plant viruses exploit the host machinery for targeting the viral genome–movement protein complex to plasmodesmata (PD). The mechanism by which the non-structural protein m (NSm) of Groundnut bud necrosis virus (GBNV) is targeted to PD was investigated using Agrobacterium mediated transient expression of NSm and its fusion proteins in Nicotiana benthamiana. GFP:NSm formed punctuate structures that colocalized with mCherry:plasmodesmata localized protein 1a (PDLP 1a) confirming that GBNV NSm localizes to PD. Unlike in other movement proteins, the C-terminal coiled coil domain of GBNV NSm was shown to be involved in the localization of NSm to PD, as deletion of this domain resulted in the cytoplasmic localization of NSm. Treatment with Brefeldin A demonstrated the role of ER in targeting GFP NSm to PD. Furthermore, mCherry:NSm co-localized with ER–GFP (endoplasmic reticulum targeting peptide (HDEL peptide fused with GFP). Co-expression of NSm with ER–GFP showed that the ER-network was transformed into vesicles indicating that NSm interacts with ER and remodels it. Mutations in the conserved hydrophobic region of NSm (residues 130–138) did not abolish the formation of vesicles. Additionally, the conserved prolines at positions 140 and 142 were found to be essential for targeting the vesicles to the cell membrane. Further, systematic deletion of amino acid residues from N- and C-terminus demonstrated that N-terminal 203 amino acids are dispensable for the vesicle formation. On the other hand, the C-terminal coiled coil domain when expressed alone could also form vesicles. These results suggest that GBNV NSm remodels the ER network by forming vesicles via its interaction through the C-terminal coiled coil domain. Interestingly, NSm interacts with NP in vitro and coexpression of these two proteins in planta resulted in the relocalization of NP to PD and this relocalization was abolished when the N-terminal unfolded region of NSm was deleted. Thus, the NSm

  20. Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

    PubMed Central

    Wünschiers, Röbbe; Batur, Mehtap; Lindblad, Peter

    2003-01-01

    Background Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. Results We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. Conclusion Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation

  1. TubZ filament assembly dynamics requires the flexible C-terminal tail

    PubMed Central

    Fuentes-Pérez, Maria E.; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A.

    2017-01-01

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY. PMID:28230082

  2. Novel C-terminally amidated opioid peptide in human phaeochromocytoma tumour.

    PubMed

    Matsuo, H; Miyata, A; Mizuno, K

    As has often been observed in hypothalamic releasing factors and gastrointestinal hormones, the carboxy-terminal amide structure is a unique feature of peptides exhibiting hormonal or physiological activities. Although a variety of opioid peptides have hitherto been identified, such a C-terminal amidated species has never before been discovered in mammals. Here we present the first identification of a novel opioid octapeptide with a C-terminal amide structure, henceforth designated as 'adrenorphin', in human phaeochromocytoma tumour derived from adrenal medulla. The complete amino acid sequence of adrenorphin was determined by microsequencing and corresponds to the sequence of the first eight amino acids of peptide E which is derived from proenkephalin A. Adrenorphin has also been identified chromatographically in normal human and bovine adrenal medulla. Adrenorphin exhibits potent opioid activity in guinea pig ileum assay, suggesting a specialized physiological function.

  3. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17

    PubMed Central

    Garcia-Doval, Carmela; van Raaij, Mark J.

    2012-01-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371–553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P212121 (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C2221 (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress. PMID:22297990

  4. Crystallization of the C-terminal domain of the bacteriophage T7 fibre protein gp17.

    PubMed

    Garcia-Doval, Carmela; van Raaij, Mark J

    2012-02-01

    Bacteriophage T7 attaches to its host using the C-terminal domains of its six fibres, which are trimers of the gp17 protein. A C-terminal fragment of gp17 consisting of amino acids 371-553 has been expressed, purified and crystallized. Crystals of two forms were obtained, belonging to space group P2(1)2(1)2(1) (unit-cell parameters a = 61.2, b = 86.0, c = 118.4 Å) and space group C222(1) (unit-cell parameters a = 68.3, b = 145.6, c = 172.1 Å). They diffracted to 1.9 and 2.0 Å resolution, respectively. Both crystals are expected to contain one trimer in the asymmetric unit. Multiwavelength anomalous dispersion phasing with a mercury derivative is in progress.

  5. Surface expression of Kv1 channels is governed by a C-terminal motif.

    PubMed

    Li, D; Takimoto, K; Levitan, E S

    2000-04-21

    Voltage-gated K(+) channel subunits must reach the plasma membrane to repolarize action potentials. Yet the efficiency of cell surface targeting varies among Kv subunits with some requiring auxiliary subunits for optimal expression. Here we identify a conserved motif located in the variable C-terminal region of Kv1 channels that controls the efficiency of functional channel expression. Variations among wild type channels in the optimal sequence VXXSL produce differences in distribution and the requirement for auxiliary subunits. Furthermore, deletion of this motif decreases subunit glycosylation and surface localization but does not prohibit subunit multimerization. Finally, the action of the essential sequence is shown to be independent of the chaperone effect of Kvbeta subunits. Thus, the newly identified C-terminal motif governs processing and cell surface expression of Kv1 voltage-gated K(+) channels.

  6. Rearrangement of the histone H2A C-terminal domain in the nucleosome

    SciTech Connect

    Usachenko, S.I.; Bavykin, S.G.; Gavin, I.M.; Bradbury, M. |

    1994-07-19

    Using zero-length covalent protein-DNA crosslinking, the authors have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. They found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. The results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.

  7. TubZ filament assembly dynamics requires the flexible C-terminal tail.

    PubMed

    Fuentes-Pérez, Maria E; Núñez-Ramírez, Rafael; Martín-González, Alejandro; Juan-Rodríguez, David; Llorca, Oscar; Moreno-Herrero, Fernando; Oliva, Maria A

    2017-02-23

    Cytomotive filaments are essential for the spatial organization in cells, showing a dynamic behavior based on nucleotide hydrolysis. TubZ is a tubulin-like protein that functions in extrachromosomal DNA movement within bacteria. TubZ filaments grow in a helical fashion following treadmilling or dynamic instability, although the underlying mechanism is unclear. We have unraveled the molecular basis for filament assembly and dynamics combining electron and atomic force microscopy and biochemical analyses. Our findings suggest that GTP caps retain the filament helical structure and hydrolysis triggers filament stiffening upon disassembly. We show that the TubZ C-terminal tail is an unstructured domain that fulfills multiple functions contributing to the filament helical arrangement, the polymer remodeling into tubulin-like rings and the full disassembly process. This C-terminal tail displays the binding site for partner proteins and we report how it modulates the interaction of the regulator protein TubY.

  8. Confirming the Revised C-Terminal Domain of the MscL Crystal Structure

    PubMed Central

    Maurer, Joshua A.; Elmore, Donald E.; Clayton, Daniel; Xiong, Li; Lester, Henry A.; Dougherty, Dennis A.

    2008-01-01

    The structure of the C-terminal domain of the mechanosensitive channel of large conductance (MscL) has generated significant controversy. As a result, several structures have been proposed for this region: the original crystal structure (1MSL) of the Mycobacterium tuberculosis homolog (Tb), a model of the Escherichia coli homolog, and, most recently, a revised crystal structure of Tb-MscL (2OAR). To understand which of these structures represents a physiological conformation, we measured the impact of mutations to the C-terminal domain on the thermal stability of Tb-MscL using circular dichroism and performed molecular dynamics simulations of the original and the revised crystal structures of Tb-MscL. Our results imply that this region is helical and adopts an α-helical bundle conformation similar to that observed in the E. coli MscL model and the revised Tb-MscL crystal structure. PMID:18326638

  9. Growth hormone secretagogues derived from NN703 with hydrazidesas c-terminal.

    PubMed

    Ankersen, M; Kramer Nielsen, K; Kruse Hansen, T; Raun, K; Sehested Hansen, B

    2000-05-01

    A series of GH secretagogues based on modifications in the C-terminal of NN703 is reported. The C-terminal N-methyl amide of NN703 has been replaced with alkylated hydrazides in order to decrease the volume of distribution and identify GH secretagogues with shorter duration of action. Most of the prepared compounds show high potency in a rat pituitary assay. Subsequent to an initial in vivo screening in dogs, four compounds were selected for further pharmacological and pharmacokinetic evaluation. The four compounds showed oral bioavailability around 35% and equipotency in vitro compared to NN703. The relationship between lipophilicity and volume of distribution is discussed and it is speculated whether the lower volume of distribution is attributed to the observed higher in vivo potency and shorter plasma elimination half-life.

  10. Multimodal Recognition of Diverse Peptides by the C-Terminal SH2 Domain of Phospholipase C-γ1 Protein.

    PubMed

    McKercher, Marissa A; Guan, Xiaoyang; Tan, Zhongping; Wuttke, Deborah S

    2017-04-11

    SH2 domains recognize phosphotyrosine (pY)-containing peptide ligands and play key roles in the regulation of receptor tyrosine kinase pathways. Each SH2 domain has individualized specificity, encoded in the amino acids neighboring the pY, for defined targets that convey their distinct functions. The C-terminal SH2 domain (PLCC) of the phospholipase C-γ1 full-length protein (PLCγ1) typically binds peptides containing small and hydrophobic amino acids adjacent to the pY, including a peptide derived from platelet-derived growth factor receptor B (PDGFRB) and an intraprotein recognition site (Y783 of PLCγ1) involved in the regulation of the protein's lipase activity. Remarkably, PLCC also recognizes unexpected peptides containing amino acids with polar or bulky side chains that deviate from this pattern. This versatility in recognition specificity may allow PLCγ1 to participate in diverse, previously unrecognized, signaling pathways in response to binding chemically dissimilar partners. We have used structural approaches, including nuclear magnetic resonance and X-ray crystallography, to elucidate the mechanisms of noncognate peptide binding to PLCC by ligands derived from receptor tyrosine kinase ErbB2 and from the insulin receptor. The high-resolution peptide-bound structures reveal that PLCC has a relatively static backbone but contains a chemically rich protein surface comprised of a combination of hydrophobic pockets and amino acids with charged side chains. We demonstrate that this expansive and chemically diverse PLCC interface, in addition to peptide conformational plasticity, permits PLCC to recognize specific noncognate peptide ligands with multimodal specificity.

  11. Structural and Functional Characterization of the C-terminal Transmembrane Region of NBCe1-A*

    PubMed Central

    Zhu, Quansheng; Kao, Liyo; Azimov, Rustam; Abuladze, Natalia; Newman, Debra; Pushkin, Alexander; Liu, Weixin; Chang, Connie; Kurtz, Ira

    2010-01-01

    NBCe1-A and AE1 both belong to the SLC4 HCO3− transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala800–Lys967. Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met858 accessible to both biotin maleimide and TAMRA and Thr926–Ala929 only to TAMRA labeling. The intracellular surface contains a highly exposed (Met813–Gly828) region and a cryptic (Met887–Arg904) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp960. Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro868–Leu967 (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1. PMID:20837482

  12. Elevated fasting and postprandial C-terminal telopeptide after Roux-en-Y gastric bypass.

    PubMed

    Maghsoodi, Negar; Alaghband-Zadeh, Jamshid; Cross, Gemma F; Werling, Malin; Fändriks, Lars; Docherty, Neil G; Olbers, Torsten; Dew, Tracy; Sherwood, Roy A; Vincent, Royce P; le Roux, Carel W

    2017-07-01

    Background Roux-en-Y gastric bypass increases circulating bile acid concentrations, known mediators of postprandial suppression of markers of bone resorption. Long-term data, however, indicate that Roux-en-Y gastric bypass confers an increased risk of bone loss on recipients. Methods Thirty-six obese individuals, median age 44 (26-64) with median body mass index at baseline of 42.5 (40.4-46) were studied before and 15 months after Roux-en-Y gastric bypass. After an overnight fast, patients received a 400 kcal mixed meal. Blood samples were collected premeal then at 30-min periods for 120 min. Pre and postmeal samples were analysed for total bile acids, parathyroid hormone and C-terminal telopeptide. Results Body weight loss post Roux-en-Y gastric bypass was associated with a median 4.9-fold increase in peak postprandial total bile acid concentration, and a median 2.4-fold increase in cumulative food evoked bile acid response. Median fasting parathyroid hormone, postprandial reduction in parathyroid hormone and total parathyroid hormone release over 120 min remained unchanged after surgery. After surgery, median fasting C-terminal telopeptide increased 2.3-fold, peak postprandial concentrations increased 3.8-fold and total release was increased 1.9-fold. Conclusions Fasting and postprandial total bile acids and C-terminal telopeptide are increased above reference range after Roux-en-Y gastric bypass. These changes occur in spite of improved vitamin D status with supplementation. These results suggest that post-Roux-en-Y gastric bypass increases in total bile acids do not effectively oppose an ongoing resorptive signal operative along the gut-bone axis. Serial measurement of C-terminal telopeptide may be of value as a risk marker for long-term skeletal pathology in patients post Roux-en-Y gastric bypass.

  13. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

    PubMed

    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  14. Dual Thermosensitive Hydrogels Assembled from the Conserved C-Terminal Domain of Spider Dragline Silk.

    PubMed

    Qian, Zhi-Gang; Zhou, Ming-Liang; Song, Wen-Wen; Xia, Xiao-Xia

    2015-11-09

    Stimuli-responsive hydrogels have great potentials in biomedical and biotechnological applications. Due to the advantages of precise control over molecular weight and being biodegradable, protein-based hydrogels and their applications have been extensively studied. However, protein hydrogels with dual thermosensitive properties are rarely reported. Here we present the first report of dual thermosensitive hydrogels assembled from the conserved C-terminal domain of spider dragline silk. First, we found that recombinant C-terminal domain of major ampullate spidroin 1 (MaSp1) of the spider Nephila clavipes formed hydrogels when cooled to approximately 2 °C or heated to 65 °C. The conformational changes and self-assembly of the recombinant protein were studied to understand the mechanism of the gelation processes using multiple methods. It was proposed that the gelation in the low-temperature regime was dominated by hydrogen bonding and hydrophobic interaction between folded protein molecules, whereas the gelation in the high-temperature regime was due to cross-linking of the exposed hydrophobic patches resulting from partial unfolding of the protein upon heating. More interestingly, genetic fusion of the C-terminal domain to a short repetitive region of N. clavipes MaSp1 resulted in a chimeric protein that formed a hydrogel with significantly improved mechanical properties at low temperatures between 2 and 10 °C. Furthermore, the formation of similar hydrogels was observed for the recombinant C-terminal domains of dragline silk of different spider species, thus demonstrating the conserved ability to form dual thermosensitive hydrogels. These findings may be useful in the design and construction of novel protein hydrogels with tunable multiple thermosensitivity for applications in the future.

  15. Study on the C-terminal beta-hairpin of protein G in AB heteropolymer model

    NASA Astrophysics Data System (ADS)

    Kim, Seung-Yeon

    2016-08-01

    The off-lattice AB heteropolymer model, consisting of the hydrophobic (A) and hydrophilic (B) polymers, is one of popular protein models. Its energy function includes the bending energy and the van der Waals interaction energy. The properties and the energy landscape of the C-terminal beta-hairpin of protein G are studied in the off-lattice AB heteropolymer model with conformational space annealing, a powerful global optimization method.

  16. Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers

    SciTech Connect

    Gnesa, Eric; Hsia, Yang; Yarger, Jeffery L.; Weber, Warner; Lin-Cereghino, Joan; Lin-Cereghino, Geoff; Tang, Simon; Agari, Kimiko; Vierra, Craig

    2012-05-24

    Spider silk is renowned for its extraordinary mechanical properties, having a balance of high tensile strength and extensibility. To date, the majority of studies have focused on the production of dragline silks from synthetic spider silk gene products. Here we report the first mechanical analysis of synthetic egg case silk fibers spun from the Latrodectus hesperus tubuliform silk proteins, TuSp1 and ECP-2. We provide evidence that recombinant ECP-2 proteins can be spun into fibers that display mechanical properties similar to other synthetic spider silks. We also demonstrate that silks spun from recombinant thioredoxin-TuSp1 fusion proteins that contain the conserved C-terminal domain exhibit increased extensibility and toughness when compared to the identical fibers spun from fusion proteins lacking the C-terminus. Mechanical analyses reveal that the properties of synthetic tubuliform silks can be modulated by altering the postspin draw ratios of the fibers. Fibers subject to increased draw ratios showed elevated tensile strength and decreased extensibility but maintained constant toughness. Wide-angle X-ray diffraction studies indicate that postdrawn fibers containing the C-terminal domain of TuSp1 have more amorphous content when compared to fibers lacking the C-terminus. Taken together, these studies demonstrate that recombinant tubuliform spidroins that contain the conserved C-terminal domain with embedded protein tags can be effectively spun into fibers, resulting in similar tensile strength but increased extensibility relative to nontagged recombinant dragline silk proteins spun from equivalently sized proteins.

  17. The C-terminal helical bundle of the tetrameric prokaryotic sodium channel accelerates the inactivation rate

    PubMed Central

    Irie, Katsumasa; Shimomura, Takushi; Fujiyoshi, Yoshinori

    2012-01-01

    Most tetrameric channels have cytosolic domains to regulate their functions, including channel inactivation. Here we show that the cytosolic C-terminal region of NavSulP, a prokaryotic voltage-gated sodium channel cloned from Sulfitobacter pontiacus, accelerates channel inactivation. The crystal structure of the C-terminal region of NavSulP grafted into the C-terminus of a NaK channel revealed that the NavSulP C-terminal region forms a four-helix bundle. Point mutations of the residues involved in the intersubunit interactions of the four-helix bundle destabilized the tetramer of the channel and reduced the inactivation rate. The four-helix bundle was directly connected to the inner helix of the pore domain, and a mutation increasing the rigidity of the inner helix also reduced the inactivation rate. These findings suggest that the NavSulP four-helix bundle has important roles not only in stabilizing the tetramer, but also in accelerating the inactivation rate, through promotion of the conformational change of the inner helix. PMID:22531178

  18. C-terminal tyrosine residues modulate the fusion activity of the Hendra virus fusion protein

    PubMed Central

    Popa, Andreea; Pager, Cara Teresia; Dutch, Rebecca Ellis

    2011-01-01

    The paramyxovirus family includes important human pathogens such as measles, mumps, respiratory syncytial virus and the recently emerged, highly pathogenic Hendra and Nipah viruses. The viral fusion (F) protein plays critical roles in infection, promoting both the viral-cell membrane fusion events needed for viral entry as well as cell-cell fusion events leading to syncytia formation. We describe the surprising finding that addition of the short epitope HA tag to the cytoplasmic tail (CT) of the Hendra virus F protein leads to a significant increase in cell-cell membrane fusion. This increase was not due to alterations in surface expression, cleavage state, or association with lipid microdomains. Addition of a Myc tag of similar length did not alter Hendra F fusion activity, indicating that the observed stimulation was not solely a result of lengthening the CT. Three tyrosine residues within the HA tag were critical for the increase in fusion, suggesting C-terminal tyrosines may modulate Hendra fusion activity. The effects of HA tag addition varied with other fusion proteins, as parainfluenza virus 5 F-HA showed decreased surface expression and no stimulation in fusion. These results indicate that additions to the C-terminal end of the F protein CT can modulate protein function in a sequence specific manner, reinforcing the need for careful analysis of epitope tagged glycoproteins. In addition, our results implicate C-terminal tyrosine residues in modulation of the membrane fusion reaction promoted by these viral glycoproteins. PMID:21175223

  19. Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity.

    PubMed

    Hierro, Aitor; Arizmendi, Jesús M; Bañuelos, Sonia; Prado, Adelina; Muga, Arturo

    2002-05-21

    The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.

  20. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  1. Probing the Impact of the EchinT C-Terminal Domain on Structure and Catalysis

    SciTech Connect

    S Bardaweel; J Pace; T Chou; V Cody; C Wagner

    2011-12-31

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2{sub 1} with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T{sub m} value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  2. Probing the impact of the echinT C-terminal domain on structure and catalysis.

    PubMed

    Bardaweel, Sanaa; Pace, James; Chou, Tsui-Fen; Cody, Vivian; Wagner, Carston R

    2010-12-10

    Histidine triad nucleotide binding protein (Hint) is considered as the ancestor of the histidine triad protein superfamily and is highly conserved from bacteria to humans. Prokaryote genomes, including a wide array of both Gram-negative bacteria and Gram-positive bacteria, typically encode one Hint gene. The cellular function of Hint and the rationale for its evolutionary conservation in bacteria have remained a mystery. Despite its ubiquity and high sequence similarity to eukaryote Hint1 [Escherichia coli Hint (echinT) is 48% identical with human Hint1], prokaryote Hint has been reported in only a few studies. Here we report the first conformational information on the full-length N-terminal and C-terminal residues of Hint from the E. coli complex with GMP. Structural analysis of the echinT-GMP complex reveals that it crystallizes in the monoclinic space group P2(1) with four homodimers in the asymmetric unit. Analysis of electron density for both the N-terminal residues and the C-terminal residues of the echinT-GMP complex indicates that the loops in some monomers can adopt more than one conformation. The observation of conformational flexibility in terminal loop regions could explain the presence of multiple homodimers in the asymmetric unit of this structure. To explore the impact of the echinT C-terminus on protein structure and catalysis, we conducted a series of catalytic radiolabeling and kinetic experiments on the C-terminal deletion mutants of echinT. In this study, we show that sequential deletion of the C-terminus likely has no effect on homodimerization and a modest effect on the secondary structure of echinT. However, we observed a significant impact on the folding structure, as reflected by a significant lowering of the T(m) value. Kinetic analysis reveals that the C-terminal deletion mutants are within an order of magnitude less efficient in catalysis compared to wild type, while the overall kinetic mechanism that proceeds through a fast step

  3. Fusogenic properties of the C-terminal domain of the Alzheimer beta-amyloid peptide.

    PubMed

    Pillot, T; Goethals, M; Vanloo, B; Talussot, C; Brasseur, R; Vandekerckhove, J; Rosseneu, M; Lins, L

    1996-11-15

    A series of natural peptides and mutants, derived from the Alzheimer beta-amyloid peptide, was synthesized, and the potential of these peptides to induce fusion of unilamellar lipid vesicles was investigated. These peptide domains were identified by computer modeling and correspond to respectively the C-terminal (e.g. residues 29-40 and 29-42) and a central domain (13-28) of the beta-amyloid peptide. The C-terminal peptides are predicted to insert in an oblique way into a lipid membrane through their N-terminal end, while the mutants are either parallel or perpendicular to the lipid bilayer. Peptide-induced vesicle fusion was demonstrated by several techniques, including lipid-mixing and core-mixing assays using pyrene-labeled vesicles. The effect of peptide elongation toward the N-terminal end of the entire beta-amyloid peptide was also investigated. Peptides corresponding to residues 22-42 and 12-42 were tested using the same techniques. Both the 29-40 and 29-42 beta-amyloid peptides were able to induce fusion of unilamellar lipid vesicles and calcein leakage, and the amyloid 29-42 peptide was the most potent fusogenic peptide. Neither the two mutants or the 13-28 beta-amyloid peptide had any fusogenic activity. Circular dichroism measurements showed an increase of the alpha-helical content of the two C-terminal peptides at increasing concentrations of trifluoroethanol, which was accompanied by an increase of the fusogenic potential of the peptides. Our data suggest that the alpha-helical content and the angle of insertion of the peptide into a lipid bilayer are critical for the fusogenic activity of the C-terminal domain of the amyloid peptide. The differences observed between the fusogenic capacity of the amyloid 29-40 and 29-42 peptides might result from differences in the degree of penetration of the peptides into the membrane and the resulting membrane destabilization. The longer peptides, residues 22-42 and 12-42, had decreased, but significant, fusogenic

  4. Three-dimensional studies of pathogenic peptides from the c-terminal of Trypanosoma cruzi ribosomal P proteins and their interaction with a monoclonal antibody structural model.

    PubMed

    Martín, Osvaldo A; Villegas, Myriam E; Aguilar, Carlos F

    2009-05-27

    The acidic C-terminal peptides from Trypanosoma cruzi ribosomal P proteins are the major target of the antibody response in patients suffering Chagas chronic heart disease. It has been proposed that the disease is triggered by the cross-reaction of these antibodies with the second extra cellular loop of the beta1-adrenoreceptor, brought about by the molecular mimicry between the acidic C-terminal peptides and the receptor's loop. To improve the understanding of the structural basis of the autoimmune response against heart receptors, the 3-dimensional structure of the C-terminal peptides of Trypanosoma cruzi ribosomal proteins P0 (EDDDDDFGMGALF) and P2beta (EEEDDDMGFGLFD) were solved using the Electrostaticaly Driven MonteCarlo method. Their structures were compared with the second extra-cellular loop of our homology model of human rhodopsin and the existing experimental NMR structures of the C-terminal peptides from human P0 (EESDDDMGFGLFD) and from Leishmania braziliensis P0 (EEADDDMGFGLFD). Docking of Trypanosoma cruzi peptides P0, P2beta and human rhodopsin loop into our anti-P2beta monoclonal antibody homology model allowed to explore their interactions.The solution structure of peptides P0 and P2beta can be briefly described as a bend. Although the global conformations of the peptides are not identical they shared a common region of four residues (3 to 6) that have a similar structure. The structural alignment of the five peptides also showed a surprising conformational similarity for the same residues. The antibody model and docking studies revealed a most remarkable feature in the active site, a positively charged, narrow and deep cavity where the acidic residues 3 to 6 were accommodated. These results suggest that the most important elements in the molecular peptide recognition by the antibody may be the shape of the loop and the presence of negative charges in positions 3-5 (P0, P2beta) or a negative charge in position 4 (rhodopsin loop). This work

  5. Three-dimensional studies of pathogenic peptides from the c-terminal of Trypanosoma cruzi ribosomal P proteins and their interaction with a monoclonal antibody structural model

    PubMed Central

    Martín, Osvaldo A; Villegas, Myriam E; Aguilar, Carlos F

    2009-01-01

    The acidic C-terminal peptides from Trypanosoma cruzi ribosomal P proteins are the major target of the antibody response in patients suffering Chagas chronic heart disease. It has been proposed that the disease is triggered by the cross-reaction of these antibodies with the second extra cellular loop of the β1-adrenoreceptor, brought about by the molecular mimicry between the acidic C-terminal peptides and the receptor's loop. To improve the understanding of the structural basis of the autoimmune response against heart receptors, the 3-dimensional structure of the C-terminal peptides of Trypanosoma cruzi ribosomal proteins P0 (EDDDDDFGMGALF) and P2β (EEEDDDMGFGLFD) were solved using the Electrostaticaly Driven MonteCarlo method. Their structures were compared with the second extra-cellular loop of our homology model of human rhodopsin and the existing experimental NMR structures of the C-terminal peptides from human P0 (EESDDDMGFGLFD) and from Leishmania braziliensis P0 (EEADDDMGFGLFD). Docking of Trypanosoma cruzi peptides P0, P2β and human rhodopsin loop into our anti-P2β monoclonal antibody homology model allowed to explore their interactions. The solution structure of peptides P0 and P2β can be briefly described as a bend. Although the global conformations of the peptides are not identical they shared a common region of four residues (3 to 6) that have a similar structure. The structural alignment of the five peptides also showed a surprising conformational similarity for the same residues. The antibody model and docking studies revealed a most remarkable feature in the active site, a positively charged, narrow and deep cavity where the acidic residues 3 to 6 were accommodated. These results suggest that the most important elements in the molecular peptide recognition by the antibody may be the shape of the loop and the presence of negative charges in positions 3–5 (P0, P2β) or a negative charge in position 4 (rhodopsin loop). This work describes

  6. Dephosphorylation of the linker regions of Smad1 and Smad2/3 by small C-terminal domain phosphatases has distinct outcomes for bone morphogenetic protein and transforming growth factor-beta pathways.

    PubMed

    Sapkota, Gopal; Knockaert, Marie; Alarcón, Claudio; Montalvo, Ermelinda; Brivanlou, Ali H; Massagué, Joan

    2006-12-29

    Smad proteins transduce bone morphogenetic protein (BMP) and transforming growth factor-beta (TGFbeta) signals upon phosphorylation of their C-terminal SXS motif by receptor kinases. The activity of Smad1 in the BMP pathway and Smad2/3 in the TGFbeta pathway is restricted by pathway cross-talk and feedback through protein kinases, including MAPK, CDK2/4, p38MAPK, JNK, and others. These kinases phosphorylate Smads 1-3 at the region that links the N-terminal DNA-binding domain and the C-terminal transcriptional domain. Phosphatases that dephosphorylate the linker region are therefore likely to play an integral part in the regulation of Smad activity. We reported previously that small C-terminal domain phosphatases 1, 2, and 3 (SCP1-3) dephosphorylate Smad1 C-terminal tail, thereby attenuating BMP signaling. Here we provide evidence that SCP1-3 also dephosphorylate the linker regions of Smad1 and Smad2/3 in vitro, in mammalian cells and in Xenopus embryos. Overexpression of SCP 1, 2, or 3 decreased linker phosphorylation of Smads 1, 2 and 3. Moreover, RNA interference-mediated knockdown of SCP1/2 increased the BMP-dependent phosphorylation of the Smad1 linker region as well as the C terminus. In contrast, SCP1/2 knockdown increased the TGFbeta-dependent linker phosphorylation of Smad2/3 but not the C-terminal phosphorylation. Consequently, SCP1/2 knockdown inhibited TGFbeta transcriptional responses, but it enhanced BMP transcriptional responses. Thus, by dephosphorylating Smad2/3 at the linker (inhibitory) but not the C-terminal (activating) site, the SCPs enhance TGFbeta signaling, and by dephosphorylating Smad1 at both sites, the SCPs reset Smad1 to the basal unphosphorylated state.

  7. Recruitment of A20 by the C-terminal domain of NEMO suppresses NF-κB activation and autoinflammatory disease

    PubMed Central

    Zilberman-Rudenko, Jevgenia; Shawver, Linda Monaco; Wessel, Alex W.; Luo, Yongquan; Pelletier, Martin; Tsai, Wanxia Li; Lee, Younglang; Vonortas, Spiridon; Cheng, Laurence; Ashwell, Jonathan D.; Orange, Jordan S.; Siegel, Richard M.; Hanson, Eric P.

    2016-01-01

    Receptor-induced NF-κB activation is controlled by NEMO, the NF-κB essential modulator. Hypomorphic NEMO mutations result in X-linked ectodermal dysplasia with anhidrosis and immunodeficiency, also referred to as NEMO syndrome. Here we describe a distinct group of patients with NEMO C-terminal deletion (ΔCT-NEMO) mutations. Individuals harboring these mutations develop inflammatory skin and intestinal disease in addition to ectodermal dysplasia with anhidrosis and immunodeficiency. Both primary cells from these patients, as well as reconstituted cell lines with this deletion, exhibited increased IκB kinase (IKK) activity and production of proinflammatory cytokines. Unlike previously described loss-of-function mutations, ΔCT-NEMO mutants promoted increased NF-κB activation in response to TNF and Toll-like receptor stimulation. Investigation of the underlying mechanisms revealed impaired interactions with A20, a negative regulator of NF-κB activation, leading to prolonged accumulation of K63-ubiquitinated RIP within the TNFR1 signaling complex. Recruitment of A20 to the C-terminal domain of NEMO represents a novel mechanism limiting NF-κB activation by NEMO, and its absence results in autoinflammatory disease. PMID:26802121

  8. Synapse associated protein 102 (SAP102) binds the C-terminal part of the scaffolding protein neurobeachin.

    PubMed

    Lauks, Juliane; Klemmer, Patricia; Farzana, Fatima; Karupothula, Ramesh; Zalm, Robbert; Cooke, Nancy E; Li, Ka Wan; Smit, August B; Toonen, Ruud; Verhage, Matthijs

    2012-01-01

    Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea's PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system.

  9. Synapse Associated Protein 102 (SAP102) Binds the C-Terminal Part of the Scaffolding Protein Neurobeachin

    PubMed Central

    Farzana, Fatima; Karupothula, Ramesh; Zalm, Robbert; Cooke, Nancy E.; Li, Ka Wan; Smit, August B.; Toonen, Ruud; Verhage, Matthijs

    2012-01-01

    Neurobeachin (Nbea) is a multidomain scaffold protein abundant in the brain, where it is highly expressed during development. Nbea-null mice have severe defects in neuromuscular synaptic transmission resulting in lethal paralysis of the newborns. Recently, it became clear that Nbea is important also for the functioning of central synapses, where it is suggested to play a role in trafficking membrane proteins to both, the pre- and post-synaptic sites. So far, only few binding partners of Nbea have been found and the precise mechanism of their trafficking remains unclear. Here, we used mass spectrometry to identify SAP102, a MAGUK protein implicated in trafficking of the ionotropic glutamate AMPA- and NMDA-type receptors during synaptogenesis, as a novel Nbea interacting protein in mouse brain. Experiments in heterologous cells confirmed this interaction and revealed that SAP102 binds to the C-terminal part of Nbea that contains the DUF, PH, BEACH and WD40 domains. Furthermore, we discovered that introducing a mutation in Nbea’s PH domain, which disrupts its interaction with the BEACH domain, abolishes this binding, thereby creating an excellent starting point to further investigate Nbea-SAP102 function in the central nervous system. PMID:22745750

  10. Importance of Glu53 in the C-terminal region of brazzein, a sweet-tasting protein.

    PubMed

    Lim, Jin-Kyung; Jang, Jin-Chul; Kong, Ji-Na; Kim, Myung-Chul; Kong, Kwang-Hoon

    2016-07-01

    The sweetness of brazzein, one of the known sweet proteins, is dependent on charges and/or structures of its specific amino acid side chains. As the residues in the C-terminus of brazzein are known to play a critical role in sweetness, the currently unknown function of Glu53 requires further study. To identify important residues responsible for the sweetness of the protein brazzein, four mutants of the Glu53 residue in the C-terminal region of des-pE1M-brazzein, which lacks the N-terminal pyroglutamate, were constructed using site-directed mutagenesis. Mutations of Glu53 substitution to Ala or Asp significantly decreased the sweetness. On the other hand, a Lys mutation resulted in a molecule with sweetness similar to that of des-pE1M-brazzein. Mutation of Glu53 to Arg resulted in a molecule significantly sweeter than des-pE1M-brazzein, which agrees with previous findings showing that mutation with positively charged residues results in a sweeter protein. Our results suggest that the residue at position 53 is crucial for the sweetness of brazzein, which may be interacting with the sweet-taste receptor. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  11. 7TM X-ray structures for class C GPCRs as new drug-discovery tools. 1. mGluR5.

    PubMed

    Topiol, Sid; Sabio, Michael

    2016-01-15

    We illustrate, with a focus on mGluR5, how the recently published, first X-ray structures of mGluR 7TM domains, specifically those of mGluR1 and mGluR5 complexed with negative allosteric modulators (NAMs), will begin to influence ligand- (e.g., drug- or sweetener-) discovery efforts involving class C GPCRs. With an extensive docking study allowing full ligand flexibility and full side chain flexibility of all residues in the ligand-binding cavity, we have predicted and analyzed the binding modes of a variety of structurally diverse mGluR5 NAM ligands, showing how the X-ray structures serve to effectively rationalize each ligand's binding characteristics. We demonstrated that the features that are inherent in our earlier overlay model are preserved in the protein structure-based docking models. We identified structurally diverse compounds, which potentially act as mGluR NAMs, and revealed binding-site differences by performing high-throughput docking using a database of approximately six million structures of commercially available compounds and the mGluR1 and mGluR5 X-ray structures. By comparing the 7TM domains of the mGluR5 and mGluR1 X-rays structures, we identified selectivity factors within group I of the mGluRs. Similarly, using homology models that we built for mGluR2 and mGluR4, we have identified the factors leading to the selectivity between group I and groups II and III for ligands occupying the deepest portion of the mGluR5 binding cavity. Finally, we have proposed a structure-based explanation of the pharmacological switching within a set of positive allosteric modulators (PAMs) and their corresponding, very close NAM analogs.

  12. The role of the C-terminal region in phosphoglycerate mutase.

    PubMed Central

    Walter, R A; Nairn, J; Duncan, D; Price, N C; Kelly, S M; Rigden, D J; Fothergill-Gilmore, L A

    1999-01-01

    Removal of the C-terminal seven residues from phosphoglycerate mutase from Saccharomyces cerevisiae by limited proteolysis is associated with loss of mutase activity, but no change in phosphatase activity. The presence of the cofactor 2, 3-bisphosphoglycerate, or of the cofactor and substrate 3-phosphoglycerate together, confers protection against proteolysis. The substrate alone offers no protection. Replacement of either or both of the two lysines at the C-terminus by glycines has only limited effects on the kinetic properties of phosphoglycerate mutase, indicating that these residues are unlikely to be involved in crucial electrostatic interactions with the substrate, intermediate or product in the reaction. However, the double-mutant form of the enzyme is more sensitive to proteolysis and is no longer protected against proteolysis by the presence of cofactor. The proteolysed wild-type and two of the mutated forms of the enzyme show a reduced response to 2-phosphoglycollate, which enhances the instability of the phospho form of the native enzyme. The phosphoglycerate mutase from Schizosaccharomyces pombe, which lacks the analogous C-terminal tail, has an inherently lower mutase activity and is also less responsive to stimulation by 2-phosphoglycollate. It is proposed that the C-terminal region of phosphoglycerate mutase helps to maintain the enzyme in its active phosphorylated form and assists in the retention of the bisphosphoglycerate intermediate at the active site. However, its role seems not to be to contribute directly to ligand binding, but rather to exert indirect effects on the transfer of the phospho group between substrate, enzyme, intermediate and product. PMID:9854029

  13. The C-terminal region of laminin beta chains modulates the integrin binding affinities of laminins.

    PubMed

    Taniguchi, Yukimasa; Ido, Hiroyuki; Sanzen, Noriko; Hayashi, Maria; Sato-Nishiuchi, Ryoko; Futaki, Sugiko; Sekiguchi, Kiyotoshi

    2009-03-20

    Laminins are major cell-adhesive proteins in basement membranes that are capable of binding to integrins. Laminins consist of three chains (alpha, beta, and gamma), in which three laminin globular modules in the alpha chain and the Glu residue in the C-terminal tail of the gamma chain have been shown to be prerequisites for binding to integrins. However, it remains unknown whether any part of the beta chain is involved in laminin-integrin interactions. We compared the binding affinities of pairs of laminin isoforms containing the beta1 or beta2 chain toward a panel of laminin-binding integrins, and we found that beta2 chain-containing laminins (beta2-laminins) bound more avidly to alpha3beta1 and alpha7X2beta1 integrins than beta1 chain-containing laminins (beta1-laminins), whereas alpha6beta1, alpha6beta4, and alpha7X1beta1 integrins did not show any preference toward beta2-laminins. Because alpha3beta1 contains the "X2-type" variable region in the alpha3 subunit and alpha6beta1 and alpha6beta4 contain the "X1-type" region in the alpha6 subunit, we hypothesized that only integrins containing the X2-type region were capable of discriminating between beta1-laminins and beta2-laminins. In support of this possibility, a putative X2-type variant of alpha6beta1 was produced and found to bind preferentially to beta2-laminins. Production of a series of swap mutants between the beta1 and beta2 chains revealed that the C-terminal 20 amino acids in the coiled-coil domain were responsible for the enhanced integrin binding by beta2-laminins. Taken together, the results provide evidence that the C-terminal region of beta chains is involved in laminin recognition by integrins and modulates the binding affinities of laminins toward X2-type integrins.

  14. BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2

    PubMed Central

    Shen, Chih-Lung; Gonzalez-Hurtado, Elsie; Zhang, Zhi-Min; Xu, Muyu; Martinez, Ernest; Peng, Chih-Wen; Song, Jikui

    2016-01-01

    Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection. PMID:26845565

  15. Vertebrate TFPI-2 C-terminal peptides exert therapeutic applications against Gram-negative infections.

    PubMed

    Kasetty, Gopinath; Smeds, Emanuel; Holmberg, Emelie; Wrange, Louise; Adikesavan, Selvi; Papareddy, Praveen

    2016-06-27

    Tissue factor pathway inhibitor-2 (TFPI-2) is a serine protease inhibitor that exerts multiple physiological and patho-physiological activities involving the modulation of coagulation, angiogenesis, tumor invasion, and apoptosis. In previous studies we reported a novel role of human TFPI-2 in innate immunity by serving as a precursor for host defense peptides. Here we employed a number of TFPI-2 derived peptides from different vertebrate species and found that their antibacterial activity is evolutionary conserved although the amino acid sequence is not well conserved. We further studied the theraputic potential of one selected TFPI-2 derived peptide (mouse) in a murine sepsis model. Hydrophobicity and net charge of many peptides play a important role in their host defence to invading bacterial pathogens. In vertebrates, the C-terminal portion of TFPI-2 consists of a highly conserved cluster of positively charged amino acids which may point to an antimicrobial activity. Thus a number of selected C-terminal TFPI-2 derived peptides from different species were synthesized and it was found that all of them exert antimicrobial activity against E. coli and P. aeruginosa. The peptide-mediated killing of E. coli was enhanced in human plasma, suggesting an involvement of the classical pathway of the complement. Under in vitro conditions the peptides displayed anti-coagulant activity by modulating the intrinsic pathway of coagulation and in vivo treatment with the mouse derived VKG24 peptide protects mice from an otherwise lethal LPS shock model. Our results suggest that the evolutionary conserved C-terminal part of TFPI-2 is an interesting agent for the development of novel antimicrobial therapies.

  16. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations.

    PubMed

    Williams, Alison A; Mehler, Vera J; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2.

  17. Roles of the C-terminal residues of calmodulin in structure and function

    PubMed Central

    Kitagawa, Chihiro; Nakatomi, Akiko; Hwang, Dasol; Osaka, Issey; Fujimori, Hiroki; Kawasaki, Hideya; Arakawa, Ryuichi; Murakami, Yota; Ohki, Shinya

    2011-01-01

    Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation. PMID:27857591

  18. Heterologous C-terminal sequences disrupt transcriptional activation and oncogenesis by p59v-rel.

    PubMed Central

    Diehl, J A; Hannink, M

    1993-01-01

    Members of the NF-kappa B/rel family of transcription factors are regulated through a trans association with members of a family of inhibitor proteins, collectively known as I kappa B proteins, that contain five to eight copies of a 33-amino-acid repeat sequence (ankyrin repeat). Certain NF-kappa B/rel proteins are also regulated by cis-acting ankyrin repeat-containing domains. The C terminus of p105NF-kappa B, the precursor of the 50-kDa subunit of NF-kappa B, contains a series of ankyrin repeats; proteolytic removal of this ankyrin domain is necessary for the manifestation of sequence-specific DNA binding and nuclear translocation of the N-terminal product. To investigate the structural requirements important for regulation of different NF-kappa B/rel family members by polypeptides containing ankyrin repeat domains, we have constructed a p59v-rel:p105NF-kappa B chimeric protein (p110v-rel-ank). The presence of C-terminal p105NF-kappa B-derived sequences in p110v-rel-ank inhibited nuclear translocation, sequence-specific DNA binding, pp40I kappa B-alpha association, and oncogenic transformation. Sequential truncation of the C-terminal ankyrin domain of p110v-rel-ank resulted in the restoration of nuclear translocation, DNA binding, and pp40I kappa B-alpha association but did not restore the oncogenic properties of p59v-rel. The presence of 67 C-terminal p105NF-kappa B-derived amino acids was sufficient to inhibit both transcriptional activation and oncogenic transformation by p59v-rel. These results support a model in which activation of gene expression by p59v-rel is required for its ability to induce oncogenic transformation. Images PMID:8230438

  19. C-terminal residues of rice translin are essential for octamer formation and nucleic acid binding.

    PubMed

    Gupta, Alka; Nair, Anuradha; Ballal, Anand; Chittela, Rajani Kant

    2017-09-01

    Translin is a DNA/RNA binding protein involved in DNA repair and RNA metabolism. Previously, we had shown that rice translin (221 amino acids) exhibits biochemical activities similar to that of the human translin protein. Here we report the role of the C-terminal random coil in rice translin function by analyzing truncation (after 215(th) residue, Tra - 215) and substitution mutant proteins (Ser216Ala, Lys217Ala, Gln218Ala, Glu219Ala). Circular Dichroism (CD) analysis of Tra-215 showed deviations in comparison to Tra-WT. Truncation abolished the DNA binding activity and octamer formation as evidenced by the absence of ring like structures from TEM analysis. CD analysis of the substitution mutant proteins showed that the secondary structure was maintained in all the mutant proteins in comparison to wild type protein. Native PAGE and TEM analysis of the substitution mutants showed that Lys217Ala mutation completely abolished the octamer formation as rings and nucleic acid binding. Glu219Ala mutation also affected oligomerization but exhibited marginal RNA binding at higher protein concentrations and interestingly, failed to bind to DNA. However, Ser216Ala and Gln218Ala substitutions did not affect above mentioned activities of translin. Our results indicate that the C-terminal residues are one of the determinants of octamer formation in rice translin, with lysine at 217(th) position being the most important. Therefore, in conclusion, although the C-terminal residues do not form any defined secondary structure in the translin monomer, they are definitely involved in octamer formation and hence important for its molecular function. We have attempted to find the critical residues in translin function, which will advance our understanding of translin in DNA repair process in general and of rice translin in particular. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  20. Apoptotic Activity of MeCP2 Is Enhanced by C-Terminal Truncating Mutations

    PubMed Central

    Mehler, Vera J.; Mueller, Christina; Vonhoff, Fernando; White, Robin; Duch, Carsten

    2016-01-01

    Methyl-CpG binding protein 2 (MeCP2) is a widely abundant, multifunctional protein most highly expressed in post-mitotic neurons. Mutations causing Rett syndrome and related neurodevelopmental disorders have been identified along the entire MECP2 locus, but symptoms vary depending on mutation type and location. C-terminal mutations are prevalent, but little is known about the function of the MeCP2 C-terminus. We employ the genetic efficiency of Drosophila to provide evidence that expression of p.Arg294* (more commonly identified as R294X), a human MECP2 E2 mutant allele causing truncation of the C-terminal domains, promotes apoptosis of identified neurons in vivo. We confirm this novel finding in HEK293T cells and then use Drosophila to map the region critical for neuronal apoptosis to a small sequence at the end of the C-terminal domain. In vitro studies in mammalian systems previously indicated a role of the MeCP2 E2 isoform in apoptosis, which is facilitated by phosphorylation at serine 80 (S80) and decreased by interactions with the forkhead protein FoxG1. We confirm the roles of S80 phosphorylation and forkhead domain transcription factors in affecting MeCP2-induced apoptosis in Drosophila in vivo, thus indicating mechanistic conservation between flies and mammalian cells. Our findings are consistent with a model in which C- and N-terminal interactions are required for healthy function of MeCP2. PMID:27442528

  1. A C-terminal Aldehyde Analog of the Insect Kinins Inhibits Diuresis in the Housefly

    DTIC Science & Technology

    2006-09-21

    p e p t i d e s 2 8 ( 2 0 0 7 ) 1 4 6 – 1 5 2A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly Ronald J. Nachman a...secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h...to stimulate Malpighian tubule fluid secretion [2,25]. In the housefly, muscakinin has been implicated in the control of diuresis in response to

  2. C-Terminal acetylene derivatized peptides via silyl-based alkyne immobilization.

    PubMed

    Strack, Martin; Metzler-Nolte, Nils; Albada, H Bauke

    2013-06-21

    A new Silyl-based Alkyne Modifying (SAM)-linker for the synthesis of C-terminal acetylene-derivatized peptides is reported. The broad scope of this SAM2-linker is illustrated by manual synthesis of peptides that are side-chain protected, fully deprotected, and disulfide-bridged. Synthesis of a 14-meric (KLAKLAK)2 derivative by microwave-assisted automated SPPS and a one-pot cleavage click procedure yielding protected 1,2,3-triazole peptide conjugates are also described.

  3. [Research advances on ubiquitin C-terminal hydrolase in oncogenesis and progression].

    PubMed

    Yu, Juan; Chen, Wei-lin

    2015-03-01

    By regulating the ubiquitination and deubiquitination of key proteins, ubiquitin-proteasome system mediates a variety of cellular activities. Ubiquitin C-terminal hydrolase (UCH) is a deubiquitinating enzyme which can remove ubiquitin chains at the end of ubiquited proteins. The abnormal expression of UCH has been found in a variety of tumor tissues, indicating that it participates in the process of tumor development. Here we review the characteristics of UCH members and current understanding about the role of UCH in tumor development, and the potential target for cancer treatment.

  4. Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain.

    PubMed

    Simon, Binto; Huang, Xuexia; Ju, Huangxian; Sun, Guoxuan; Yang, Min

    2017-02-21

    Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

  5. Functional Mechanism of C-Terminal Tail in the Enzymatic Role of Porcine Testicular Carbonyl Reductase: A Combined Experiment and Molecular Dynamics Simulation Study of the C-Terminal Tail in the Enzymatic Role of PTCR

    PubMed Central

    Park, Chanin; Lee, Yuno; Kwon, Seul Gi; Kim, Sam Woong; Kim, Chul Wook; Lee, Keun Woo

    2014-01-01

    Porcine testicular carbonyl reductase, PTCR which is one of the short chain dehydrogenases/reductases (SDR) superfamily catalyzes the NADPH-dependent reduction of carbonyl compounds including steroids and prostaglandins. Previously we reported C- terminal tail of PTCR was deleted due to a nonsynonymous single nucleotide variation (nsSNV). Here we identified from kinetic studies that the enzymatic properties for 5α-dihydrotestosterone (5α-DHT) were different between wild-type and C-terminal-deleted PTCRs. Compared to wild-type PTCR, C-terminal-deleted PTCR has much higher reduction rate. To investigate structural difference between wild-type and C-terminal-deleted PTCRs upon 5α-DHT binding, we performed molecular dynamics simulations for two complexes. Using trajectories, molecular interactions including hydrogen bonding patterns, distance between 5α-DHT and catalytic Tyr193, and interaction energies are analyzed and compared. During the MD simulation time, the dynamic behavior of C-terminal tail in wild-type PTCR is also examined using essential dynamics analysis. The results of our simulations reveal that the binding conformation of 5α-DHT in C-terminal-deleted PTCR is more favorable for reduction reaction in PTCR, which shows strong agreement with kinetic data. These structural findings provide valuable information to understand substrate specificity of PTCR and further kinetic properties of enzymes belonging to the SDR superfamily. PMID:24646606

  6. Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces cerevisiae Mating Pheromone a-Factor.

    PubMed

    Diaz-Rodriguez, Veronica; Ganusova, Elena; Rappe, Todd M; Becker, Jeffrey M; Distefano, Mark D

    2015-11-20

    Peptides containing C-terminal esters are an important class of bioactive molecules that includes a-factor, a farnesylated dodecapeptide, involved in the mating of Saccharomyces cerevisiae. Here, results that expand the scope of solid-phase peptide synthetic methodology that uses trityl side-chain anchoring for the preparation of peptides with C-terminal cysteine alkyl esters are described. In this method, Fmoc-protected C-terminal cysteine esters are anchored to trityl chloride resin and extended by standard solid-phase procedures followed by acidolytic cleavage and HPLC purification. Analysis using a Gly-Phe-Cys-OMe model tripeptide revealed minimal epimerization of the C-terminal cysteine residue under basic conditions used for Fmoc deprotection. (1)H NMR analysis of the unfarnesylated a-factor precursor peptide confirmed the absence of epimerization. The side-chain anchoring method was used to produce wild-type a-factor that contains a C-terminal methyl ester along with ethyl-, isopropyl-, and benzyl-ester analogs in good yield. Activity assays using a yeast-mating assay demonstrate that while the ethyl and isopropyl esters manifest near-wild-type activity, the benzyl ester-containing analog is ca. 100-fold less active. This simple method opens the door to the synthesis of a variety of C-terminal ester-modified peptides that should be useful in studies of protein prenylation and other structurally related biological processes.

  7. Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes.

    PubMed

    Cai, Bishuang; Giridharan, Sai Srinivas Panapakkam; Zhang, Jing; Saxena, Sugandha; Bahl, Kriti; Schmidt, John A; Sorgen, Paul L; Guo, Wei; Naslavsky, Naava; Caplan, Steve

    2013-10-18

    Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.

  8. Role of the C-terminal basic amino acids and the lipid anchor of the Gγ2 protein in membrane interactions and cell localization.

    PubMed

    Noguera-Salvà, Maria A; Guardiola-Serrano, Francisca; Martin, M Laura; Marcilla-Etxenike, Amaia; Bergo, Martin O; Busquets, Xavier; Escribá, Pablo V

    2017-02-21

    Heterotrimeric G proteins are peripheral membrane proteins that frequently localize to the plasma membrane where their presence in molar excess over G protein coupled receptors permits signal amplification. Their distribution is regulated by protein-lipid interactions, which has a clear influence on their activity. Gβγ dimer drives the interaction between G protein heterotrimers with cell membranes. We focused our study on the role of the C-terminal region of the Gγ2 protein in G protein interactions with cell membranes. The Gγ2 subunit is modified at cysteine (Cys) 68 by the addition of an isoprenyl lipid, which is followed by the proteolytic removal of the last three residues that leaves an isoprenylated and carboxyl methylated Cys-68 as the terminal amino acid. The role of Cys isoprenylation of the CAAX box has been defined for other proteins, yet the importance of proteolysis and carboxyl methylation of isoprenylated proteins is less clear. Here, we showed that not only geranylgeranylation but also proteolysis and carboxyl methylation are essential for the correct localization of Gγ2 in the plasma membrane. Moreover, we showed the importance of electrostatic interactions between the inner leaflet of the plasma membrane and the positively charged C-terminal domain of the Gγ2 subunit (amino acids Arg-62, Lys-64 and Lys-65) as a second signal to reach the plasma membrane. Indeed, single or multiple point mutations at Gγ2 C-terminal amino acids have a significant effect on Gγ2 protein-plasma membrane interactions and its localization to charged Ld (liquid disordered) membrane microdomains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo Escríba-Ruíz.

  9. Small C-terminal domain phosphatases dephosphorylate the regulatory linker regions of Smad2 and Smad3 to enhance transforming growth factor-beta signaling.

    PubMed

    Wrighton, Katharine H; Willis, Danielle; Long, Jianyin; Liu, Fang; Lin, Xia; Feng, Xin-Hua

    2006-12-15

    Transforming growth factor-beta (TGF-beta) controls a diverse set of cellular processes, and its canonical signaling is mediated via TGF-beta-induced phosphorylation of receptor-activated Smads (2 and 3) at the C-terminal SXS motif. We recently discovered that PPM1A can dephosphorylate Smad2/3 at the C-terminal SXS motif, implicating a critical role for phosphatases in regulating TGF-beta signaling. Smad2/3 activity is also regulated by phosphorylation in the linker region (and N terminus) by a variety of intracellular kinases, making it a critical platform for cross-talk between TGF-beta and other signaling pathways. Using a functional genomic approach, we identified the small C-terminal domain phosphatase 1 (SCP1) as a specific phosphatase for Smad2/3 dephosphorylation in the linker and N terminus. A catalytically inactive SCP1 mutant (dnSCP1) had no effect on Smad2/3 phosphorylation in vitro or in vivo. Of the other FCP/SCP family members SCP2 and SCP3, but not FCP1, could also dephosphorylate Smad2/3 in the linker/N terminus. Depletion of SCP1/2/3 enhanced Smad2/3 linker phosphorylation. SCP1 increased TGF-beta-induced transcriptional activity in agreement with the idea that phosphorylation in the Smad2/3 linker must be removed for a full transcriptional response. SCP1 overexpression also counteracts the inhibitory effect of epidermal growth factor on TGF-beta-induced p15 expression. Taken together, this work identifies the first example of a Smad2/3 linker phosphatase(s) and reveals an important new substrate for SCPs.

  10. Functional analysis of the C-terminal region of the vacuolar cadmium-transporting rice OsHMA3.

    PubMed

    Kumagai, Saori; Suzuki, Tatsuya; Tezuka, Kouichi; Satoh-Nagasawa, Namiko; Takahashi, Hidekazu; Sakurai, Kenji; Watanabe, Akio; Fujimura, Tatsuhito; Akagi, Hiromori

    2014-03-03

    Rice OsHMA3 is a vacuolar cadmium (Cd) transporter belonging to the P1B-ATPase family and has a long (273aa) C-terminal region. We analyzed the function of the region related to Cd using the transgenic Arabidopsis Col-0 ecotype, which is sensitive to Cd. The OsHMA3 variant containing a truncated (58aa) C-terminal region did not confer Cd tolerance, whereas an OsHMA3 variant containing a longer truncated (105aa) C-terminal region conferred Cd tolerance to transgenic Arabidopsis. We conclude that the C-terminal region, particularly the region containing the first 105aa, has an important role in OsHMA3 activity. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  11. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed Central

    Chen, J M; Fortunato, M; Barrett, A J

    2000-01-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation. PMID:11085925

  12. Activation of human prolegumain by cleavage at a C-terminal asparagine residue.

    PubMed

    Chen, J M; Fortunato, M; Barrett, A J

    2000-12-01

    The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56 kDa prolegumain. The 56 kDa form was enzymically inactive but stable at neutral pH, unlike the 35 kDa mature pig legumain; it could be converted into a 46 kDa active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replacement of Asn(323) at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation.

  13. Molecular Understanding of USP7 Substrate Recognition and C-Terminal Activation.

    PubMed

    Rougé, Lionel; Bainbridge, Travis W; Kwok, Michael; Tong, Raymond; Di Lello, Paola; Wertz, Ingrid E; Maurer, Till; Ernst, James A; Murray, Jeremy

    2016-08-02

    The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Presynaptic C-terminal truncated tau is released from cortical synapses in Alzheimer's disease

    PubMed Central

    Sokolow, Sophie; Henkins, Kristen M.; Bilousova, Tina; Gonzalez, Bianca; Vinters, Harry V.; Miller, Carol A.; Cornwell, Lindsey; Poon, Wayne W.; Gylys, Karen H.

    2015-01-01

    The microtubule-associated protein tau has primarily been associated with axonal location and function; however, recent work shows tau release from neurons and suggests an important role for tau in synaptic plasticity. In our study, we measured synaptic levels of total tau using synaptosomes prepared from cryopreserved human postmortem Alzheimer's disease (AD) and control samples. Flow cytometry data show that a majority of synaptic terminals are highly immunolabeled with the total tau antibody (HT7) in both AD and control samples. Immunoblots of synaptosomal fractions reveal increases in a 20 kDa tau fragment and in tau dimers in AD synapses, and terminal-specific antibodies show that in many synaptosome samples tau lacks a C-terminus. Flow cytometry experiments to quantify the extent of C-terminal truncation reveal that only 15-25% of synaptosomes are positive for intact C-terminal tau. Potassium-induced depolarization demonstrates release of tau and tau fragments from presynaptic terminals, with increased release from AD compared to control samples. This study indicates that tau is normally highly localized to synaptic terminals in cortex where it is well-positioned to affect synaptic plasticity. Tau cleavage may facilitate tau aggregation as well as tau secretion and propagation of tau pathology from the presynaptic compartment in AD. PMID:25393609

  15. Effect of C-terminal domain truncation of Thermus thermophilus trehalose synthase on its substrate specificity.

    PubMed

    Cho, Chang-Bae; Park, Da-Yeon; Lee, Soo-Bok

    2017-01-01

    The C-terminal domain of the three-domain-comprising trehalose synthase from Thermus thermophilus was truncated in order to study the effect on the enzyme's activity and substrate specificity. Compared with the wild-type (WT) enzyme, the two truncated enzymes (DM1 and DM2) showed lower maltose- and trehalose-converting activities and a different transglycosylation reaction mechanism. In the mutants, the glucose moiety cleaved from the maltose substrate was released from the enzyme and intercepted by external glucose oxidase, preventing the production of trehalose. The WT enzyme, however, retained the glucose in the active site to effectively produce trehalose. In addition, DM1 synthesized much higher amounts of mannose-containing disaccharide trehalose analog (Man-TA) than did the WT and DM2. The results suggest that the C-terminal domain in the WT enzyme is important for retaining the glucose moiety within the active site. The mutant enzymes could be used to produce Man-TA, a postulated inhibitor of gut disaccharidases. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene

    PubMed Central

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene. PMID:28107454

  17. C-terminal interactions of apolipoprotein E4 respond to the postprandial state.

    PubMed

    Tetali, Sarada D; Budamagunta, Madhu S; Voss, John C; Rutledge, John C

    2006-07-01

    Increased triglyceride-rich lipoproteins (TGRLs) in the postprandial state are associated with atherosclerosis. We investigated whether the postprandial state induced structural changes at the apolipoprotein E4 (apoE4) C terminus, its principal lipid binding domain, using electron paramagnetic resonance (EPR) spectroscopy of a site-directed spin label attached to the cysteine of apoE4-W264C. Spin coupling between labels located in the C termini was followed after mixing with preprandial and postprandial human plasma samples. Our results indicate that postprandial plasma triggers a reorganization of the protein such that the dipolar broadening is diminished, indicating a reduction in C-terminal interaction. The loss of spectral broadening was directly correlated with an increase in postprandial plasma triglycerides and was reduced with delipidated plasma. The spin-labeled apoE4 displayed a lipid preference of VLDL > LDL > HDL in the preprandial and postprandial states. The apoE4 shift to VLDL during the postprandial state was accompanied by a loss in spectral broadening of the protein. These findings suggest that apoE4 associated with LDL maintains self-association via its C terminus and that this association is diminished in VLDL-associated protein. Lipolyzed TGRL reflected a depletion of the C-terminal interaction of apoE4. Addition of palmitate to VLDL gave a similar response as lipolyzed TGRL, suggesting that lipolysis products play a major role in reorganizing apoE4 during the postprandial state.

  18. Structure of the C-terminal domain of nsp4 from feline coronavirus

    SciTech Connect

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-08-01

    The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

  19. The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

    PubMed

    de Abreu da Silva, Isabel Caetano; Carneiro, Vitor Coutinho; Vicentino, Amanda Roberta Revoredo; Aguilera, Estefania Anahi; Mohana-Borges, Ronaldo; Thiengo, Silvana; Fernandez, Monica Ammon; Fantappié, Marcelo Rosado

    2016-04-01

    The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  20. Identification of Novel Short C-Terminal Transcripts of Human SERPINA1 Gene.

    PubMed

    Matamala, Nerea; Aggarwal, Nupur; Iadarola, Paolo; Fumagalli, Marco; Gomez-Mariano, Gema; Lara, Beatriz; Martinez, Maria Teresa; Cuesta, Isabel; Stolk, Jan; Janciauskiene, Sabina; Martinez-Delgado, Beatriz

    2017-01-01

    Human SERPINA1 gene is located on chromosome 14q31-32.3 and is organized into three (IA, IB, and IC) non-coding and four (II, III, IV, V) coding exons. This gene produces α1-antitrypsin (A1AT), a prototypical member of the serpin superfamily of proteins. We demonstrate that human peripheral blood leukocytes express not only a product corresponding to the transcript coding for the full-length A1AT protein but also two short transcripts (ST1C4 and ST1C5) of A1AT. In silico sequence analysis revealed that the last exon of the short transcripts contains an Open Reading Frame (ORF) and thus putatively can produce peptides. We found ST1C4 expression across different human tissues whereas ST1C5 was mainly restricted to leukocytes, specifically neutrophils. A high up-regulation (10-fold) of short transcripts was observed in isolated human blood neutrophils after activation with lipopolysaccharide. Parallel analyses by liquid chromatography-mass spectrometry identified peptides corresponding to C-terminal region of A1AT in supernatants of activated but not naïve neutrophils. Herein we report for the first time a tissue specific expression and regulation of short transcripts of SERPINA1 gene, and the presence of C-terminal peptides in supernatants from activated neutrophils, in vitro. This gives a novel insight into the studies on the transcription of SERPINA1 gene.

  1. Crystallization and halide phasing of the C-terminal domain of human KIN17

    SciTech Connect

    Maire, Albane le; Schiltz, Marc; Braud, Sandrine; Gondry, Muriel; Charbonnier, Jean-Baptiste; Zinn-Justin, Sophie; Stura, Enrico

    2006-03-01

    Expression, purification, crystallization and phasing procedure are reported for the C-terminal domain of human KIN17. Here, the crystallization and initial phasing of the C-terminal domain of human KIN17, a 45 kDa protein mainly expressed in response to ionizing radiation and overexpressed in certain tumour cell lines, are reported. Crystals diffracting to 1.4 Å resolution were obtained from 10% ethylene glycol, 27% PEG 6000, 500 mM LiCl and 100 mM sodium acetate pH 6.3 in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 45.75, b = 46.31, c = 60.80 Å and one molecule in the asymmetric unit. Since this domain has a basic pI, heavy-atom derivatives were obtained by soaking the crystals with negatively charged ions such as tungstate and iodine. The replacement of LiCl by KI in the cryosolution allowed the determination of phases from iodide ions to give an interpretable electron-density map.

  2. The phage λ CII transcriptional activator carries a C-terminal domain signaling for rapid proteolysis

    PubMed Central

    Kobiler, Oren; Koby, Simi; Teff, Dinah; Court, Donald; Oppenheim, Amos B.

    2002-01-01

    ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the λ CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the λ CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage λ carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein. PMID:12397182

  3. PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

    PubMed Central

    Saijo, Eri; Hughson, Andrew G.; Raymond, Gregory J.; Suzuki, Akio; Horiuchi, Motohiro

    2016-01-01

    ABSTRACT Understanding the structure of PrPSc and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrPSc, we purified proteinase-resistant PrPSc (PrPRES) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrPSc specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrPRES, even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrPSc-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrPSc multimers. IMPORTANCE It has long been apparent that prion strains can have different conformations near the N terminus of the PrPSc protease-resistant core. Here, we show that a C-terminal conformational PrPSc-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrPSc also contribute to the phenotypic distinction between prion strains. PMID:26937029

  4. Involvement of C-Terminal Histidines in Soybean PM1 Protein Oligomerization and Cu2+ Binding.

    PubMed

    Liu, Guobao; Liu, Ke; Gao, Yang; Zheng, Yizhi

    2017-04-06

    Late embryogenesis abundant (LEA) proteins are widely distributed among plant species, where they contribute to abiotic stress tolerance. LEA proteins can be classified into seven groups according to conserved sequence motifs. The PM1 protein from soybean, which belongs to the Pfam LEA_1 group, has been shown previously to be at least partially natively unfolded, to bind metal ions and potentially to stabilize proteins and membranes. Here, we investigated the role of the PM1 C-terminal domain and in particular the multiple histidine residues in this half of the protein. We constructed recombinant plasmids expressing full-length PM1 and two truncated forms, PM1-N and PM1-C, which represent the N- and C-terminal halves of the protein, respectively. Immunoblotting and cross-linking experiments showed that full-length PM1 forms oligomers and high molecular weight (HMW) complexes in vitro and in vivo, while PM1-C, but not PM1-N, also formed oligomers and HMW complexes in vitro. When the histidine residues in PM1 and PM1-C were chemically modified, oligomerization was abolished, suggesting that histidines play a key role in this process. Furthermore, we demonstrated that high Cu2+ concentrations promote oligomerization and induce PM1 and PM1-C to form HMW complexes. Therefore, we speculate that PM1 proteins not only maintain ion homeostasis in the cytoplasm, but also potentially stabilize and protect other proteins during abiotic stress by forming a large, oligomeric molecular shield around biological targets.

  5. Characterization of the C-terminal ER membrane anchor of PTP1B

    SciTech Connect

    Anderie, Ines Schulz, Irene; Schmid, Andreas

    2007-09-10

    The tyrosine phosphatase PTP1B is an important regulator of cell function. In living cells PTP1B activity is restricted to the vicinity of the endoplasmic reticulum (ER) by post-translational C-terminal attachment of PTP1B to the ER membrane network. In our study we investigated the membrane anchor of PTP1B by use of EGFP fusion proteins. We demonstrate that the membrane anchor of PTP1B cannot be narrowed down to a unique amino acid sequence with a defined start and stop point but rather is moveable within several amino acids. Removal of up to seven amino acids from the C-terminus, as well as exchange of single amino acids in the putative transmembrane sequence did not influence subcellular localization of PTP1B. With the method of bimolecular fluorescence complementation we could demonstrate dimerization of PTP1B in vivo. Homodimerization was, in contrast to other tail-anchored proteins, not dependent on the membrane anchor. Our data demonstrate that the C-terminal membrane anchor of PTP1B is formed by a combination of a single stretch transmembrane domain (TMD) followed by a tail. TMD and tail length are variable and there are no sequence-specific features. Our data for PTP1B are consistent with a concept that explains the ER membrane anchor of tail-anchored proteins as a physicochemical structure.

  6. The C-terminal region of OVGP1 remodels the zona pellucida and modifies fertility parameters

    PubMed Central

    Algarra, B.; Han, L.; Soriano-Úbeda, C.; Avilés, M.; Coy, P.; Jovine, L.; Jiménez-Movilla, M.

    2016-01-01

    OVGP1 is the major non-serum glycoprotein in the oviduct fluid at the time of fertilization and early embryo development. Its activity differs among species. Here, we show that the C-terminal region of recombinant OVGP1 regulates its binding to the extracellular zona pellucida and affects its activity during fertilization. While porcine OVGP1 penetrates two-thirds of the thickness of the zona pellucida, shorter OVGP1 glycoproteins, including rabbit OVGP1, are restricted to the outer one-third of the zona matrix. Deletion of the C-terminal region reduces the ability of the glycoprotein to penetrate through the zona pellucida and prevents OVGP1 endocytosis. This affects the structure of the zona matrix and increases its resistance to protease digestion. However, only full-length porcine OVGP1 is able to increase the efficiency rate of in vitro fertilization. Thus, our findings document that the presence or absence of conserved regions in the C-terminus of OVGP1 modify its association with the zona pellucida that affects matrix structure and renders the zona matrix permissive to sperm penetration and OVGP1 endocytosis into the egg. PMID:27601270

  7. Structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    PubMed

    Kim, Sun-Yong; Hakoshima, Toshio; Kitano, Ken

    2013-11-21

    Bloom syndrome is a rare genetic disorder characterized by genomic instability and cancer predisposition. The disease is caused by mutations of the Bloom syndrome protein (BLM). Here we report the crystal structure of a RecQ C-terminal (RQC) domain from human BLM. The structure reveals three novel features of BLM RQC which distinguish it from the previous structures of the Werner syndrome protein (WRN) and RECQ1. First, BLM RQC lacks an aromatic residue at the tip of the β-wing, a key element of the RecQ-family helicases used for DNA-strand separation. Second, a BLM-specific insertion between the N-terminal helices exhibits a looping-out structure that extends at right angles to the β-wing. Deletion mutagenesis of this insertion interfered with binding to Holliday junction. Third, the C-terminal region of BLM RQC adopts an extended structure running along the domain surface, which may facilitate the spatial positioning of an HRDC domain in the full-length protein.

  8. Molecular architecture of the nucleoprotein C-terminal domain from the Ebola and Marburg viruses.

    PubMed

    Baker, Laura E; Ellena, Jeffrey F; Handing, Katarzyna B; Derewenda, Urszula; Utepbergenov, Darkhan; Engel, Daniel A; Derewenda, Zygmunt S

    2016-01-01

    The Filoviridae family of negative-sense, single-stranded RNA (ssRNA) viruses is comprised of two species of Marburgvirus (MARV and RAVV) and five species of Ebolavirus, i.e. Zaire (EBOV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV) and Bundibugyo (BDBV). In each of these viruses the ssRNA encodes seven distinct proteins. One of them, the nucleoprotein (NP), is the most abundant viral protein in the infected cell and within the viral nucleocapsid. It is tightly associated with the viral RNA in the nucleocapsid, and during the lifecycle of the virus is essential for transcription, RNA replication, genome packaging and nucleocapsid assembly prior to membrane encapsulation. The structure of the unique C-terminal globular domain of the NP from EBOV has recently been determined and shown to be structurally unrelated to any other known protein [Dziubańska et al. (2014), Acta Cryst. D70, 2420-2429]. In this paper, a study of the C-terminal domains from the NP from the remaining four species of Ebolavirus, as well as from the MARV strain of Marburgvirus, is reported. As expected, the crystal structures of the BDBV and TAFV proteins show high structural similarity to that from EBOV, while the MARV protein behaves like a molten globule with a core residual structure that is significantly different from that of the EBOV protein.

  9. The spt5 C-terminal region recruits yeast 3' RNA cleavage factor I.

    PubMed

    Mayer, Andreas; Schreieck, Amelie; Lidschreiber, Michael; Leike, Kristin; Martin, Dietmar E; Cramer, Patrick

    2012-04-01

    During transcription elongation, RNA polymerase II (Pol II) binds the general elongation factor Spt5. Spt5 contains a repetitive C-terminal region (CTR) that is required for cotranscriptional recruitment of the Paf1 complex (D. L. Lindstrom et al., Mol. Cell. Biol. 23:1368-1378, 2003; Z. Zhang, J. Fu, and D. S. Gilmour, Genes Dev. 19:1572-1580, 2005). Here we report a new role of the Spt5 CTR in the recruitment of 3' RNA-processing factors. Chromatin immunoprecipitation (ChIP) revealed that the Spt5 CTR is required for normal recruitment of pre-mRNA cleavage factor I (CFI) to the 3' ends of Saccharomyces cerevisiae genes. RNA contributes to CFI recruitment, as RNase treatment prior to ChIP further decreases CFI ChIP signals. Genome-wide ChIP profiling detected occupancy peaks of CFI subunits around 100 nucleotides downstream of the polyadenylation (pA) sites of genes. CFI recruitment to this defined region may result from simultaneous binding to the Spt5 CTR, to nascent RNA containing the pA sequence, and to the elongating Pol II isoform that is phosphorylated at serine 2 (S2) residues in its C-terminal domain (CTD). Consistent with this model, the CTR interacts with CFI in vitro but is not required for pA site recognition and transcription termination in vivo.

  10. Structure of the C-terminal Domain of Transcription Facto IIB from Trypanosoma brucei

    SciTech Connect

    Ibrahim, B.; Kanneganti, N; Rieckhof, G; Das, A; Laurents, D; Palenchar, J; Bellofatto, V; Wah, D

    2009-01-01

    In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 A resolution structure of the C-terminal domain of tTFIIB (tTFIIBC). The tTFIIBC structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIBC contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms.

  11. Evaluation of plasma C-terminal atrial natriuretic peptide in healthy cats and cats with heart disease.

    PubMed

    Hori, Y; Yamano, S; Iwanaga, K; Kano, T; Tanabe, M; Uechi, M; Kanai, K; Nakao, R; Hoshi, F; Higuchi, S

    2008-01-01

    The clinical implications of evaluating C-terminal atrial natriuretic peptide (ANP) concentration in cats are still controversial. The objective of this study was to investigate the relationship between plasma C-terminal ANP concentration and left atrial pressure (LAP) in healthy cats with volume overload (study 1), and to compare plasma C-terminal ANP in normal cats and cats with cardiomyopathy (study 2). Five healthy adult cats were used in study 1, and clinically healthy cats (n=8) and cats with cardiomyopathy (n=14) were used in study 2. In study 1, cats were anesthetized and given acetated Ringer's solution (100 mL/kg/h for 60 minute) via the cephalic vein. Hemodynamic measurements and blood samples, collected from the jugular vein, were performed at 10-min intervals. In study 2, blood samples from normal cats and cats with cardiomyopathy were collected from the cephalic vein. The plasma C-terminal ANP concentration was determined by radioimmunoassay for human alpha-ANP. In study 1, volume overload significantly increased the C-terminal ANP concentration and LAP from baseline. The C-terminal ANP concentration was strongly correlated with the mean LAP. In study 2, age, E wave velocity, and the ratios of the left atrium to aorta were significantly higher in the cats with cardiomyopathy compared with the normal cats. The C-terminal ANP concentration was significantly higher in the cats with cardiomyopathy compared with the normal cats. Our results suggest that the measurement of plasma C-terminal ANP in cats may provide additional information for the diagnosis of heart disease.

  12. Iron-sulfur cluster biosynthesis: functional characterization of the N- and C-terminal domains of human NFU.

    PubMed

    Liu, Yushi; Qi, Wenbin; Cowan, J A

    2009-02-10

    Human NFU (also known as HIRIP5) has been implicated in cellular iron-sulfur cluster biosynthesis. Bacterial and yeast forms are smaller than the human protein and are homologous to the C-terminal domain of the latter. This C-terminal domain contains a pair of redox active cysteines and demonstrates thioredoxin-like activity by mediating persulfide bond cleavage of sulfur-loaded NifS (an IscS-type protein), the sulfide donor for [2Fe-2S] cluster assembly on ISU-type scaffold proteins. Herein, the affinity of full-length human NFU and the individual N- and C-terminal domains for sulfide donor and cluster scaffold proteins is assessed. The influence of the N-terminal domain on C-terminal NFU binding to NifS and persulfide reductase activity is also examined. Only the C-terminal domain is required for persulfide reductase activity, while complex formation of NifS with full-length NFU is similar to that of the C-terminal domain alone (K(D) approximately 9.7 +/- 0.7 and 10.1 +/- 0.6 microM, respectively). There is negligible affinity between the isolated C- and N-terminal domains, while the N-terminal domain has negligible affinity for either sulfide donor or cluster scaffold proteins. The temperature dependence of the binding enthalpy for formation of the complex between NifS and the C-terminal domain of NFU yields a change in molar heat capacity (DeltaC(p) approximately 138 cal mol(-1) K(-1)) that suggests bonding at the protein-protein interface is dominated by electrostatic interactions. This is consistent with electrostatic potential maps for bacterial homologues of the N- and C-terminal domains of human NFU, which most likely reflect the structural characteristics expected for full-length human NFU.

  13. The Atlastin C-terminal Tail Is an Amphipathic Helix That Perturbs the Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion

    PubMed Central

    Faust, Joseph E.; Desai, Tanvi; Verma, Avani; Ulengin, Idil; Sun, Tzu-Lin; Moss, Tyler J.; Betancourt-Solis, Miguel A.; Huang, Huey W.; Lee, Tina; McNew, James A.

    2015-01-01

    Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion. PMID:25555915

  14. The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

    PubMed

    Klug, Yoel A; Kapach, Gal; Rotem, Etai; Dubreuil, Benjamin; Shai, Yechiel

    2016-04-01

    For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

  15. Direct binding of the PDZ domain of Dishevelled to a conserved internal sequence in the C-terminal region of Frizzled.

    PubMed

    Wong, Hing-C; Bourdelas, Audrey; Krauss, Anke; Lee, Ho-Jin; Shao, Youming; Wu, Dianqing; Mlodzik, Marek; Shi, De-Li; Zheng, Jie

    2003-11-01

    The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl.

  16. The C-terminal MIR-containing region in the Pmt1 O-mannosyltransferase restrains sporulation and is dispensable for virulence in Beauveria bassiana.

    PubMed

    He, Zhangjiang; Luo, Linli; Keyhani, Nemat O; Yu, Xiaodong; Ying, Shenghua; Zhang, Yongjun

    2017-02-01

    Protein O-mannosyltransferases (Pmts) belong to a highly conserved protein family responsible for the initiation of O-glycosylation of many proteins. Pmts contain one dolichyl-phosphate-mannose-protein mannosyltransferases (PMT) domain and three MIR motifs (mannosyltransferase, inositol triphosphate, and ryanodine receptor) that are essential for activity in yeast. We report that in the insect fungal pathogen, Beauveria bassiana, deletion of the C-terminal Pmt1 MIR-containing region (Pmt1∆ (311-902)) does not alter O-mannosyltransferase activity, but does increase total cell wall protein O-mannosylation levels and results in phenotypic changes in fungal development and cell wall stability. B. bassiana mutants harboring the Pmt1 ∆ (311-902) mutation displayed a significant increase in conidiation with up-regulation of conidiation-associated genes and an increase in biomass accumulation as compared to the wild-type parent. However, decreased vegetative growth and blastospore production was noted, and Pmt1 ∆ (311-902) mutants were altered in cell wall composition and cell surface features. Insect bioassays revealed little effect on virulence for the Pmt1 ∆ (311-902) strain via cuticle infection or intrahemocoel injection assays, although differences in hyphal body differentiation in the host hemolymph and up-regulation of virulence-associated genes were noted. These data suggest novel roles for Pmt1 in negatively regulating conidiation and demonstrate that the C-terminal Pmt1 MIR-containing region is dispensable for enzymatic activity and organismal virulence.

  17. Direct Binding of the PDZ Domain of Dishevelled to a Conserved Internal Sequence in the C-Terminal Region of Frizzled

    PubMed Central

    Wong, Hing-C.; Bourdelas, Audrey; Krauss, Anke; Lee, Ho-Jin; Shao, Youming; Wu, Dianqing; Mlodzik, Marek; Shi, De-Li; Zheng, Jie

    2015-01-01

    Summary The cytoplasmic protein Dishevelled (Dvl) and the associated membrane-bound receptor Frizzled (Fz) are essential in canonical and noncanonical Wnt signaling pathways. However, the molecular mechanisms underlying this signaling are not well understood. By using NMR spectroscopy, we determined that an internal sequence of Fz binds to the conventional peptide binding site in the PDZ domain of Dvl; this type of site typically binds to C-terminal binding motifs. The C-terminal region of the Dvl inhibitor Dapper (Dpr) and Frodo bound to the same site. In Xenopus, Dvl binding peptides of Fz and Dpr/Frodo inhibited canonical Wnt signaling and blocked Wnt-induced secondary axis formation in a dose-dependent manner, but did not block noncanonical Wnt signaling mediated by the DEP domain. Together, our results identify a missing molecular connection within the Wnt pathway. Differences in the binding affinity of the Dvl PDZ domain and its binding partners may be important in regulating signal transduction by Dvl. PMID:14636582

  18. Diverse C-terminal sequences involved in Flavobacterium johnsoniae protein secretion.

    PubMed

    Kulkarni, Surashree S; Zhu, Yongtao; Brendel, Colton J; McBride, Mark J

    2017-04-10

    Flavobacterium johnsoniae and many related bacteria secrete proteins across the outer membrane using the type IX secretion system (T9SS). Proteins secreted by T9SSs have amino-terminal signal peptides for export across the cytoplasmic membrane by the Sec system and carboxy-terminal domains (CTDs) targeting them for secretion across the outer membrane by the T9SS. Most but not all T9SS CTDs belong to family TIGR04183 (type-A CTDs). We functionally characterized diverse CTDs for secretion by the F. johnsoniae T9SS. Attachment of the CTDs from F. johnsoniae RemA, AmyB, and ChiA to the foreign protein sfGFP that had a signal peptide at the amino terminus resulted in secretion across the outer membrane. In each case approximately 80 to 100 amino acids from the extreme carboxy-termini was needed for efficient secretion. Several type-A CTDs from distantly related members of the phylum Bacteroidetes functioned in F. johnsoniae, supporting secretion of sfGFP by the F. johnsoniae T9SS. F. johnsoniae SprB requires the T9SS for secretion but lacks a type-A CTD. It has a conserved C-terminal domain belonging to family TIGR04131, which we refer to as a type-B CTD. The CTD of SprB was required for its secretion, but attachment of C-terminal regions of SprB of up to 1182 amino acids to sfGFP failed to result in secretion. Additional features outside of the C-terminal region of SprB may be required for its secretion.Importance Type IX protein secretion systems (T9SSs) are common in but limited to members of the phylum Bacteroidetes Most proteins that are secreted by T9SSs have conserved carboxy-terminal domains that belong to either protein domain family TIGR04183 (type-A CTDs) or TIGR04131 (type-B CTDs). Here we identify features of T9SS CTDs of F. johnsoniae that are required for protein secretion and demonstrate that type-A CTDs from distantly related members of the phylum function with the F. johnsoniae T9SS to secrete the foreign protein sfGFP. In contrast, type-B CTDs failed

  19. C-terminal amide to alcohol conversion changes the cardiovascular effects of endomorphins in anesthetized rats.

    PubMed

    Yu, Ye; Wang, Chang-lin; Cui, Yun; Fan, Ying-zhe; Liu, Jing; Shao, Xuan; Liu, Hong-mei; Wang, Rui

    2006-01-01

    Endomorphin1-ol (Tyr-Pro-Trp-Phe-ol, EM1-ol) and endomorphin2-ol (Tyr-Pro-Phe-Phe-ol, EM2-ol), with C-terminal alcohol (-ol) containing, have been shown to exhibit higher affinity and lower intrinsic efficacy in vitro than endomorphins. In the present study, in order to investigate the alterations of systemic hemodynamic effects induced by C-terminal amide to alcohol conversion, responses to intravenous (i.v.) or intracerebroventricular (i.c.v.) injection of EM1-ol, EM2-ol and their parents were compared in the system arterial pressure (SAP) and heart rate (HR) of anesthetized rats. Both EM1-ol and EM2-ol induced dose-related decrease in SAP and HR when injected in doses of 3-100 nmol/kg, i.v. In terms of relative vasodepressor activity, it is interesting to note that EM2-ol was more potent than endomorphin2 [the dose of 25% decrease in SAP (DD25) = 6.01+/-3.19 and 13.99+/-1.56 nmol/kg, i.v., respectively] at a time when responses to EM1-ol were less potent than endomorphin1. Moreover, decreases in SAP in response to EM1-ol and EM2-ol were reduced by naloxone, atropine sulfate, L-NAME and bilateral vagotomy. It indicated that the vasodepressor responses were possibly mediated by a naloxone-sensitive, nitric oxide release, vagus-activated mechanism. It is noteworthy that i.c.v. injections of -ol derivatives produced dose-related decreases in SAP and HR, which were significantly less potent than endomorphins and were attenuated by naloxone and atropine sulfate. In summary, the results of the present study indicated that the C-terminal amide to alcohol conversion produced different effects on the vasodepressor activity of endomorphin1 and endomorphin2 and endowed EM2-ol distinctive hypotension characters in peripheral (i.v.) and central (i.c.v.) tissues. Moreover, these results provided indirect evidence that amidated C-terminus might play an important role in the regulation of the cardiovascular system.

  20. Conformationally restricted C-terminal peptides of substance P. Synthesis, mass spectral analysis and pharmacological properties.

    PubMed

    Theodoropoulos, D; Poulos, C; Gatos, D; Cordopatis, P; Escher, E; Mizrahi, J; Regoli, D; Dalietos, D; Furst, A; Lee, T D

    1985-10-01

    Four cyclic analogues of the C-terminal hepta- or hexapeptide of substance P were prepared by the solution method. The cyclizations were obtained by substituting with cysteine the residues normally present in positions 5 or 6 or 11 of substance P and by subsequent disulfide bond formation. The final products were identified by ordinary analytical procedures and advanced mass spectroscopy. The biological activities were determined on three bioassays: the guinea pig ileum, the guinea pig trachea and the rabbit mesenteric vein. Results obtained with these assays indicate that all peptides with a disulfide bridgehead in position 11 are inactive and that a cycle between positions 5 and 6 already strongly reduces the biological activity. The acyclic precursors containing thiol protection groups display weak biological activities. These results further underline the importance of the side chain in position 11 of substance P and suggest that optimal biological activities may require a linear peptide sequence.

  1. The C-terminal region of E1A: a molecular tool for cellular cartography.

    PubMed

    Yousef, Ahmed F; Fonseca, Gregory J; Cohen, Michael J; Mymryk, Joe S

    2012-04-01

    The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.

  2. Development of a tertiary-structure model of the C-terminal domain of DPP IV.

    PubMed

    Brandt, W

    2000-01-01

    Based on the recently published structure of prolyl oligopeptidase (POP) a model of the C-terminal part of dipeptidyl peptidase IV (DPP IV) which contains the active site has been developed. The structure of the model of DPP IV shows considerable similarity to the structure of POP particularly in the active site. A hydrophobic pocket (Tyr666, Tyr670, Tyr 631, Val556) forms the S1-binding site for recognition of proline. Tyr547 may stabilise the oxyanion formed in the tetrahedral intermediates by a strong hydrogen bond. The positively charged N-terminus of ligands of DPP IV is recognised by forming a salt bridge with the acidic side chain Glu668. A second hydrophobic pocket (S2' to S5') may represent an important binding site for HIV-1 Tat-protein derivatives, chemokines and others.

  3. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  4. Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone

    PubMed Central

    Coggins, John R.; Kray, William; Shaw, Elliott

    1974-01-01

    Methods are described for the synthesis of peptides terminating in Lys-CH2Cl. The products were examined as affinity labels for several enzymes of trypsin-like specificity which are resistant to Tos-Lys-CH2Cl. In part, the inertness of the latter may be due to the sulphonamide group, since Z-Lys-CH2Cl was more effective. However, a number of tripeptides with C-terminal Lys-CH2Cl were superior in their ability to inactivate subtilisin, thrombin and plasma kallikrein. The possibility of developing enzyme-specific reagents selective for members within the trypsin-like group is demonstrated by Ala-Phe-Lys-CH2Cl, which readily inactivates plasma kallikrein but not thrombin. PMID:4422496

  5. Ubiquitin C-terminal hydrolase L1 deficiency decreases bone mineralization.

    PubMed

    Shim, Sehwan; Kwon, Young-Bae; Yoshikawa, Yasuhiro; Kwon, Jungkee

    2008-06-01

    Ubiquitin C-terminal hydrolase L1 is a component of the ubiquitin proteasome system, which evidences unique biological activities. In this study, we report the pattern of UCH-L1 expression, and show that it regulates bone mineralization in osteogenesis. UCH-L1 was expressed in osteoblasts, osteoclasts, and hematopoietic precursor cells of bone marrow in the metaphysis and diaphysis of the femora. To further assess the involvement of UCH-L1 in the regulation of bone mineralization, we evaluated the bone mineral density (BMD) rate of gad mice, using the Latheta computed tomography system. Male gad mice evidenced a significantly decreased BMD rate in the metaphysis and diaphysis of the femora. These findings of decreased BMD rate in the bones of gad mice may suggest that UCH-L1 function regulates bone mineralization during osteogenesis.

  6. Structure of the C-Terminal Domain of Lettuce Necrotic Yellows Virus Phosphoprotein

    PubMed Central

    Martinez, Nicolas; Ribeiro, Euripedes A.; Leyrat, Cédric; Tarbouriech, Nicolas; Ruigrok, Rob W. H.

    2013-01-01

    Lettuce necrotic yellows virus (LNYV) is a prototype of the plant-adapted cytorhabdoviruses. Through a meta-prediction of disorder, we localized a folded C-terminal domain in the amino acid sequence of its phosphoprotein. This domain consists of an autonomous folding unit that is monomeric in solution. Its structure, solved by X-ray crystallography, reveals a lollipop-shaped structure comprising five helices. The structure is different from that of the corresponding domains of other Rhabdoviridae, Filoviridae, and Paramyxovirinae; only the overall topology of the polypeptide chain seems to be conserved, suggesting that this domain evolved under weak selective pressure and varied in size by the acquisition or loss of functional modules. PMID:23785215

  7. C-Terminal-oriented Immobilization of Enzymes Using Sortase A-mediated Technique.

    PubMed

    Hata, Yuto; Matsumoto, Takuya; Tanaka, Tsutomu; Kondo, Akihiko

    2015-10-01

    In the present study, sortase A-mediated immobilization of enzymes was used for the preparation of immobilized enzymes. Thermobifida fusca YX β-glucosidase (BGL) or Streptococcus bovis 148 α-amylase (AmyA) were produced with C-terminal sortase A recognition sequences. The resulting fusion proteins were successfully immobilized on nanoparticle surfaces using sortase A. Some properties (activity, stability, and reusability) of the immobilized fusion proteins were evaluated. Both immobilized BGL and immobilized AmyA prepared by the sortase A-mediated technique retained their catalytic activity, exhibiting activities 3.0- or 1.5-fold (respectively) of those seen with the same enzymes immobilized by chemical crosslinking. Immobilized enzymes prepared by the sortase A-mediated technique did not undergo dramatic changes in stability compared with the respective free enzymes. Thus, the sortase A-mediated technique provides a promising method for immobilization of active, stable enzymes.

  8. C-terminal truncation of GSK-3β enhances its dephosphorylation by PP2A.

    PubMed

    Jin, Nana; Wu, Yue; Xu, Wen; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2017-03-07

    Glycogen synthase kinase-3β (GSK-3β) is the major tau kinase. Its phosphorylation at Ser9 suppresses the activity. In Alzheimer's disease (AD) brain, GSK-3β is truncated at the C-terminus by over-activated calpain I, leading to an increase in its activity. However, the effect of truncation on its phosphorylation is unknown. We found here that in AD brain and in cultured cells, C-terminally truncated GSK-3β is less phosphorylated at Ser9 than the full-length enzyme. The truncation promotes GSK-3β nuclear translocation and enhances its interaction with protein phosphatase 2A (PP2A), leading to dephosphorylation. Thus, the truncation of GSK-3β may enhance its activity through Ser9 dephosphorylation by PP2A. Our findings shed new light onto the role of calpain - GSK-3β - PP2A in tau pathogenesis of AD. This article is protected by copyright. All rights reserved.

  9. Control of cytoplasmic dynein force production and processivity by its C-terminal domain

    PubMed Central

    Nicholas, Matthew P.; Höök, Peter; Brenner, Sibylle; Wynne, Caitlin L.; Vallee, Richard B.; Gennerich, Arne

    2015-01-01

    Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function. PMID:25670086

  10. Folding of the C-terminal bacterial binding domain in statherin upon adsorption onto hydroxyapatite crystals

    PubMed Central

    Goobes, Gil; Goobes, Rivka; Schueler-Furman, Ora; Baker, David; Stayton, Patrick S.; Drobny, Gary P.

    2006-01-01

    Statherin is an enamel pellicle protein that inhibits hydroxyapatite (HAP) nucleation and growth, lubricates the enamel surface, and is recognized by oral bacteria in periodontal diseases. We report here from solid-state NMR measurements that the protein's C-terminal region folds into an α-helix upon adsorption to HAP crystals. This region contains the binding sites for bacterial fimbriae that mediate bacterial cell adhesion to the surface of the tooth. The helical segment is shown through long-range distance measurements to fold back onto the intermediate region (residues Y16–P28) defining the global fold of the protein. Statherin, previously shown to be unstructured in solution, undergoes conformation selection on its substrate mineral surface. This surface-induced folding of statherin can be related to its functionality in inhibiting HAP crystal growth and can explain how oral pathogens selectively recognize HAP-bound statherin. PMID:17060618

  11. Control of cytoplasmic dynein force production and processivity by its C-terminal domain

    NASA Astrophysics Data System (ADS)

    Nicholas, Matthew P.; Höök, Peter; Brenner, Sibylle; Wynne, Caitlin L.; Vallee, Richard B.; Gennerich, Arne

    2015-02-01

    Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a ‘cap’ over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

  12. Design and biological testing of peptidic dimerization inhibitors of human Hsp90 that target the C-terminal domain.

    PubMed

    Bopp, Bertan; Ciglia, Emanuele; Ouald-Chaib, Anissa; Groth, Georg; Gohlke, Holger; Jose, Joachim

    2016-06-01

    Small molecules targeting the dimerization interface of the C-terminal domain of Hsp90, a validated target for cancer treatment, have yet to be identified. Three peptides were designed with the aim to inhibit the dimerization of Hsp90. Computational and biophysical methods examined the α-helical structure for the three peptides. Based on the Autodisplay technology, a novel flow cytometer dimerization assay was developed to test inhibition of Hsp90 dimerization. Microscale thermophoresis was used to determine the K(D) of the peptides towards the C-terminal domain of Hsp90. MD simulations and CD spectroscopy indicated an α-helical structure for two of the three peptides. By flow cytometer analysis, IC(50) values of 2.08 μM for peptide H2 and 8.96 μM for peptide H3 were determined. Dimer formation of the C-terminal dimerization domain was analyzed by microscale thermophoresis, and a K(D) of 1.29 nM was determined. Furthermore, microscale thermophoresis studies demonstrated a high affinity binding of H2 and H3 to the C-terminal domain, with a K(D) of 1.02 μM and 1.46 μM, respectively. These results revealed the first peptidic inhibitors of Hsp90 dimerization targeting the C-terminal domain. Furthermore, it has been shown that these peptides bind to the C-terminal domain with a low micromolar affinity. These results can be used to design and screen for small molecules that inhibit the dimerization of the C-terminal domain of Hsp90, which could open a new route for cancer therapy. Copyright © 2016. Published by Elsevier B.V.

  13. C-terminal region of Mad2 plays an important role during mitotic spindle checkpoint in fission yeast Schizosaccharomyces pombe.

    PubMed

    Singh, Gaurav Kumar; Karade, Sharanbasappa Shrimant; Ranjan, Rajeev; Ahamad, Nafees; Ahmed, Shakil

    2017-02-01

    The mitotic arrest deficiency 2 (Mad2) protein is an essential component of the spindle assembly checkpoint that interacts with Cdc20/Slp1 and inhibit its ability to activate anaphase promoting complex/cyclosome (APC/C). In bladder cancer cell line the C-terminal residue of the mad2 gene has been found to be deleted. In this study we tried to understand the role of the C-terminal region of mad2 on the spindle checkpoint function. To envisage the role of C-terminal region of Mad2, we truncated 25 residues of Mad2 C-terminal region in fission yeast S.pombe and characterized its effect on spindle assembly checkpoint function. The cells containing C-terminal truncation of Mad2 exhibit sensitivity towards microtubule destabilizing agent suggesting perturbation of spindle assembly checkpoint. Further, the C-terminal truncation of Mad2 exhibit reduced viability in the nda3-KM311 mutant background at non-permissive temperature. Truncation in mad2 gene also affects its foci forming ability at unattached kinetochore suggesting that the mad2-∆CT mutant is unable to maintain spindle checkpoint activation. However, in response to the defective microtubule, only brief delay of mitotic progression was observed in Mad2 C-terminal truncation mutant. In addition we have shown that the deletion of two β strands of Mad2 protein abolishes its ability to interact with APC activator protein Slp1/Cdc20. We purpose that the truncation of two β strands (β7 and β8) of Mad2 destabilize the safety belt and affect the Cdc20-Mad2 interaction leading to defects in the spindle checkpoint activation.

  14. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production.

    PubMed

    Poksay, Karen S; Sheffler, Douglas J; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E; Cosford, Nicholas D P; John, Varghese

    2017-01-01

    Alzheimer's disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds - identified here using cells and tissues expressing wt human APP - in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects.

  15. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I‧ region

    NASA Astrophysics Data System (ADS)

    Anderson, Benjamin A.; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-01

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I‧ band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D2O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  16. Autoinhibition of Bacteriophage T4 Mre11 by Its C-terminal Domain*

    PubMed Central

    Gao, Yang; Nelson, Scott W.

    2014-01-01

    Mre11 and Rad50 form a stable complex (MR) and work cooperatively in repairing DNA double strand breaks. In the bacteriophage T4, Rad50 (gene product 46) enhances the nuclease activity of Mre11 (gene product 47), and Mre11 and DNA in combination stimulate the ATPase activity of Rad50. The structural basis for the cross-activation of the MR complex has been elusive. Various crystal structures of the MR complex display limited protein-protein interfaces that mainly exist between the C terminus of Mre11 and the coiled-coil domain of Rad50. To test the role of the C-terminal Rad50 binding domain (RBD) in Mre11 activation, we constructed a series of C-terminal deletions and mutations in bacteriophage T4 Mre11. Deletion of the RBD in Mre11 eliminates Rad50 binding but only has moderate effect on its intrinsic nuclease activity; however, the additional deletion of the highly acidic flexible linker that lies between RBD and the main body of Mre11 increases the nuclease activity of Mre11 by 20-fold. Replacement of the acidic residues in the flexible linker with alanine elevates the Mre11 activity to the level of the MR complex when combined with deletion of RBD. Nuclease activity kinetics indicate that Rad50 association and deletion of the C terminus of Mre11 both enhance DNA substrate binding. Additionally, a short peptide that contains the flexible linker and RBD of Mre11 acts as an inhibitor of Mre11 nuclease activity. These results support a model where the Mre11 RBD and linker domain act as an autoinhibitory domain when not in complex with Rad50. Complex formation with Rad50 alleviates this inhibition due to the tight association of the RBD and the Rad50 coiled-coil. PMID:25077970

  17. Screening for Small Molecule Inhibitors of Statin-Induced APP C-terminal Toxic Fragment Production

    PubMed Central

    Poksay, Karen S.; Sheffler, Douglas J.; Spilman, Patricia; Campagna, Jesus; Jagodzinska, Barbara; Descamps, Olivier; Gorostiza, Olivia; Matalis, Alex; Mullenix, Michael; Bredesen, Dale E.; Cosford, Nicholas D. P.; John, Varghese

    2017-01-01

    Alzheimer’s disease (AD) is characterized by neuronal and synaptic loss. One process that could contribute to this loss is the intracellular caspase cleavage of the amyloid precursor protein (APP) resulting in release of the toxic C-terminal 31-amino acid peptide APP-C31 along with the production of APPΔC31, full-length APP minus the C-terminal 31 amino acids. We previously found that a mutation in APP that prevents this caspase cleavage ameliorated synaptic loss and cognitive impairment in a murine AD model. Thus, inhibition of this cleavage is a reasonable target for new therapeutic development. In order to identify small molecules that inhibit the generation of APP-C31, we first used an APPΔC31 cleavage site-specific antibody to develop an AlphaLISA to screen several chemical compound libraries for the level of N-terminal fragment production. This antibody was also used to develop an ELISA for validation studies. In both high throughput screening (HTS) and validation testing, the ability of compounds to inhibit simvastatin- (HTS) or cerivastatin- (validation studies) induced caspase cleavage at the APP-D720 cleavage site was determined in Chinese hamster ovary (CHO) cells stably transfected with wildtype (wt) human APP (CHO-7W). Several compounds, as well as control pan-caspase inhibitor Q-VD-OPh, inhibited APPΔC31 production (measured fragment) and rescued cell death in a dose-dependent manner. The effective compounds fell into several classes including SERCA inhibitors, inhibitors of Wnt signaling, and calcium channel antagonists. Further studies are underway to evaluate the efficacy of lead compounds – identified here using cells and tissues expressing wt human APP – in mouse models of AD expressing mutated human APP, as well as to identify additional compounds and determine the mechanisms by which they exert their effects. PMID:28261092

  18. Kinetic and inhibition studies on substrate channelling in the bifunctional enzyme catalysing C-terminal amidation.

    PubMed Central

    Moore, A B; May, S W

    1999-01-01

    A series of experiments has been conducted to investigate the possibility that substrate channelling might occur in the bifunctional forms of enzymes carrying out C-terminal amidation, a post-translational modification essential to the biological activity of many neuropeptides. C-terminal amidation entails sequential action by peptidylglycine mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5), with the mono-oxygenase catalysing conversion of a glycine-extended pro-peptide into the corresponding alpha-hydroxyglycine derivative, which is then converted by the lyase into amidated peptide plus glyoxylate. Since the mono-oxygenase and lyase reactions exhibit tandem reaction stereospecificities, channelling of the alpha-hydroxy intermediate might occur, as is the case for some other multifunctional enzymes. Selective inhibition of the mono-oxygenase domain by competitive ester inhibitors, as well as mechanism-based mono-oxygenase inactivation by the novel olefinic inhibitor 5-acetamido-4-oxo-6-phenylhex-2-enoate (N-acetylphenylalanyl acrylate), has little to no effect on the kinetic parameters of the lyase domain of the AE from Xenopus laevis. Similarly, inhibition of the lyase domain by the potent dioxo inhibitor 2,4-dioxo-5-acetamido-6-phenylhexanoate has little effect on the activity of the monooxygenase domain in the bifunctional enzyme. A series of experiments on intermediate accumulation and conversion were also carried out, along with kinetic investigations of the reactivities of the monofunctional and bifunctional forms of PAM and PGL towards substrates and inhibitors. Taken together, the results demonstrate the kinetic independence of the mono-oxygenase and lyase domains, and provide no evidence for substrate channelling between these domains in the bifunctional amidating enzyme. PMID:10377242

  19. Mutational analysis of the C-terminal FATC domain of Saccharomyces cerevisiae Tra1

    PubMed Central

    Hoke, Stephen M. T.; Irina Mutiu, A.; Genereaux, Julie; Kvas, Stephanie; Buck, Michael; Yu, Michael; Gloor, Gregory B.

    2010-01-01

    Tra1 is a component of the Saccharomyces cerevisiae SAGA and NuA4 complexes and a member of the PIKK family, which contain a C-terminal phosphatidylinositol 3-kinase-like (PI3K) domain followed by a 35-residue FATC domain. Single residue changes of L3733A and F3744A, within the FATC domain, resulted in transcriptional changes and phenotypes that were similar but not identical to those caused by mutations in the PI3K domain or deletions of other SAGA or NuA4 components. The distinct nature of the FATC mutations was also apparent from the additive effect of tra1-L3733A with SAGA, NuA4, and tra1 PI3K domain mutations. Tra1-L3733A associates with SAGA and NuA4 components and with the Gal4 activation domain, to the same extent as wild-type Tra1; however, steady-state levels of Tra1-L3733A were reduced. We suggest that decreased stability of Tra1-L3733A accounts for the phenotypes since intragenic suppressors of tra1-L3733A restored Tra1 levels, and reducing wild-type Tra1 led to comparable growth defects. Also supporting a key role for the FATC domain in the structure/function of Tra1, addition of a C-terminal glycine residue resulted in decreased association with Spt7 and Esa1, and loss of cellular viability. These findings demonstrate the regulatory potential of mechanisms targeting the FATC domains of PIKK proteins. Electronic supplementary material The online version of this article (doi:10.1007/s00294-010-0313-3) contains supplementary material, which is available to authorized users. PMID:20635087

  20. Temperature dependence of C-terminal carboxylic group IR absorptions in the amide I' region.

    PubMed

    Anderson, Benjamin A; Literati, Alex; Ball, Borden; Kubelka, Jan

    2015-01-05

    Studies of structural changes in peptides and proteins using IR spectroscopy often rely on subtle changes in the amide I' band as a function of temperature. However, these changes can be obscured by the overlap with other absorptions, namely the side-chain and terminal carboxylic groups. The former were the subject of our previous report (Anderson et al., 2014). In this paper we investigate the IR spectra of the asymmetric stretch of α-carboxylic groups for amino acids representing all major types (Gly, Ala, Val, Leu, Ser, Thr, Asp, Glu, Lys, Asn, His, Trp, Pro) as well as the C-terminal groups of three dipeptides (Gly-Gly, Gly-Ala, Ala-Gly) in D₂O at neutral pH. Experimental temperature dependent IR spectra were analyzed by fitting of both symmetric and asymmetric pseudo-Voigt functions. Qualitatively the spectra exhibit shifts to higher frequency, loss in intensity and narrowing with increased temperature, similar to that observed previously for the side-chain carboxylic groups of Asp. The observed dependence of the band parameters (frequency, intensity, width and shape) on temperature is in all cases linear: simple linear regression is therefore used to describe the spectral changes. The spectral parameters vary between individual amino acids and show systematic differences between the free amino acids and dipeptides, particularly in the absolute peak frequencies, but the temperature variations are comparable. The relative variations between the dipeptide spectral parameters are most sensitive to the C-terminal amino acid, and follow the trends observed in the free amino acid spectra. General rules for modeling the α-carboxylic IR absorption bands in peptides and proteins as the function of temperature are proposed.

  1. A proprotein convertase subtilisin-like/kexin type 9 (PCSK9) C-terminal domain antibody antigen-binding fragment inhibits PCSK9 internalization and restores low density lipoprotein uptake.

    PubMed

    Ni, Yan G; Condra, Jon H; Orsatti, Laura; Shen, Xun; Di Marco, Stefania; Pandit, Shilpa; Bottomley, Matthew J; Ruggeri, Lionello; Cummings, Richard T; Cubbon, Rose M; Santoro, Joseph C; Ehrhardt, Anka; Lewis, Dale; Fisher, Timothy S; Ha, Sookhee; Njimoluh, Leila; Wood, Dana D; Hammond, Holly A; Wisniewski, Douglas; Volpari, Cinzia; Noto, Alessia; Lo Surdo, Paola; Hubbard, Brian; Carfí, Andrea; Sitlani, Ayesha

    2010-04-23

    PCSK9 binds to the low density lipoprotein receptor (LDLR) and leads to LDLR degradation and inhibition of plasma LDL cholesterol clearance. Consequently, the role of PCSK9 in modulating circulating LDL makes it a promising therapeutic target for treating hypercholesterolemia and coronary heart disease. Although the C-terminal domain of PCSK9 is not involved in LDLR binding, the location of several naturally occurring mutations within this region suggests that it has an important role for PCSK9 function. Using a phage display library, we identified an anti-PCSK9 Fab (fragment antigen binding), 1G08, with subnanomolar affinity for PCSK9. In an assay measuring LDL uptake in HEK293 and HepG2 cells, 1G08 Fab reduced 50% the PCSK9-dependent inhibitory effects on LDL uptake. Importantly, we found that 1G08 did not affect the PCSK9-LDLR interaction but inhibited the internalization of PCSK9 in these cells. Furthermore, proteolysis and site-directed mutagenesis studies demonstrated that 1G08 Fab binds a region of beta-strands encompassing Arg-549, Arg-580, Arg-582, Glu-607, Lys-609, and Glu-612 in the PCSK9 C-terminal domain. Consistent with these results, 1G08 fails to bind PCSK9DeltaC, a truncated form of PCSK9 lacking the C-terminal domain. Additional studies revealed that lack of the C-terminal domain compromised the ability of PCSK9 to internalize into cells, and to inhibit LDL uptake. Together, the present study demonstrate that the PCSK9 C-terminal domain contribute to its inhibition of LDLR function mainly through its role in the cellular uptake of PCSK9 and LDLR complex. 1G08 Fab represents a useful new tool for delineating the mechanism of PCSK9 uptake and LDLR degradation.

  2. SERS characterization of neuropeptide Y and its C-terminal fragments deposited onto colloidal gold nanoparticle surface.

    PubMed

    Domin, Helena; Piergies, Natalia; Święch, Dominika; Pięta, Ewa; Proniewicz, Edyta

    2017-01-01

    It has been suggested that the family of neuropeptide Y (NPY) peptides is a promising target for the neuroprotective therapy; therefore, knowledge of the structure of these biologically active compounds and their behavior at solid/liquid interface is important in order to design new analogues. Because there is still a lack of detailed information on the behavior of NPY and its mutated analogues at the solid/liquid interfaces, in this work surface-enhanced Raman spectroscopy (SERS) analysis was used to investigate NPY and its native NPY(3-36), NPY(13-36), and NPY(22-36) and mutated acetyl-(Leu(28,31))-NPY(24-36)C-terminal fragments, acting on Y2 receptors (Y2R), in order to determine their possible metal surface/molecule interactions. In these studies, colloidal gold nanoparticle surface served as a solid surface, whereas an aqueous solution was used as a liquid medium. The observed differences in the band intensities, wavenumbers, and widths allowed us to draw conclusions on an adsorption mode of NPY and on changes in this mode upon the shortening of the peptide chain and increase in solution pH (from pH 3 to pH 11). Briefly, three different species of Tyr were identified onto the colloidal gold surface depending upon the length of the peptide chain and solution pH. Tyrosine (TyrOH) is present in a basic medium. Tyrosinate (TyrO(-)) is present in an acidic solution, whereas phenoxyl radical (Tyr(*)) appears at neutral pH for peptides having relatively short peptide chain (acetyl-(Leu(28,31))-NPY(24-36)). The elongation of the peptide chain partially (NPY(13-36) and NPY(22-36)) or completely (NPY(3-36) and NPY) protects the Tyr residue against conversion to the radical form. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain.

    PubMed

    Poirier, Steve; Hamouda, Hocine Ait; Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9.

  4. Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

    PubMed Central

    Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

    2016-01-01

    PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

  5. An antibody against the C-terminal domain of PCSK9 lowers LDL cholesterol levels in vivo.

    PubMed

    Schiele, Felix; Park, John; Redemann, Norbert; Luippold, Gerd; Nar, Herbert

    2014-02-20

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with autosomal dominant hypercholesterolemia, a state of elevated levels of LDL (low-density lipoprotein) cholesterol. Autosomal dominant hypercholesterolemia can result in severe implications such as stroke and coronary heart disease. The inhibition of PCSK9 function by therapeutic antibodies that block interaction of PCSK9 with the epidermal growth factor-like repeat A domain of LDL receptor (LDLR) was shown to successfully lower LDL cholesterol levels in clinical studies. Here we present data on the identification, structural and biophysical characterization and in vitro and in vivo pharmacology of a PCSK9 antibody (mAb1). The X-ray structure shows that mAb1 binds the module 1 of the C-terminal domain (CTD) of PCSK9. It blocks access to an area bearing several naturally occurring gain-of-function and loss-of-function mutations. Although the antibody does not inhibit binding of PCSK9 to epidermal growth factor-like repeat A, it partially reverses PCSK9-induced reduction of the LDLR and LDL cholesterol uptake in a cellular assay. mAb1 is also effective in lowering serum levels of LDL cholesterol in cynomolgus monkeys in vivo. Complete loss of PCSK9 is associated with insufficient liver regeneration and increased risk of hepatitis C infections. Blocking of the CTD is sufficient to partially inhibit PCSK9 function. Antibodies binding the CTD of PCSK9 may thus be advantageous in patients that do not tolerate complete inhibition of PCSK9.

  6. The ROXY1 C-terminal L**LL motif is essential for the interaction with TGA transcription factors.

    PubMed

    Li, Shutian; Gutsche, Nora; Zachgo, Sabine

    2011-12-01

    Glutaredoxins (GRXs) are small, ubiquitous, glutathione-dependent oxidoreductases that participate in redox-regulated processes associated with stress responses. Recently, GRXs have been shown to exert crucial functions during flower developmental processes. GRXs modulate their target protein activities by the reduction of protein disulfide bonds or deglutathionylation reactions. The Arabidopsis (Arabidopsis thaliana) GRX ROXY1 participates in petal primordia initiation and further petal morphogenesis. ROXY1 belongs to a land plant-specific class of GRXs with a CC-type active site motif, deviating from the ubiquitously occurring CPYC and CGFS GRX classes. ROXY1 was previously shown to interact with floral TGA transcription factors in the nucleus, and this interaction is a prerequisite for ROXY1 to exert its activity required for Arabidopsis petal development. Deletion analysis further identified the importance of the ROXY1 C terminus for the ROXY1/TGA protein interactions and for the ROXY1 function in petal development. Here, by dissecting the ROXY1 C terminus, an α-helical L**LL motif immediately adjacent to the ROXY1 C-terminal eight amino acids was identified that is essential for the interaction with TGA transcription factors and crucial for the ROXY1 function in planta. Similar to the α-helical L**LL motifs binding to transcriptional coactivators with liganded nuclear receptors in animals, a hydrophobic face formed by the conserved leucines in the L**LL motif of ROXY1 possibly mediates the interaction with TGA transcription factors. Thus, the α-helical L**LL sequence is a conserved protein-protein interaction motif in both animals and plants. Furthermore, two separate TGA domains were identified by deletion experiments as being essential for mediating TGA protein interactions with ROXYs.

  7. SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes

    PubMed Central

    Rainbow, RD; Lodwick, D; Hudman, D; Davies, NW; Norman, RI; Standen, NB

    2004-01-01

    C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K+ channels (KATP). To investigate the protective role of KATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A-fragment, reduced sarcolemmal KATP currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 ± 1.22; and A-fragment 0.94 ± 0.07 pA pF−1, n = 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 pA pF−1, n = 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca2+ loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca2+ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca2+ loading, was abolished by the A-fragment. Our data are consistent with a role for KATP channels in causing action potential failure and reduced Ca2+ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal KATP channel, but we also discuss the possibility of mitochondrial effects. PMID:15020694

  8. C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction

    PubMed Central

    Spring, Ashlyn M.; Brusich, Douglas J.; Frank, C. Andrew

    2016-01-01

    Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals–but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating

  9. Carboxypeptidase D is the only enzyme responsible for antibody C-terminal lysine cleavage in Chinese hamster ovary (CHO) cells.

    PubMed

    Hu, Zhilan; Zhang, Henry; Haley, Benjamin; Macchi, Frank; Yang, Feng; Misaghi, Shahram; Elich, Joseph; Yang, Renee; Tang, Yun; Joly, John C; Snedecor, Bradley R; Shen, Amy

    2016-10-01

    Heterogeneity of C-terminal lysine levels often observed in therapeutic monoclonal antibodies is believed to result from the proteolysis by endogenous carboxypeptidase(s) during cell culture production. Identifying the responsible carboxypeptidase(s) for C-terminal lysine cleavage in CHO cells would provide valuable insights for antibody production cell culture processes development and optimization. In this study, five carboxypeptidases, CpD, CpM, CpN, CpB, and CpE, were studied for message RNA (mRNA) expression by qRT-PCR analysis in two most commonly used blank hosts (DUXB-11 derived DHFR-deficient DP12 host and DHFR-positive CHOK1 host), used for therapeutic antibody production, as well an antibody-expressing cell line derived from each host. Our results showed that CpD had the highest mRNA expression. When CpD mRNA levels were reduced by RNAi (RNA interference) technology, C-terminal lysine levels increased, whereas there was no obvious change in C-terminal lysine levels when a different carboxypeptidase mRNA level was knocked down suggesting that carboxypeptidase D is the main contributor for C-terminal lysine processing. Most importantly, when CpD expression was knocked out by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology, C-terminal lysine cleavage was completely abolished in CpD knockout cells based on mass spectrometry analysis, demonstrating that CpD is the only endogenous carboxypeptidase that cleaves antibody heavy chain C-terminal lysine in CHO cells. Hence, our work showed for the first time that the cleavage of antibody heavy chain C-terminal lysine is solely mediated by the carboxypeptidase D in CHO cells and our finding provides one solution to eliminating C-terminal lysine heterogeneity for therapeutic antibody production by knocking out CpD gene expression. Biotechnol. Bioeng. 2016;113: 2100-2106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. Requirement of the C-terminal proline residue for stability of the Ca(2+)-activated photoprotein aequorin.

    PubMed Central

    Watkins, N J; Campbell, A K

    1993-01-01

    cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine. Images Figure 1 Figure 2 PMID:8101077

  11. The C-terminal region of alpha-crystallin: involvement in protection against heat-induced denaturation

    NASA Technical Reports Server (NTRS)

    Takemoto, L.; Emmons, T.; Horwitz, J.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    Recent studies have demonstrated that the alpha-crystallins can protect other proteins against heat-induced denaturation and aggregation. To determine the possible involvement of the C-terminal region in this activity, the alpha-crystallins were subjected to limited tryptic digestion, and the amount of cleavage from the N-terminal and C-terminal regions of the alpha-A and alpha-B crystallin chains was assessed using antisera specific for these regions. Limited tryptic digestion resulted in cleavage only from the C-terminal region of alpha-A crystallin. This trypsin-treated alpha-A crystallin preparation showed a decreased ability to protect proteins from heat-induced aggregation using an in vitro assay. Together, these results demonstrate that the C-terminal region of alpha-A crystallin is important for its ability to protect against heat-induced aggregation, which is consistent with the hypothesis that post-translational changes that are known to occur at the C-terminal region may have significant effects on the ability of alpha-A crystallin to protect against protein denaturation in vivo.

  12. YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal*

    PubMed Central

    Login, Frédéric H.; Wolf-Watz, Hans

    2015-01-01

    All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca2+-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the “classical” N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. PMID:26338709

  13. YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal.

    PubMed

    Login, Frédéric H; Wolf-Watz, Hans

    2015-10-23

    All type III secretion systems (T3SS) harbor a member of the YscU/FlhB family of proteins that is characterized by an auto-proteolytic process that occurs at a conserved cytoplasmic NPTH motif. We have previously demonstrated that YscUCC, the C-terminal peptide generated by auto-proteolysis of Yersinia pseudotuberculosis YscU, is secreted by the T3SS when bacteria are grown in Ca(2+)-depleted medium at 37 °C. Here, we investigated the secretion of this early T3S-substrate and showed that YscUCC encompasses a specific C-terminal T3S signal within the 15 last residues (U15). U15 promoted C-terminal secretion of reporter proteins like GST and YopE lacking its native secretion signal. Similar to the "classical" N-terminal secretion signal, U15 interacted with the ATPase YscN. Although U15 is critical for YscUCC secretion, deletion of the C-terminal secretion signal of YscUCC did neither affect Yop secretion nor Yop translocation. However, these deletions resulted in increased secretion of YscF, the needle subunit. Thus, these results suggest that YscU via its C-terminal secretion signal is involved in regulation of the YscF secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Trypanosoma evansi: identification and characterization of a variant surface glycoprotein lacking cysteine residues in its C-terminal domain.

    PubMed

    Jia, Yonggen; Zhao, Xinxin; Zou, Jingru; Suo, Xun

    2011-01-01

    African trypanosomes are flagellated unicellular parasites which proliferate extracellularly in the mammalian host blood-stream and tissue spaces. They evade the hosts' antibody-mediated lyses by sequentially changing their variant surface glycoprotein (VSG). VSG tightly coats the entire parasite body, serving as a physical barrier. In Trypanosoma brucei and the closely related species Trypanosoma evansi, Trypanosoma equiperdum, each VSG polypeptide can be divided into N- and C-terminal domains, based on cysteine distribution and sequence homology. N-terminal domain, the basis of antigenic variation, is hypervariable and contains all the exposed epitopes; C-terminal domain is relatively conserved and a full set of four or eight cysteines were generally observed. We cloned two genes from two distinct variants of T. evansi, utilizing RT-PCR with VSG-specific primers. One contained a VSG type A N-terminal domain followed a C-terminal domain lacking cysteine residues. To confirm that this gene is expressed as a functional VSG, the expression and localization of the corresponding gene product were characterized using Western blotting and immunofluorescent staining of living trypanosomes. Expression analysis showed that this protein was highly expressed, variant-specific, and had a ubiquitous cellular surface localization. All these results indicated that it was expressed as a functional VSG. Our finding showed that cysteine residues in VSG C-terminal domain were not essential; the conserved C-terminal domain generally in T. brucei like VSGs would possibly evolve for regulating the VSG expression.

  15. An autoinhibitory helix in the C-terminal region of phospholipase C-[beta] mediates G[alpaha subscript q] activation

    SciTech Connect

    Lyon, Angeline M.; Tesmer, Valerie M.; Dhamsania, Vishan D.; Thal, David M.; Gutierrez, Joanne; Chowdhury, Shoaib; Suddala, Krishna C.; Northup, John K.; Tesmer, John J.G.

    2012-03-16

    The enzyme phospholipase C-{beta} (PLC{beta}) is a crucial regulator of intracellular calcium levels whose activity is controlled by heptahelical receptors that couple to members of the G{sub q} family of heterotrimeric G proteins. We have determined atomic structures of two invertebrate homologs of PLC{beta} (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction in human PLC{beta}3 considerably increase basal activity and diminish stimulation by G{alpha}{sub q}. G{alpha}{sub q} binding requires displacement of the autoinhibitory helix from the catalytic core, thus providing an allosteric mechanism for activation of PLC{beta}.

  16. In contrast to agonist monoclonal antibodies, both C-terminal truncated form and full length form of Pleiotrophin failed to activate vertebrate ALK (anaplastic lymphoma kinase)?

    PubMed

    Mathivet, Thomas; Mazot, Pierre; Vigny, Marc

    2007-12-01

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development in specific regions of the central and peripheral nervous system. ALK expression persists at a lower level in the adult brain. Thus, it might play an important role in both the normal development and function of the nervous system. The nature of the cognate ligand of this receptor in vertebrates is still a matter of debate. Pleiotrophin and midkine have been proposed as ligands of ALK but several independent studies do not confirm this hypothesis. Interestingly, a recent study proposed that a C-terminal truncated form of Pleiotrophin (Pleiotrophin.15) and not the full length form (Pleiotrophin.18) promotes glioblastoma proliferation in an ALK-dependent fashion. These data were obviously a strong basis to conciliate the conflicting results so far reported in the literature. In the present study, we first purified to homogeneity the two forms of Pleiotrophin secreted by HEK 293 cells. In contrast to agonist monoclonal antibodies, both Pleiotrophin.15 and Pleiotrophin.18 failed to activate ALK in neuroblastoma and glioblastoma cells expressing this receptor. Thus, for our point of view, ALK is still an orphan receptor in vertebrates.

  17. Hsp90 N- and C-terminal double inhibition synergistically suppresses Bcr-Abl-positive human leukemia cells.

    PubMed

    Chen, Chun; Zhuang, Yingting; Chen, Xianling; Chen, Xiaole; Li, Ding; Fan, Yingjuan; Xu, Jianhua; Chen, Yuanzhong; Wu, Lixian

    2017-02-07

    Heat shock protein 90 (Hsp90) contains amino (N)-terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states.

  18. Correct processing of the kiwifruit protease actinidin in transgenic tobacco requires the presence of the C-terminal propeptide.

    PubMed Central

    Paul, W; Amiss, J; Try, R; Praekelt, U; Scott, R; Smith, H

    1995-01-01

    A 355 cauliflower mosaic virus promoter and a tapetum-specific promoter were used to direct the synthesis in tobacco of preproactinidin and a derivative that lacked a C-terminal extension. Preproactinidin was processed into a form that migrated identically on protein gels with mature actinidin extracted from kiwifruit. This protein was proteolytically active in vitro, and high-level accumulation of this protein appeared to be detrimental to plant growth. Plants expressing an actinidin cDNA construct that lacked the sequence encoding the C-terminal propeptide were phenotypically normal but accumulated N-proactinidin, which was proteolytically active in vitro but did not self-cleave to mature actinidin. In transgenic tobacco, the C-terminal extension of actinidin is therefore required for correct processing. PMID:7784505

  19. Mre11 nuclease and C-terminal tail-mediated DDR functions are required for initiating yeast telomere healing.

    PubMed

    Bhattacharyya, M K; Matthews, K M; Lustig, A J

    2008-08-01

    Mre11 is a central factor in creating an optimal substrate for telomerase loading and elongation. We have used a G2/M synchronized telomere-healing assay as a tool to separate different functions of Mre11 that are not apparent in null alleles. An analysis of healing efficiencies of several mre11 alleles revealed that both nuclease and C-terminal mutations led to a loss of healing. Interestingly, trans-complementation of the 49 amino acid C-terminal deletion (DeltaC49) and the D16A mutant, deficient in nuclease activity and partially defective in MRX complex formation, restores healing. DeltaC49 provokes Rad53 phosphorylation after treatment with the radiomimetic agent MMS exclusively through the Tel1 pathway, suggesting that a Tel1-mediated function is initiated through the C-terminal tail.

  20. A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER.

    PubMed

    Reithinger, Johannes H; Yim, Chewon; Park, Kwangjin; Björkholm, Patrik; von Heijne, Gunnar; Kim, Hyun

    2013-11-01

    Sdh3/Shh3, a subunit of mitochondrial succinate dehydrogenase, contains transmembrane domains with a hydrophobicity comparable to that of endoplasmic reticulum (ER) proteins. Here, we show that a C-terminal reporter fusion to Sdh3/Shh3 results in partial mis-targeting of the protein to the ER. This mis-targeting is mediated by the signal recognition particle (SRP) and depends on the length of the C-terminal tail. These results imply that if nuclear-encoded mitochondrial proteins contain strongly hydrophobic transmembrane domains and a long C-terminal tail, they have the potential to be recognized by SRP and mis-targeted to the ER. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  1. The polyanionic C-terminal tail of human Rad17 regulates interaction with the 9-1-1 complex.

    PubMed

    Fukumoto, Yasunori; Nakayama, Yuji; Yamaguchi, Naoto

    2017-09-02

    In the activation and maintenance of ATR-dependent DNA damage checkpoint, the interaction between the Rad17-RFC2-5 and 9-1-1 complexes is essential, however, the regulatory mechanism of the interaction is not known. Here we show that vertebrate Rad17 proteins contain a polyanionic 12-amino acid sequence in the C-terminal ends that is important for the 9-1-1 interaction. We demonstrate that the C-terminal tail contains a conserved sequence designated iVERGE that must be intact for the 9-1-1 interaction and contains potential posttranslational modification sites. Our data raise a possibility that the Rad17 C-terminal tail is a molecular switch that regulates the 9-1-1 interaction and the ATR pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Binding of a C-terminal fragment (residues 369 to 435) of vitamin D-binding protein to actin.

    PubMed

    Buch, Stefan; Gremm, Dagmar; Wegner, Albrecht; Mannherz, Hans Georg

    2002-10-01

    The vitamin D-binding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The full-length protein and the N- and C-terminal halves of DBP remained insoluble probably because the protein did not fold to its native three-dimensional structure due to formation of accidental intra- and inter-molecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a C-terminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the C-terminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than full-length DBP. This suggests that in addition to the C-terminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The C-terminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentration-dependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the C-terminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

  3. Biological variability of plasma intact and C-terminal FGF23 measurements.

    PubMed

    Smith, Edward R; Cai, Michael M; McMahon, Lawrence P; Holt, Stephen G

    2012-09-01

    FGF23 measurement may have a role in the management of patients with chronic kidney disease (CKD). Our objective was to study the biological variability of plasma intact FGF23 (iFGF23) and C-terminal FGF23 (cFGF23) concentrations. Plasma samples were taken from 12 healthy adults at multiple time points on 2 consecutive days to assess diurnal variability of FGF23 concentrations. Early-morning fasting and nonfasting samples were also taken over a 6-wk period to estimate components of biological variation. Samples from 170 volunteers were used to define reference intervals. FGF23 concentrations were measured by commercial ELISA. Western blotting was used to analyze FGF23 species from the plasma of healthy adults and patients with predialysis CKD and those undergoing dialysis. A total of 180 healthy adults and 18 adults with stage 3-5D CKD participated in this study at a hospital research unit. Estimates were made of the biological variability of plasma FGF23 concentrations. iFGF23, but not cFGF23, showed significant diurnal variation. cFGF23 had a significantly lower intra-individual variation than iFGF23 (8.3 vs. 18.3%) but higher inter-individual variation than iFGF23 (28.9 vs. 19.2%). Fourteen samples would be needed to estimate an individual's homeostatic set point (within 10%) for iFGF23 compared with only three samples for cFGF23. Using Western blotting, C-terminal FGF23 fragments were detected in the plasma of individuals with normal renal function and at all stages of renal disease. The percent cFGF23 was significantly higher in those without renal impairment (P < 0.001) and was positively correlated with plasma phosphate concentration in those with normal renal function. The high intra-individual biological variability of iFGF23 may limit its clinical use as a diagnostic or management tool. Risk-related thresholds may be more appropriate for clinical decision making based on cFGF23 measurements than conventional reference intervals. FGF23 cleavage pathways

  4. Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

    PubMed Central

    Dar, Mohd J; Monel, Blandine; Krishnan, Lavanya; Shun, Ming-Chieh; Di Nunzio, Francesca; Helland, Dag E; Engelman, Alan

    2009-01-01

    Background The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. Results Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. Conclusion HIV-11-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as

  5. Protein identification with N and C-terminal sequence tags in proteome projects.

    PubMed

    Wilkins, M R; Gasteiger, E; Tonella, L; Ou, K; Tyler, M; Sanchez, J C; Gooley, A A; Walsh, B J; Bairoch, A; Appel, R D; Williams, K L; Hochstrasser, D F

    1998-05-08

    Genome sequences are available for increasing numbers of organisms. The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis. Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels. The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed. Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied. Sequence tags of five amino acid residues were found to be even more specific. To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent (http://www.expasy.ch/www/tools.html), which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database. We demonstrate the utility of this identification approach with sequence tags generated from 91 different E. coli proteins purified by 2D gel electrophoresis. Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data. We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available. The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein

  6. 2-Phenylethyl ester and 2-phenylethyl amide derivative analogues of the C-terminal hepta- and octapeptide of cholecystokinin.

    PubMed

    Fulcrand, P; Rodriguez, M; Galas, M C; Lignon, M F; Laur, J; Aumelas, A; Martinez, J

    1988-11-01

    Syntheses of analogues of the C-terminal octa- and heptapeptide of cholecystokinin are described. These analogues were obtained by replacing the C-terminal phenylalanine residue by 2-phenylethyl alcohol or by 2-phenylethylamine derivatives and by replacing the tryptophan residue by a D-tryptophan. The CCK-derivatives were tested for their ability to inhibit binding of labeled CCK-8 to rat pancreatic acini and to guinea pig brain membranes, and for their action on stimulation of amylase release from rat pancreatic acini. Some of these derivatives appeared to exhibit only part of the CCK-activity on amylase release, the D-Trp analogues behaving as CCK-antagonists.

  7. [Covalent C-terminal fixation of cyanogen bromide peptides in the liquid-phase-sequenator (author's transl)].

    PubMed

    Braunitzer, G; Pfletschinger, J

    1978-08-01

    This paper describes the covalent fixation and hydrophilisation of homoserin lactone peptides enabling complete C-terminal sequencing in the squenator. Dimethylformamide, dimethylsulfoxide and 6M guanidine hydrochloride in water were used as solvents, ethylendiamine, hexamethylendiamine and histamine base as amino components. The diamine peptide derivative was reacted with the hydrophilic isothiocyanates I and IV, the fixed peptide was sequenced to the C-terminal amino acid, Histamine reacted particularly well and the program with 0.1N quadrol and the hydrophobic buffers was especially suitable for this derivative. The phenylthiohydantoin derivative of homoserine was proven in good yields. The application of this method is suggested.

  8. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    NASA Astrophysics Data System (ADS)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  9. The C-terminal helix of Bcl-xL mediates Bax retrotranslocation from the mitochondria

    PubMed Central

    Todt, F; Cakir, Z; Reichenbach, F; Youle, R J; Edlich, F

    2013-01-01

    The proapoptotic Bcl-2 protein Bax can commit a cell to apoptosis by translocation from the cytosol to the mitochondria and permeabilization of the outer mitochondrial membrane. Prosurvival Bcl-2 family members, such as Bcl-xL, control Bax activity. Bcl-xL recognizes Bax after a conformational change in the N-terminal segment of Bax on the mitochondria and retrotranslocates it back into the cytoplasm, stabilizing the inactive form of Bax. Here we show that Bax retrotranslocation depends on the C-terminal helix of Bcl-xL. Deletion or substitution of this segment reduces Bax retrotranslocation and correlates with the accumulation of GFP-tagged or endogenous Bax on the mitochondria of non-apoptotic cells. Unexpectedly, the substitution of the Bcl-xL membrane anchor by the corresponding Bax segment reverses the Bax retrotranslocation activity of Bcl-xL, but not that of Bcl-xL shuttling. Bax retrotranslocation depends on interaction to the Bcl-xL membrane anchor and interaction between the Bax BH3 domain and the Bcl-xL hydrophobic cleft. Interference with either interaction increases mitochondrial levels of endogenous Bax. In healthy cells, mitochondrial Bax does not permeabilize the outer mitochondrial membrane, but increases cell death after apoptosis induction. PMID:23079612

  10. Microtubule C-Terminal Tails Can Change Characteristics of Motor Force Production.

    PubMed

    Shojania Feizabadi, Mitra; Janakaloti Narayanareddy, Babu Reddy; Vadpey, Omid; Jun, Yonggun; Chapman, Dail; Rosenfeld, Steven; Gross, Steven P

    2015-10-01

    Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties--if at all--is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin-1, and also a mitotic kinesin likely optimized for group function, kinesin-5, moving along either bovine brain or MCF7(breast cancer) MTs. We found that kinesin-1 functioned similarly on the two sets of MTs--in particular, its mean force production was approximately the same, though due to its previously reported decreased processivity, the mean duration of kinesin-1 force production was slightly decreased on MCF7 MTs. In contrast, kinesin-5's function changed dramatically on MCF7 MTs: its average detachment force was reduced and its force-velocity curve was different. In spite of the reduced detachment force, the force-velocity alteration surprisingly improved high-load group function for kinesin-5 on the cancer-cell MTs, potentially contributing to functions such as spindle-mediated chromosome separation. Significant differences were previously reported for C-terminal tubulin tails in MCF7 versus bovine brain tubulin. Consistent with this difference being functionally important, elimination of the tails made transport along the two sets of MTs similar. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region

    PubMed Central

    Faraj, Santiago E.; González-Lebrero, Rodolfo M.; Roman, Ernesto A.; Santos, Javier

    2016-01-01

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics. PMID:26856628

  12. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    NASA Astrophysics Data System (ADS)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  13. Pharmacologic study of C-terminal fragments of frog skin calcitonin gene-related peptide.

    PubMed

    Ladram, Ali; Besné, Isabelle; Breton, Lionel; de Lacharrière, Olivier; Nicolas, Pierre; Amiche, Mohamed

    2008-07-01

    The calcitonin gene-related peptide from the skin of the frog Phyllomedusa bicolor (pbCGRP) is a 37-residue neuropeptide that differs from human alpha CGRP (halphaCGRP) at 16 positions. The affinities of the C-terminal fragments of pbCGRP and halphaCGRP were evaluated in SK-N-MC cells: pbCGRP(8-37) (K(i)=0.2nM) and pbCGRP(27-37) (K(i)=95nM) were, respectively, 3 times and 20 times more potent than the human fragments halphaCGRP(8-37) and halphaCGRP(27-37). Their antagonistic potencies were measured in SK-N-MC and Col 29 cells, and the rat vas deferens. pbCGRP(8-37) inhibited the halphaCGRP-stimulated production of cAMP by SK-N-MC and Col 29 cells 3 to 4 times more strongly than halphaCGRP(8-37). Thus pbCGRP(8-37) is the most potent CGRP-1 competitive antagonist of all the natural sequences reported to date. pbCGRP(27-37) was also as potent as [D(31), A(34), F(35)] halphaCGRP(27-37), a prototypic antagonist analog derived from structure-activity relationship studies of halphaCGRP(8-37).

  14. RGS19 enhances cell proliferation through its C-terminal PDZ motif.

    PubMed

    Tso, Prudence H; Wang, Yingchun; Wong, Sivia Y S; Poon, Lydia S W; Chan, Anthony S L; Wong, Yung H

    2010-11-01

    Regulator of G protein signaling 19 (RGS19), also known as Galpha-interacting protein (GAIP), is a GTPase activating protein (GAP) for Galpha(i) subunits. Apart from its GAP function, RGS19 has been implicated in growth factor signaling through binding to GAIP-interacting protein C-terminus (GIPC) via its C-terminal PDZ-binding motif. To gain additional insight on its function, we have stably expressed RGS19 in a number of mammalian cell lines and examined its effect on cell proliferation. Interestingly, overexpression of RGS19 stimulated the growth of HEK293, PC12, Caco2, and NIH3T3 cells. This growth promoting effect was not shared by other RGS proteins including RGS4, RGS10 and RGS20. Despite its ability to stimulate cell proliferation, RGS19 failed to induce neoplastic transformation in NIH3T3 cells as determined by focus formation and soft-agar assays, and it did not induce tumor growth in athymic nude mice. Deletion mutants of RGS19 lacking the PDZ-binding motif failed to complex with GIPC and did not exhibit any growth promoting effect. Overexpression of GIPC alone in HEK293 cells stimulated cell proliferation whereas its knockdown in H1299 non-small cell lung carcinomas suppressed cell proliferation. This study demonstrates that RGS19, in addition to acting as a GAP, is able to stimulate cell proliferation in a GIPC-dependent manner. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  15. Design, synthesis and biological evaluation of alkylamino biphenylamides as Hsp90 C-terminal inhibitors.

    PubMed

    Garg, Gaurav; Zhao, Huiping; Blagg, Brian S J

    2017-01-15

    Hsp90 is a promising therapeutic target for the development of anti-cancer agents due to its integral role in the stability and function of proteins associated with all ten hallmarks of cancer. Novobiocin, a coumarin antibiotic, was the first natural product identified that targeted the Hsp90 C-terminal domain and manifested anti-proliferative activity (SKBr3 IC50∼700μM). Subsequent structural investigations on novobiocin led to analogues with significantly improved anti-proliferative activity against multiple cancer cell lines. In an effort to develop more efficacious and diverse analogues, it was recently found that the coumarin ring of novobiocin could be replaced with the biphenyl core without compromising activity. Based on these prior studies, a series of alkylamino biphenylamides was designed, synthesized and evaluated for anti-proliferative activity against two breast cancer cell lines. SAR studies demonstrated that the incorporation of an alkylamino side chain onto the biphenyl core improved anti-proliferative activity and resulted in compounds that exhibit sub-micromolar to mid-nanomolar activity through Hsp90 inhibition. Importantly, these studies indicate the presence of a hydrophilic region about the central core that can be exploited for the design of new inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. The C-terminal domain promotes the hemorrhagic damage caused by Vibrio vulnificus metalloprotease.

    PubMed

    Miyoshi, S; Kawata, K; Tomochika, K; Shinoda, S; Yamamoto, S

    2001-12-01

    Vibrio vulnificus, an opportunistic human pathogen, produces a 45-kDa zinc metalloprotease (V. vulnificus protease; VVP) as an important virulence determinant. VVP injected intradermally into the dorsal skin causes the hemorrhagic damage through specific degradation of type IV collage in the vascular basement membrane. The N-terminal 35-kDa polypeptide (VVP-N), the catalytic domain, also evoked the hemorrhagic skin reaction within minutes. However, the hemorrhagic activity of VVP-N was one-third of that of VVP. Besides, the proteolytic activity of VVP-N toward the reconstituted basement membrane or type IV collagen was found to be about 50 % of VVP. VVP-N, like VVP, was quickly inactivated by an equimolar amount of alpha(2)-macroglobulin, a broad-spectrum plasma protease inhibitor. These findings indicate that the C-terminal 10-kDa polypeptide, the substrate-binding domain mediating the effective binding to protein substrates, functions to augment the hemorrhagic reaction of VVP.

  17. C-terminal Amidation of an Osteocalcin-derived Peptide Promotes Hydroxyapatite Crystallization*

    PubMed Central

    Hosseini, Samaneh; Naderi-Manesh, Hossein; Mountassif, Driss; Cerruti, Marta; Vali, Hojatollah; Faghihi, Shahab

    2013-01-01

    Genesis of natural biocomposite-based materials, such as bone, cartilage, and teeth, involves interactions between organic and inorganic systems. Natural biopolymers, such as peptide motif sequences, can be used as a template to direct the nucleation and crystallization of hydroxyapatite (HA). In this study, a natural motif sequence consisting of 13 amino acids present in the first helix of osteocalcin was selected based on its calcium binding ability and used as substrate for nucleation of HA crystals. The acidic (acidic osteocalcin-derived peptide (OSC)) and amidic (amidic osteocalcin-derived peptide (OSN)) forms of this sequence were synthesized to investigate the effects of different C termini on the process of biomineralization. Electron microscopy analyses show the formation of plate-like HA crystals with random size and shape in the presence of OSN. In contrast, spherical amorphous calcium phosphate is formed in the presence of OSC. Circular dichroism experiments indicate conformational changes of amidic peptide to an open and regular structure as a consequence of interaction with calcium and phosphate. There is no conformational change detectable in OSC. It is concluded that HA crystal formation, which only occurred in OSN, is attributable to C-terminal amidation of a natural peptide derived from osteocalcin. It is also proposed that natural peptides with the ability to promote biomineralization have the potential to be utilized in hard tissue regeneration. PMID:23362258

  18. Evolutionary diversification of an ancient gene family (rhs) through C-terminal displacement.

    PubMed

    Jackson, Andrew P; Thomas, Gavin H; Parkhill, Julian; Thomson, Nicholas R

    2009-12-07

    Rhs genes are prominent features of bacterial genomes that have previously been implicated in genomic rearrangements in E. coli. By comparing rhs repertoires across the Enterobacteriaceae, this study provides a robust explanation of rhs diversification and evolution, and a mechanistic model of how rhs diversity is gained and lost. Rhs genes are ubiquitous and comprise six structurally distinct lineages within the Enterobacteriaceae. There is considerable intergenomic variation in rhs repertoire; for instance, in Salmonella enterica, rhs are restricted to mobile elements, while in Escherichia coli one rhs lineage has diversified through transposition as older lineages have been deleted. Overall, comparative genomics reveals frequent, independent gene gains and losses, as well as occasional lateral gene transfer, in different genera. Furthermore, we demonstrate that Rhs 'core' domains and variable C-termini are evolutionarily decoupled, and propose that rhs diversity is driven by homologous recombination with circular intermediates. Existing C-termini are displaced by laterally acquired alternatives, creating long arrays of dissociated 'tips' that characterize the appearance of rhs loci. Rhs repertoires are highly dynamic among Enterobacterial genomes, due to repeated gene gains and losses. In contrast, the primary structures of Rhs genes are evolutionarily conserved, indicating that rhs sequence diversity is driven, not by rapid mutation, but by the relatively slow evolution of novel core/tip combinations. Hence, we predict that a large pool of dissociated rhs C-terminal tips exists episomally and these are potentially transmitted across taxonomic boundaries.

  19. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function.

    PubMed

    Blaurock, Nancy; Schmerler, Diana; Hünniger, Kerstin; Kurzai, Oliver; Ludewig, Katrin; Baier, Michael; Brunkhorst, Frank Martin; Imhof, Diana; Kiehntopf, Michael

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis.

  20. Identification and characterization of the role of c-terminal Src kinase in dengue virus replication

    PubMed Central

    Kumar, Rinki; Agrawal, Tanvi; Khan, Naseem Ahmed; Nakayama, Yuji; Medigeshi, Guruprasad R.

    2016-01-01

    We screened a siRNA library targeting human tyrosine kinases in Huh-7 cells and identified c-terminal Src kinase (Csk) as one of the kinases involved in dengue virus replication. Knock-down of Csk expression by siRNAs or inhibition of Csk by an inhibitor reduced dengue virus RNA levels but did not affect viral entry. Csk partially colocalized with viral replication compartments. Dengue infection was drastically reduced in cells lacking the three ubiquitous src family kinases, Src, Fyn and Yes. Csk knock-down in these cells failed to block dengue virus replication suggesting that the effect of Csk is via regulation of Src family kinases. Csk was found to be hyper-phosphorylated during dengue infection and inhibition of protein kinase A led to a block in Csk phosphorylation and dengue virus replication. Overexpression studies suggest an important role for the kinase and SH3 domains in this process. Our results identified a novel role for Csk as a host tyrosine kinase involved in dengue virus replication and provide further insights into the role of host factors in dengue replication. PMID:27457684

  1. Dynamic condensation of linker histone C-terminal domain regulates chromatin structure.

    PubMed

    Luque, Antoni; Collepardo-Guevara, Rosana; Grigoryev, Sergei; Schlick, Tamar

    2014-07-01

    The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region.

    PubMed

    Faraj, Santiago E; González-Lebrero, Rodolfo M; Roman, Ernesto A; Santos, Javier

    2016-02-09

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich's Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.

  3. Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation ▿

    PubMed Central

    García, Alicia; Rosonina, Emanuel; Manley, James L.; Calvo, Olga

    2010-01-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle. PMID:20823273

  4. A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

    PubMed Central

    Aitken, Stuart; Alexander, Ross D.; Beggs, Jean D.

    2013-01-01

    The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3′ end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics. PMID:23804443

  5. Immunometric assay of BN 52080, a heptapeptide C-terminal analogue of sorbin.

    PubMed

    Ezan, E; Tarrade, T; Cazenave, C; Ardouin, T; Genet, R; Grassi, J; Grognet, J M; Pradelles, P

    1995-01-01

    A novel type of enzyme immunometric assay has been developed for a heptapeptide, BN 52080. This compound is a short C-terminal analogue of sorbin and is under clinical evaluation for treatment of chronic diarrhea. In this solid-phase immobilized epitope immunoassay (SPIE-IA), the peptide is first immunologically bound to polyclonal antibodies adsorbed to a solid phase and then, after covalent immobilization with glutaraldehyde, is released from the antibody paratope by NaOH. The peptide linked to the solid phase is further quantified with a tracer consisting of the same antibodies purified by affinity chromatography and coupled to acetylcholinesterase. This assay has a detection limit of 10 pg/ml and is therefore five times more sensitive than competitive enzyme immunoassay using the same antibodies and BN 52080 coupled to acetylcholinesterase as tracer. The assay is specific and allows direct measurement of peptide in human plasma after subcutaneous or intravenous administration of 200 micrograms of BN 52080 to volunteers.

  6. C-Terminal Alpha-1 Antitrypsin Peptide: A New Sepsis Biomarker with Immunomodulatory Function

    PubMed Central

    Blaurock, Nancy; Schmerler, Diana; Hünniger, Kerstin; Kurzai, Oliver; Ludewig, Katrin; Baier, Michael; Brunkhorst, Frank Martin; Imhof, Diana; Kiehntopf, Michael

    2016-01-01

    Systemic inflammatory response syndrome (SIRS) is a life threatening condition and the leading cause of death in intensive care units. Although single aspects of pathophysiology have been described in detail, numerous unknown mediators contribute to the progression of this complex disease. The aim of this study was to elucidate the pathophysiological role of CAAP48, a C-terminal alpha-1 antitrypsin fragment, that we found to be elevated in septic patients and to apply this peptide as diagnostic marker for infectious and noninfectious etiologies of SIRS. Incubation of human polymorphonuclear neutrophils with synthetic CAAP48, the SNP-variant CAAP47, and several control peptides revealed intense neutrophil activation, induction of neutrophil chemotaxis, reduction of neutrophil viability, and release of cytokines. We determined the abundance of CAAP48 in patients with severe sepsis, severe SIRS of noninfectious origin, and viral infection. CAAP48 levels were 3-4-fold higher in patients with sepsis compared to SIRS of noninfectious origin and allowed discrimination of those patients with high sensitivity and specificity. Our results suggest that CAAP48 is a promising discriminatory sepsis biomarker with immunomodulatory functions, particularly on human neutrophils, supporting its important role in the host response and pathophysiology of sepsis. PMID:27382189

  7. Structure of the C-terminal domain of nsp4 from feline coronavirus

    PubMed Central

    Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

    2009-01-01

    Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions. PMID:19622868

  8. Role of ubiquitin C-terminal hydrolase-L1 in antipolyspermy defense of mammalian oocytes.

    PubMed

    Susor, Andrej; Liskova, Lucie; Toralova, Tereza; Pavlok, Antonin; Pivonkova, Katerina; Karabinova, Pavla; Lopatarova, Miloslava; Sutovsky, Peter; Kubelka, Michal

    2010-06-01

    The ubiquitin-proteasome system regulates many cellular processes through rapid proteasomal degradation of ubiquitin-tagged proteins. Ubiquitin C-terminal hydrolase-L1 (UCHL1) is one of the most abundant proteins in mammalian oocytes. It has weak hydrolytic activity as a monomer and acts as a ubiquitin ligase in its dimeric or oligomeric form. Recently published data show that insufficiency in UCHL1 activity coincides with polyspermic fertilization; however, the mechanism by which UCHL1 contributes to this process remains unclear. Using UCHL1-specific inhibitors, we induced a high rate of polyspermy in bovine zygotes after in vitro fertilization. We also detected decreased levels in the monomeric ubiquitin and polyubiquitin pool. The presence of UCHL1 inhibitors in maturation medium enhanced formation of presumptive UCHL1 oligomers and subsequently increased abundance of K63-linked polyubiquitin chains in oocytes. We analyzed the dynamics of cortical granules (CGs) in UCHL1-inhibited oocytes; both migration of CGs toward the cortex during oocyte maturation and fertilization-induced extrusion of CGs were impaired. These alterations in CG dynamics coincided with high polyspermy incidence in in vitro-produced UCHL1-inhibited zygotes. These data indicate that antipolyspermy defense in bovine oocytes may rely on UCHL1-controlled functioning of CGs.

  9. The region-specific functions of two ubiquitin C-terminal hydrolase isozymes along the epididymis.

    PubMed

    Kwon, Jungkee; Sekiguchi, Satoshi; Wang, Yu-Lai; Setsuie, Rieko; Yoshikawa, Yasuhiro; Wada, Keiji

    2006-01-01

    We previously showed that gad mice, which are deficient for ubiquitin C-terminal hydrolase L1 (UCH-L1), have a significantly increased number of defective spermatozoa, suggesting that UCH-L1 functions in sperm quality control during epididymal maturation. The epididymis is the site of spermatozoa maturation, transport and storage. Region-specific functions along the epididymis are essential for establishing the environment required for sperm maturation. We analyzed the region-specific expression of UCH-L1 and UCH-L3 along the epididymis, and also assessed the levels of ubiquitin, which has specificity for UCH-L1. In wild-type mice, western blot analysis demonstrated a high level of UCH-L1 expression in the caput epididymis, consistent with ubiquitin expression, whereas UCH-L3 expression was high in the cauda epididymis. We also investigated the function of UCH-L1 and UCH-L3 in epididymal apoptosis induced by efferent duct ligation. The caput epididymides of gad mice were resistant to apoptotic stress induced by efferent duct ligation, whereas Uchl3 knockout mice showed a marked increase in apoptotic cells following ligation. In conclusion, the response of gad and Uchl3 knockout mice to androgen withdrawal suggests a reciprocal function of the two UCH enzymes in the caput epididymis.

  10. Effects of ubiquitin C-terminal hydrolase L1 deficiency on mouse ova.

    PubMed

    Koyanagi, Sayaka; Hamasaki, Hiroko; Sekiguchi, Satoshi; Hara, Kenshiro; Ishii, Yoshiyuki; Kyuwa, Shigeru; Yoshikawa, Yasuhiro

    2012-03-01

    Maternal proteins are rapidly degraded by the ubiquitin-proteasome system during oocyte maturation in mice. Ubiquitin C-terminal hydrolase L1 (UCHL1) is highly and specifically expressed in mouse ova and is involved in the polyspermy block. However, the role of UCHL1 in the underlying mechanism of polyspermy block is poorly understood. To address this issue, we performed a comprehensive proteomic analysis to identify maternal proteins that were relevant to the role of UCHL1 in mouse ova using UCHL1-deficient gad. Furthermore, we assessed morphological features in gad mouse ova using transmission electron microscopy. NACHT, LRR, and PYD domain-containing (NALP) family proteins and endoplasmic reticulum (ER) chaperones were identified by proteomic analysis. We also found that the 'maternal antigen that embryos require' (NLRP5 (MATER)) protein level increased significantly in gad mouse ova compared with that in wild-type mice. In an ultrastructural study, gad mouse ova contained less ER in the cortex than in wild-type mice. These results provide new insights into the role of UCHL1 in the mechanism of polyspermy block in mouse ova.

  11. The Rrp6 C-terminal domain binds RNA and activates the nuclear RNA exosome

    PubMed Central

    Wasmuth, Elizabeth V.; Lima, Christopher D.

    2017-01-01

    The eukaryotic RNA exosome is an essential, multi-subunit complex that catalyzes RNA turnover, maturation, and quality control processes. Its non-catalytic donut-shaped core includes 9 subunits that associate with the 3′ to 5′ exoribonucleases Rrp6, and Rrp44/Dis3, a subunit that also catalyzes endoribonuclease activity. Although recent structures and biochemical studies of RNA bound exosomes from S. cerevisiae revealed that the Exo9 central channel guides RNA to either Rrp6 or Rrp44 using partially overlapping and mutually exclusive paths, several issues related to RNA recruitment remain. Here, we identify activities for the highly basic Rrp6 C-terminal tail that we term the ‘lasso’ because it binds RNA and stimulates ribonuclease activities associated with Rrp44 and Rrp6 within the 11-subunit nuclear exosome. Stimulation is dependent on the Exo9 central channel, and the lasso contributes to degradation and processing activities of exosome substrates in vitro and in vivo. Finally, we present evidence that the Rrp6 lasso may be a conserved feature of the eukaryotic RNA exosome. PMID:27899565

  12. Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels.

    PubMed

    Do, Thanh D; LaPointe, Nichole E; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C; Teplow, David B; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T

    2016-01-20

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

  13. Amyloid β-Protein C-terminal Fragments: Formation of Cylindrins and β-barrels

    PubMed Central

    Do, Thanh D.; LaPointe, Nichole E.; Nelson, Rebecca; Krotee, Pascal; Hayden, Eric Y.; Ulrich, Brittany; Quan, Sarah; Feinstein, Stuart C.; Teplow, David B.; Eisenberg, David; Shea, Joan-Emma; Bowers, Michael T.

    2016-01-01

    In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer’s disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross-sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using di-glycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins. PMID:26700445

  14. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    PubMed Central

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-01-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring. PMID:28155880

  15. Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism

    PubMed Central

    Petrlova, Jitka; Hansen, Finja C.; van der Plas, Mariena J. A.; Mörgelin, Matthias; Malmsten, Martin; Bond, Peter J.; Schmidtchen, Artur

    2017-01-01

    Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation. PMID:28473418

  16. Human Frataxin Folds Via an Intermediate State. Role of the C-Terminal Region

    NASA Astrophysics Data System (ADS)

    Faraj, Santiago E.; González-Lebrero, Rodolfo M.; Roman, Ernesto A.; Santos, Javier

    2016-02-01

    The aim of this study is to investigate the folding reaction of human frataxin, whose deficiency causes the neurodegenerative disease Friedreich’s Ataxia (FRDA). The characterization of different conformational states would provide knowledge about how frataxin can be stabilized without altering its functionality. Wild-type human frataxin and a set of mutants, including two highly destabilized FRDA-associated variants were studied by urea-induced folding/unfolding in a rapid mixing device and followed by circular dichroism. The analysis clearly indicates the existence of an intermediate state (I) in the folding route with significant secondary structure content but relatively low compactness, compared with the native ensemble. However, at high NaCl concentrations I-state gains substantial compaction, and the unfolding barrier is strongly affected, revealing the importance of electrostatics in the folding mechanism. The role of the C-terminal region (CTR), the key determinant of frataxin stability, was also studied. Simulations consistently with experiments revealed that this stretch is essentially unstructured, in the most compact transition state ensemble (TSE2). The complete truncation of the CTR drastically destabilizes the native state without altering TSE2. Results presented here shed light on the folding mechanism of frataxin, opening the possibility of mutating it to generate hyperstable variants without altering their folding kinetics.

  17. The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles

    PubMed Central

    De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

    2016-01-01

    Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite’s genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite. PMID:27270330

  18. Functional Characteristics of C-terminal Lysine to Cysteine Mutant Form of CTLA-4Ig

    PubMed Central

    Kim, Bongi; Shin, Jun-Seop

    2013-01-01

    CTLA-4Ig is regarded as an inhibitory agent of the T cell proliferation via blocking the costimulatory signal which is essential for full T cell activation. To improve applicability, we developed the CTLA-4Ig-CTKC in which the c-terminal lysine had been replaced by cysteine through single amino acid change. The single amino acid mutation of c-terminus of CTLA-4Ig was performed by PCR and was checked by in vitro transcription and translation. DNA construct of mutant form was transfected to Chinese hamster ovary (CHO) cells by electroporation. The purified proteins were confirmed by Western blot and B7-1 binding assay for their binding ability. The suppressive capacity of CTLA-4Ig-CTKC was evaluated by the mixed lymphocyte reaction (MLR) and in the allogeneic pancreatic islet transplantation model. CTLA-4Ig-CTKC maintained binding ability to B7-1 molecule and effectively inhibits T cell proliferation in MLR. In the murine allogeneic pancreatic islet transplantation, short-term treatment of CTLA-4Ig-CTKC prolonged the graft survival over 100 days. CTLA-4Ig-CTKC effectively inhibits immune response both in MLR and in allogeneic islet transplantation model, indicating that single amino acid mutation does not affect the inhibitory function of CTLA-4Ig. CTLA-4Ig-CTKC can be used in vehicle-mediated drug delivery system such as liposome conjugation. PMID:23559896

  19. The E. coli thioredoxin folding mechanism: the key role of the C-terminal helix.

    PubMed

    Vazquez, Diego S; Sánchez, Ignacio E; Garrote, Ana; Sica, Mauricio P; Santos, Javier

    2015-02-01

    In this work, the unfolding mechanism of oxidized Escherichia coli thioredoxin (EcTRX) was investigated experimentally and computationally. We characterized seven point mutants distributed along the C-terminal α-helix (CTH) and the preceding loop. The mutations destabilized the protein against global unfolding while leaving the native structure unchanged. Global analysis of the unfolding kinetics of all variants revealed a linear unfolding route with a high-energy on-pathway intermediate state flanked by two transition state ensembles TSE1 and TSE2. The experiments show that CTH is mainly unfolded in TSE1 and the intermediate and becomes structured in TSE2. Structure-based molecular dynamics are in agreement with these experiments and provide protein-wide structural information on transient states. In our model, EcTRX folding starts with structure formation in the β-sheet, while the protein helices coalesce later. As a whole, our results indicate that the CTH is a critical module in the folding process, restraining a heterogeneous intermediate ensemble into a biologically active native state and providing the native protein with thermodynamic and kinetic stability. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Dynein's C-terminal Domain Plays a Novel Role in Regulating Force Generation

    NASA Astrophysics Data System (ADS)

    Gennerich, Arne; Nicholas, Matthew; Brenner, Sibylle; Lazar, Caitlin; Weil, Sarah; Vallee, Richard; Hook, Peter; Gennerich Lab Collaboration; Vallee Lab Collaboration

    2014-03-01

    Cytoplasmic dynein is a microtubule motor involved in a wide range of low and high force requiring functions in metazoans. In contrast, yeast dynein is involved in a single, nonessential function, nuclear positioning. Interestingly, the single-molecule function of yeast dynein is also unique: whereas mammalian dyneins generate forces of 1-2 pN, S. cerevisiae dynein stalls at 5-7 pN. The basis for this functional difference is unknown. However, the major structural difference between mammalian and yeast dyneins is a ~30 kDa C-terminal extension (CT) present in higher eukaryotic dyneins, but missing in yeast. To test whether the CT accounts for the differences in function, we produced recombinant rat dynein motor domains (MD) with (WT-MD) and without (ΔCT-MD) the CT, using baculovirus expression. To define motor function, we performed single-molecule optical trapping studies. Dimerized WT-MD stalls at ~1 pN and detaches from microtubules after brief stalls, in agreement with previous studies on native mammalian dynein. In sharp contrast, but similar to yeast dynein, ΔCT-MD stalls at ~6 pN, with stall durations up to minutes. These results identify the CT as a new regulatory element for controlling dynein force generation. Supported by NIH GM094415 (A.G.) and GM102347 (R.B.V.)

  1. The C-terminal region of Trypanosoma cruzi MASPs is antigenic and secreted via exovesicles.

    PubMed

    De Pablos, Luis Miguel; Díaz Lozano, Isabel María; Jercic, Maria Isabel; Quinzada, Markela; Giménez, Maria José; Calabuig, Eva; Espino, Ana Margarita; Schijman, Alejandro Gabriel; Zulantay, Inés; Apt, Werner; Osuna, Antonio

    2016-06-08

    Trypanosoma cruzi is the etiological agent of Chagas disease, a neglected and emerging tropical disease, endemic to South America and present in non-endemic regions due to human migration. The MASP multigene family is specific to T. cruzi, accounting for 6% of the parasite's genome and plays a key role in immune evasion. A common feature of MASPs is the presence of two conserved regions: an N-terminal region codifying for signal peptide and a C-terminal (C-term) region, which potentially acts as GPI-addition signal peptide. Our aim was the analysis of the presence of an immune response against the MASP C-term region. We found that this region is highly conserved, released via exovesicles (EVs) and has an associated immune response as revealed by epitope affinity mapping, IFA and inhibition of the complement lysis assays. We also demonstrate the presence of a fast IgM response in Balb/c mice infected with T. cruzi. Our results reveal the presence of non-canonical secreted peptides in EVs, which can subsequently be exposed to the immune system with a potential role in evading immune system targets in the parasite.

  2. Characterization of the surfactin synthetase C-terminal thioesterase domain as a cyclic depsipeptide synthase.

    PubMed

    Tseng, Claire C; Bruner, Steven D; Kohli, Rahul M; Marahiel, Mohamed A; Walsh, Christopher T; Sieber, Stephan A

    2002-11-12

    The C-terminal thioesterase domain of the nonribosomal peptide synthetase producing the lipopetide surfactin (Srf TE) retains autonomous ability to generate the cyclic peptidolactone skeleton of surfactin when provided with a soluble beta-hydroxy-butyryl-heptapeptidyl thioester substrate. Utilizing the recently solved crystal structure [Bruner, S. D., et al. (2002) Structure 10, 301-310], the active-site nucleophile, Ser80, was changed to Cys, and the other members of the catalytic triad, Asp107 and His207, were changed to Ala, with the resulting mutants lacking detectable activity. Two cationic side chains in the active site, Lys111 and Arg120, were changed to Ala, causing an increased partitioning of the product to hydrolysis, as did a P26G mutant, mimicking the behavior of lipases. To evaluate recognition elements in substrates used by Srf TE, alterations to the fatty acyl group, the heptapeptide, and the thioester leaving group were made, and the resulting substrates were characterized for kinetic competency and flux of product to cyclization or hydrolysis. Alterations that could be accepted for cyclization were identified in all three parts of the substrate, although tolerance limits for changes varied. In addition, cocrystal structures of Srf TE with dipeptidyl boronate inhibitors were solved, illustrating the critical binding determinants of the substrate. On the basis of the structures and biochemical data, the cyclizing conformation of the surfactin peptide was modeled into the enzyme active site.

  3. Polycomb Group Targeting through Different Binding Partners of RING1B C-Terminal Domain

    PubMed Central

    Wang, Renjing; Taylor, Alexander B.; Leal, Belinda Z.; Chadwell, Linda V.; Ilangovan, Udayar; Robinson, Angela K.; Schirf, Virgil; Hart, P. John; Lafer, Eileen M.; Demeler, Borries; Hinck, Andrew P.; McEwen, Donald G.; Kim, Chongwoo A.

    2015-01-01

    SUMMARY RING1B, a Polycomb Group (PcG) protein, binds methylated chromatin through its association with another PcG protein called Polycomb (Pc). However, RING1B can associate with nonmethylated chromatin suggesting an alternate mechanism for RING1B interaction with chromatin. Here, we demonstrate that two proteins with little sequence identity between them, the Pc cbox domain and RYBP, bind the same surface on the C-terminal domain of RING1B (C-RING1B). Pc cbox and RYBP each fold into a nearly identical, intermolecular beta sheet with C-RING1B and a loop structure which are completely different in the two proteins. Both the beta sheet and loop are required for stable binding and transcription repression. Further, a mutation engineered to disrupt binding on the Drosophila dRING1 protein prevents chromatin association and PcG function in vivo. These results suggest that PcG targeting to different chromatin locations relies, in part, on binding partners of C-RING1B that are diverse in sequence and structure. PMID:20696397

  4. Solution structure of the RecQ C-terminal domain of human Bloom syndrome protein.

    PubMed

    Park, Chin-Ju; Ko, Junsang; Ryu, Kyoung-Seok; Choi, Byong-Seok

    2014-02-01

    RecQ C-terminal (RQC) domain is known as the main DNA binding module of RecQ helicases such as Bloom syndrome protein (BLM) and Werner syndrome protein (WRN) that recognizes various DNA structures. Even though BLM is able to resolve various DNA structures similarly to WRN, BLM has different binding preferences for DNA substrates from WRN. In this study, we determined the solution structure of the RQC domain of human BLM. The structure shares the common winged-helix motif with other RQC domains. However, half of the N-terminal has unstructured regions (α1-α2 loop and α3 region), and the aromatic side chain on the top of the β-hairpin, which is important for DNA duplex strand separation in other RQC domains, is substituted with a negatively charged residue (D1165) followed by the polar residue (Q1166). The structurally distinctive features of the RQC domain of human BLM suggest that the DNA binding modes of the BLM RQC domain may be different from those of other RQC domains.

  5. Intrinsic ssDNA annealing activity in the C-terminal region of WRN.

    PubMed

    Muftuoglu, Meltem; Kulikowicz, Tomasz; Beck, Gad; Lee, Jae Wan; Piotrowski, Jason; Bohr, Vilhelm A

    2008-09-30

    Werner syndrome (WS) is a rare autosomal recessive disorder in humans characterized by premature aging and genetic instability. WS is caused by mutations in the WRN gene, which encodes a member of the RecQ family of DNA helicases. Cellular and biochemical studies suggest that WRN plays roles in DNA replication, DNA repair, telomere maintenance, and homologous recombination and that WRN has multiple enzymatic activities including 3' to 5' exonuclease, 3' to 5' helicase, and ssDNA annealing. The goal of this study was to map and further characterize the ssDNA annealing activity of WRN. Enzymatic studies using truncated forms of WRN identified a C-terminal 79 amino acid region between the RQC and the HRDC domains (aa1072-1150) that is required for ssDNA annealing activity. Deletion of the region reduced or eliminated ssDNA annealing activity of the WRN protein. Furthermore, the activity appears to correlate with DNA binding and oligomerization status of the protein.

  6. Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function

    PubMed Central

    Snead, David; Lai, Alex L.; Wragg, Rachel T.; Parisotto, Daniel A.; Ramlall, Trudy F.; Dittman, Jeremy S.; Freed, Jack H.; Eliezer, David

    2017-01-01

    Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo. PMID:28596722

  7. Sub1 globally regulates RNA polymerase II C-terminal domain phosphorylation.

    PubMed

    García, Alicia; Rosonina, Emanuel; Manley, James L; Calvo, Olga

    2010-11-01

    The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3'-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

  8. PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

    SciTech Connect

    Zheng Wenfu; Wang Lijun; Hong Yuankai; Sha Yinlin

    2009-02-06

    PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP{sup C} to scrapie isoform PrP{sup Sc}). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126{sub CONH2} and PrP106-126{sub COOH}) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126{sub COOH} displayed a higher peptide-lipid binding affinity than PrP106-126{sub CONH2} ({approx}2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.

  9. Serpin A1 C-Terminal Peptides as Collagen Turnover Modulators.

    PubMed

    Pascarella, Simona; Tiberi, Caterina; Sabatino, Giuseppina; Nuti, Francesca; Papini, Anna Maria; Giovannelli, Lisa; Rovero, Paolo

    2016-08-19

    The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Piezo1 ion channel pore properties are dictated by C-terminal region.

    PubMed

    Coste, Bertrand; Murthy, Swetha E; Mathur, Jayanti; Schmidt, Manuela; Mechioukhi, Yasmine; Delmas, Patrick; Patapoutian, Ardem

    2015-05-26

    Piezo1 and Piezo2 encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. Structural features of Piezos remain unknown. Mouse Piezo1 is bioinformatically predicted to have 30-40 transmembrane (TM) domains. Here, we find that nine of the putative inter-transmembrane regions are accessible from the extracellular side. We use chimeras between mPiezo1 and dPiezo to show that ion-permeation properties are conferred by C-terminal region. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Our results describe important structural motifs of this channel family and lay the groundwork for a mechanistic understanding of how Piezos are mechanically gated and conduct ions.

  11. Piezo1 ion channel pore properties are dictated by C-terminal region

    PubMed Central

    Coste, Bertrand; Murthy, Swetha E.; Mathur, Jayanti; Schmidt, Manuela; Mechioukhi, Yasmine; Delmas, Patrick; Patapoutian, Ardem

    2015-01-01

    Piezo1 and Piezo2 encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. Structural features of Piezos remain unknown. Mouse Piezo1 is bioinformatically predicted to have 30–40 transmembrane (TM) domains. Here, we find that nine of the putative inter-transmembrane regions are accessible from the extracellular side. We use chimeras between mPiezo1 and dPiezo to show that ion-permeation properties are conferred by C-terminal region. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Our results describe important structural motifs of this channel family and lay the groundwork for a mechanistic understanding of how Piezos are mechanically gated and conduct ions. PMID:26008989

  12. Piezo1 ion channel pore properties are dictated by C-terminal region

    NASA Astrophysics Data System (ADS)

    Coste, Bertrand; Murthy, Swetha E.; Mathur, Jayanti; Schmidt, Manuela; Mechioukhi, Yasmine; Delmas, Patrick; Patapoutian, Ardem

    2015-05-01

    Piezo1 and Piezo2 encode mechanically activated cation channels that function as mechanotransducers involved in vascular system development and touch sensing, respectively. Structural features of Piezos remain unknown. Mouse Piezo1 is bioinformatically predicted to have 30-40 transmembrane (TM) domains. Here, we find that nine of the putative inter-transmembrane regions are accessible from the extracellular side. We use chimeras between mPiezo1 and dPiezo to show that ion-permeation properties are conferred by C-terminal region. We further identify a glutamate residue within a conserved region adjacent to the last two putative TM domains of the protein, that when mutated, affects unitary conductance and ion selectivity, and modulates pore block. We propose that this amino acid is either in the pore or closely associates with the pore. Our results describe important structural motifs of this channel family and lay the groundwork for a mechanistic understanding of how Piezos are mechanically gated and conduct ions.

  13. Endogenous C-terminal Tagging by CRISPR/Cas9 in Trypanosoma cruzi.

    PubMed

    Lander, Noelia; Chiurillo, Miguel A; Vercesi, Aníbal E; Docampo, Roberto

    2017-05-20

    To achieve the C-terminal tagging of endogenous proteins in T. cruzi we use the Cas9/pTREX-n vector (Lander et al., 2015) to insert a specific tag sequence (3xHA or 3xc-Myc) at the 3' end of a specific gene of interest (GOI). Chimeric sgRNA targeting the 3' end of the GOI is PCR-amplified and cloned into Cas9/pTREX-n vector. Then a DNA donor molecule to induce DNA repair by homologous recombination is amplified. This donor sequence contains the tag sequence and a marker for antibiotic resistance, plus 100 bp homology arms corresponding to regions located right upstream of the stop codon and downstream of the Cas9 target site at the GOI locus. Vectors pMOTag23M (Oberholzer et al., 2006) or pMOHX1Tag4H (Lander et al., 2016b) are used as PCR templates for DNA donor amplification. Epimastigotes co-transfected with the sgRNA/Cas9/pTREX-n construct and the DNA donor cassette are then cultured for 5 weeks with antibiotics for selection of double resistant parasites. Endogenous gene tagging is finally verified by PCR and Western blot analysis.

  14. Crystallization of the C-terminal globular domain of avian reovirus fibre

    PubMed Central

    van Raaij, Mark J.; Hermo Parrado, X. Lois; Guardado Calvo, Pablo; Fox, Gavin C.; Llamas-Saiz, Antonio L.; Costas, Celina; Martínez-Costas, José; Benavente, Javier

    2005-01-01

    Avian reovirus fibre, a homotrimer of the σC protein, is responsible for primary host-cell attachment. Using the protease trypsin, a C-terminal σC fragment containing amino acids 156–326 has been generated which was subsequently purified and crystallized. Two different crystal forms were obtained, one grown in the absence of divalent cations and belonging to space group P6322 (unit-cell parameters a = 75.6, c = 243.1 Å) and one grown in the presence of either zinc or cadmium sulfate and belonging to space group P321 (unit-cell parameters a = 74.7, c = 74.5 Å and a = 73.1, c = 69.9 Å for the ZnII- and CdII-grown crystals, respectively). The first crystal form diffracted synchrotron radiation to 3.0 Å resolution and the second form to 2.2–2.3 Å. Its closest related structure, the C-­terminal fragment of mammalian reovirus fibre, has only 18% sequence identity and molecular-replacement attempts were unsuccessful. Therefore, a search is under way for suitable heavy-atom derivatives and attempts are being made to grow protein crystals containing selenomethionine instead of methionine. PMID:16511119

  15. Proline isomerization in the C-terminal region of HSP27.

    PubMed

    Alderson, T Reid; Benesch, Justin L P; Baldwin, Andrew J

    2017-07-01

    In mammals, small heat-shock proteins (sHSPs) typically assemble into interconverting, polydisperse oligomers. The dynamic exchange of sHSP oligomers is regulated, at least in part, by molecular interactions between the α-crystallin domain and the C-terminal region (CTR). Here we report solution-state nuclear magnetic resonance (NMR) spectroscopy investigations of the conformation and dynamics of the disordered and flexible CTR of human HSP27, a systemically expressed sHSP. We observed multiple NMR signals for residues in the vicinity of proline 194, and we determined that, while all observed forms are highly disordered, the extra resonances arise from cis-trans peptidyl-prolyl isomerization about the G193-P194 peptide bond. The cis-P194 state is populated to near 15% at physiological temperatures, and, although both cis- and trans-P194 forms of the CTR are flexible and dynamic, both states show a residual but differing tendency to adopt β-strand conformations. In NMR spectra of an isolated CTR peptide, we observed similar evidence for isomerization involving proline 182, found within the IPI/V motif. Collectively, these data indicate a potential role for cis-trans proline isomerization in regulating the oligomerization of sHSPs.

  16. Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast.

    PubMed

    Sprague, E R; Redd, M J; Johnson, A D; Wolberger, C

    2000-06-15

    The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.

  17. Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures.

    PubMed

    Zhang, Xintao; Tang, Hongping; Sun, Ya-Ting; Liu, Xuping; Tan, Wen-Song; Fan, Li

    2015-08-01

    C-terminal lysine variants are commonly observed in monoclonal antibodies (mAbs) and found sensitive to process conditions, especially specific components in culture medium. The potential roles of media arginine (Arg) and lysine (Lys) in mAb heavy chain C-terminal lysine processing were investigated by monitoring the lysine variant levels under various Arg and Lys concentrations. Both Arg and Lys were found to significantly affect lysine variant level. Specifically, lysine variant level increased from 18.7 to 31.8 % when Arg and Lys concentrations were increased from 2 to 10 mM. Since heterogeneity of C-terminal lysine residues is due to the varying degree of proteolysis by basic carboxypeptidases (Cps), enzyme (basic Cps) level, pH conditions, and product (Arg and Lys) inhibition, which potentially affect the enzymatic reaction, were investigated under various Arg and Lys conditions. Enzyme level and pH conditions were found not to account for the different lysine variant levels, which was evident from the minimal variation in transcription level and intracellular pH. On the other hand, product inhibition effect of Arg and Lys on basic Cps was evident from the notable intracellular and extracellular Arg and Lys concentrations comparable with Ki values (inhibition constant) of basic Cps and further confirmed by cell-free assays. Additionally, a kinetic study of lysine variant level during the cell culture process enabled further characterization of the C-terminal lysine processing.

  18. Topology and dynamics of the 10 kDa C-terminal domain of DnaK in solution.

    PubMed Central

    Bertelsen, E. B.; Zhou, H.; Lowry, D. F.; Flynn, G. C.; Dahlquist, F. W.

    1999-01-01

    Hsp70 molecular chaperones contain three distinct structural domains, a 44 kDa N-terminal ATPase domain, a 17 kDa peptide-binding domain, and a 10 kDa C-terminal domain. The ATPase and peptide binding domains are conserved in sequence and are functionally well characterized. The function of the 10 kDa variable C-terminal domain is less well understood. We have characterized the secondary structure and dynamics of the C-terminal domain from the Escherichia coli Hsp70, DnaK, in solution by high-resolution NMR. The domain was shown to be comprised of a rigid structure consisting of four helices and a flexible C-terminal subdomain of approximately 33 amino acids. The mobility of the flexible region is maintained in the context of the full-length protein and does not appear to be modulated by the nucleotide state. The flexibility of this region appears to be a conserved feature of Hsp70 architecture and may have important functional implications. We also developed a method to analyze 15N nuclear spin relaxation data, which allows us to extract amide bond vector directions relative to a unique diffusion axis. The extracted angles and rotational correlation times indicate that the helices form an elongated, bundle-like structure in solution. PMID:10048327

  19. SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity

    PubMed Central

    Muramatsu, Tomonari; Takemoto, Chie; Kim, Yong-Tae; Wang, Hongfei; Nishii, Wataru; Terada, Takaho; Shirouzu, Mikako

    2016-01-01

    The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4–P1 and P1′. In this study, we determined the crystal structure of 3CLpro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3′ position [Phe(P3′)] is snugly accommodated in the S3′ pocket. Mutations of Phe(P3′) impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3′ subsite is formed by Phe(P2)-induced conformational changes of 3CLpro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3′ subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3′). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4–P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3′) is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2). PMID:27799534

  20. Occurrence of C-Terminal Residue Exclusion in Peptide Fragmentation by ESI and MALDI Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Dupré, Mathieu; Cantel, Sonia; Martinez, Jean; Enjalbal, Christine

    2012-02-01

    By screening a data set of 392 synthetic peptides MS/MS spectra, we found that a known C-terminal rearrangement was unexpectedly frequently occurring from monoprotonated molecular ions in both ESI and MALDI tandem mass spectrometry upon low and high energy collision activated dissociations with QqTOF and TOF/TOF mass analyzer configuration, respectively. Any residue localized at the C-terminal carboxylic acid end, even a basic one, was lost, provided that a basic amino acid such arginine and to a lesser extent histidine and lysine was present in the sequence leading to a fragment ion, usually depicted as (bn-1 + H2O) ion, corresponding to a shortened non-scrambled peptide chain. Far from being an epiphenomenon, such a residue exclusion from the peptide chain C-terminal extremity gave a fragment ion that was the base peak of the MS/MS spectrum in certain cases. Within the frame of the mobile proton model, the ionizing proton being sequestered onto the basic amino acid side chain, it is known that the charge directed fragmentation mechanism involved the C-terminal carboxylic acid function forming an anhydride intermediate structure. The same mechanism was also demonstrated from cationized peptides. To confirm such assessment, we have prepared some of the peptides that displayed such C-terminal residue exclusion as a C-terminal backbone amide. As expected in this peptide amide series, the production of truncated chains was completely suppressed. Besides, multiply charged molecular ions of all peptides recorded in ESI mass spectrometry did not undergo such fragmentation validating that any mobile ionizing proton will prevent such a competitive C-terminal backbone rearrangement. Among all well-known nondirect sequence fragment ions issued from non specific loss of neutral molecules (mainly H2O and NH3) and multiple backbone amide ruptures (b-type internal ions), the described C-terminal residue exclusion is highly identifiable giving raise to a single fragment ion in

  1. Charged residues in the C-terminus of the P2Y1 receptor constitute a basolateral-sorting signal

    PubMed Central

    Wolff, Samuel C.; Qi, Ai-Dong; Harden, T. Kendall; Nicholas, Robert A.

    2010-01-01

    The P2Y1 receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y1 receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins. PMID:20592187

  2. Charged residues in the C-terminus of the P2Y1 receptor constitute a basolateral-sorting signal.

    PubMed

    Wolff, Samuel C; Qi, Ai-Dong; Harden, T Kendall; Nicholas, Robert A

    2010-07-15

    The P2Y(1) receptor is localized to the basolateral membrane of polarized Madin-Darby canine kidney (MDCK) cells. In the present study, we identified a 25-residue region within the C-terminal tail (C-tail) of the P2Y(1) receptor that directs basolateral sorting. Deletion of this sorting signal caused redirection of the receptor to the apical membrane, indicating that the region from the N-terminus to transmembrane domain 7 (TM7) contains an apical-sorting signal that is overridden by a dominant basolateral signal in the C-tail. Location of the signal relative to TM7 is crucial, because increasing its distance from the end of TM7 resulted in loss of basolateral sorting. The basolateral-sorting signal does not use any previously established basolateral-sorting motifs, i.e. tyrosine-containing or di-hydrophobic motifs, for function, and it is functional even when inverted or when its amino acids are scrambled, indicating that the signal is sequence independent. Mutagenesis of different classes of amino acids within the signal identified charged residues (five basic and four acidic amino acids in 25 residues) as crucial determinants for sorting function, with amidated amino acids having a lesser role. Mutational analyses revealed that whereas charge balance (+1 overall) of the signal is unimportant, the total number of charged residues (nine), either positive or negative, is crucial for basolateral targeting. These data define a new class of targeting signal that relies on total charge and might provide a common mechanism for polarized trafficking of epithelial proteins.

  3. Structure predictions of two Bauhinia variegata lectins reveal patterns of C-terminal properties in single chain legume lectins.

    PubMed

    Moreira, Gustavo M S G; Conceição, Fabricio R; McBride, Alan J A; Pinto, Luciano da S

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and -II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins.

  4. Structure Predictions of Two Bauhinia variegata Lectins Reveal Patterns of C-Terminal Properties in Single Chain Legume Lectins

    PubMed Central

    Moreira, Gustavo M. S. G.; Conceição, Fabricio R.; McBride, Alan J. A.; Pinto, Luciano da S.

    2013-01-01

    Bauhinia variegata lectins (BVL-I and BVL-II) are single chain lectins isolated from the plant Bauhinia variegata. Single chain lectins undergo post-translational processing on its N-terminal and C-terminal regions, which determines their physiological targeting, carbohydrate binding activity and pattern of quaternary association. These two lectins are isoforms, BVL-I being highly glycosylated, and thus far, it has not been possible to determine their structures. The present study used prediction and validation algorithms to elucidate the likely structures of BVL-I and -II. The program Bhageerath-H was chosen from among three different structure prediction programs due to its better overall reliability. In order to predict the C-terminal region cleavage sites, other lectins known to have this modification were analysed and three rules were created: (1) the first amino acid of the excised peptide is small or hydrophobic; (2) the cleavage occurs after an acid, polar, or hydrophobic residue, but not after a basic one; and (3) the cleavage spot is located 5-8 residues after a conserved Leu amino acid. These rules predicted that BVL-I and –II would have fifteen C-terminal residues cleaved, and this was confirmed experimentally by Edman degradation sequencing of BVL-I. Furthermore, the C-terminal analyses predicted that only BVL-II underwent α-helical folding in this region, similar to that seen in SBA and DBL. Conversely, BVL-I and -II contained four conserved regions of a GS-I association, providing evidence of a previously undescribed X4+unusual oligomerisation between the truncated BVL-I and the intact BVL-II. This is the first report on the structural analysis of lectins from Bauhinia spp. and therefore is important for the characterisation C-terminal cleavage and patterns of quaternary association of single chain lectins. PMID:24260572

  5. The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch.

    PubMed

    Persuh, M; Turgay, K; Mandic-Mulec, I; Dubnau, D

    1999-08-01

    ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains. The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.

  6. Structural basis for the recognition of RNA polymerase II C-terminal domain by CREPT and p15RS.

    PubMed

    Mei, Kunrong; Jin, Zhe; Ren, Fangli; Wang, Yinying; Chang, Zhijie; Wang, Xinquan

    2014-01-01

    CREPT and p15RS are two recently identified homologous proteins that regulate cell proliferation in an opposite way and are closely related to human cancer development. Both CREPT and p15RS consist of an N-terminal RPR domain and a C-terminal domain with high sequence homology. The transcription enhancement by CREPT is attributed to its interaction with RNA polymerase II (Pol II). Here we provide biochemical and structural evidence to support and extend this molecular mechanism. Through fluorescence polarization analysis, we show that the RPR domains of CREPT and p15RS (CREPT-RPR and p15RS-RPR) bind to different Pol II C-terminal domain (CTD) phosphoisoforms with similar affinity and specificity. We also determined the crystal structure of p15RS-RPR. Sequence and structural comparisons with RPR domain of Rtt103, a homolog of CREPT and p15RS in yeast, reveal structural basis for the similar binding profile of CREPT-RPR and p15RS-RPR with Pol II CTD. We also determined the crystal structure of the C-terminal domain of CREPT (CREPT-CTD), which is a long rod-like dimer and each monomer adopts a coiled-coil structure. We propose that dimerization through the C-terminal domain enhances the binding strength between CREPT or p15RS with Pol II by increasing binding avidity. Our results collectively reveal the respective roles of N-terminal RPR domain and C-terminal domain of CREPT and p15RS in recognizing RNA Pol II.

  7. Pore-forming activity of BAD is regulated by specific phosphorylation and structural transitions of the C-terminal part.

    PubMed

    Polzien, Lisa; Baljuls, Angela; Roth, Heide-Marie; Kuper, Jochen; Benz, Roland; Schweimer, Kristian; Hekman, Mirko; Rapp, Ulf R

    2011-02-01

    BAD protein (Bcl-2 antagonist of cell death) belongs to the BH3-only subfamily of proapoptotic proteins and is proposed to function as the sentinel of the cellular health status. Physiological activity of BAD is regulated by phosphorylation, association with 14-3-3 proteins, binding to membrane lipids and pore formation. Since the functional role of the BAD C-terminal part has not been considered so far, we have investigated here the interplay of the structure and function of this region. The structure of the regulatory C-terminal part of human BAD was analyzed by CD spectroscopy. The channel-forming activity of full-length BAD and BAD peptides was carried out by lipid bilayer measurements. Interactions between proteins and peptides were monitored by the surface plasmon resonance technique. In aqueous solution, C-terminal part of BAD exhibits a well-ordered structure and stable conformation. In a lipid environment, the helical propensity considerably increases. The interaction of the C-terminal segment of BAD with the isolated BH3 domain results in the formation of permanently open pores whereby the phosphorylation of serine 118 within the BH3 domain is necessary for effective pore formation. In contrast, phosphorylation of serine 99 in combination with 14-3-3 association suppresses formation of channels. C-terminal part of BAD controls BAD function by structural transitions, lipid binding and phosphorylation. Conformational changes of this region upon membrane interaction in conjunction with phosphorylation of the BH3 domain suggest a novel mechanism for regulation of BAD. Multiple signaling pathways mediate inhibition and activation of cell death via BAD. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. The C- terminal region of the Major Outer Sheath Protein (Msp) of Treponema denticola inhibits neutrophil chemotaxis.

    PubMed

    Jones, Megan M; Vanyo, Stephen T; Visser, Michelle B

    2017-03-13

    Treponema denticola is an oral spirochete strongly associated with severe periodontal disease. A prominent virulence factor, the major outer sheath protein (Msp), disorients neutrophil chemotaxis by altering the cellular phosphoinositide balance, leading to impairment of downstream chemotactic events including actin rearrangement, Rac1 activation and Akt activation in response to chemoattractant stimulation. The specific regions of Msp responsible for interactions with neutrophils remain unknown. In this study, we investigated the inhibitory effect of truncated Msp regions on neutrophil chemotaxis and associated signaling pathways. Murine neutrophils were treated with recombinant protein truncations followed by assessment of chemotaxis and associated signal pathway activation. Chemotaxis assays indicate sequences within the C-terminal region; particularly the first 130 amino acids, have the strongest inhibitory effect on neutrophil chemotaxis. Neutrophils incubated with the C-terminal region protein also demonstrated the greatest inhibition of Rac1 activation, increased phosphoinositide phosphatase activity, and decreased Akt activation; orchestrating impairment of chemotaxis. Furthermore, incubation with antibodies specific to only the C-terminal region blocked the Msp induced inhibition of chemotaxis and denaturing the protein restored Rac1 activation. Msp from the strain OTK, with numerous amino acid substitutions throughout the polypeptide, including the C-terminal region compared to strain 35405, showed increased ability to impair neutrophil chemotaxis. Collectively, these results indicate the C-terminal region of Msp is the most potent region to modulate neutrophil chemotactic signaling and that specific sequences and structure is likely required. Knowledge of how spirochetes dampen neutrophil response is limited and Msp may represent a novel therapeutic target for periodontal disease. This article is protected by copyright. All rights reserved.

  9. New proangiogenic activity on vascular endothelial cells for C-terminal mechano growth factor.

    PubMed

    Deng, Moyuan; Wang, Yuanliang; Zhang, Bingbing; Liu, Peng; Xiao, Hualiang; Zhao, Jianhua

    2012-04-01

    Angiogenesis is crucial in wound healing. The administration of the C-terminal 24-a.a. peptide of mechano growth factor (MGF24E) has been previously demonstrated to induce more blood vessels in regenerating bone around defective areas compared with the control. Accordingly, this study aims to determine whether MGF24E promotes bone defect healing through MGF24E-increased angiogenesis and whether MGF24E has positive effects on angiogenesis in vitro. The roles of MGF24E on angiogenesis and the underlying mechanisms were investigated. The cell proliferation, migration, and tubulogenesis of the human vascular endothelial EA.hy926 cells co-treated with 2% serum and MGF24E were determined to assess angiogenesis in comparison with 100 ng/ml of vascular endothelial growth factor 165 (VEGF(165))-positive control or vehicle control (phosphate-buffered saline). MGF24E treatment (10 ng/ml) significantly promoted the biological processes of angiogenesis on EA.hy926 cells compared with the vehicle control. The suppression of vascular endothelial growth factor and angiopoietin-I expressions by 2% serum starvation was reversed by the addition of 10 ng/ml of MGF24E in 2% serum medium. This result suggests that MGF24E has a protective effect on angiogenesis. Moreover, the inhibition of ERK due to PD98050 pretreatment completely abolished and mostly blocked MGF24E-induced proliferation and migration, respectively, whereas the MGF24-induced tubulogenesis and the angiogenic factor expression were only partially inhibited. These new findings suggest that MGF24E promotes angiogenesis by enhancing the expression of angiogenic cytokines which involves the MAPK/ERK-signaling pathway.

  10. A helix-turn motif in the C-terminal domain of histone H1.

    PubMed Central

    Vila, R.; Ponte, I.; Jiménez, M. A.; Rico, M.; Suau, P.

    2000-01-01

    The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. PMID:10794405

  11. Cdc15 Phosphorylates the C-terminal Domain of RNA Polymerase II for Transcription during Mitosis.

    PubMed

    Singh, Amit Kumar; Rastogi, Shivangi; Shukla, Harish; Asalam, Mohd; Rath, Srikanta Kumar; Akhtar, Md Sohail

    2017-03-31

    In eukaryotes, the basal transcription in interphase is orchestrated through the regulation by kinases (Kin28, Bur1, and Ctk1) and phosphatases (Ssu72, Rtr1, and Fcp1), which act through the post-translational modification of the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD comprises the repeated Tyr-Ser-Pro-Thr-Ser-Pro-Ser motif with potential epigenetic modification sites. Despite the observation of transcription and periodic expression of genes during mitosis with entailing CTD phosphorylation and dephosphorylation, the associated CTD specific kinase(s) and its role in transcription remains unknown. Here we have identified Cdc15 as a potential kinase phosphorylating Ser-2 and Ser-5 of CTD for transcription during mitosis in the budding yeast. The phosphorylation of CTD by Cdc15 is independent of any prior Ser phosphorylation(s). The inactivation of Cdc15 causes reduction of global CTD phosphorylation during mitosis and affects the expression of genes whose transcript levels peak during mitosis. Cdc15 also influences the complete transcription of clb2 gene and phosphorylates Ser-5 at the promoter and Ser-2 toward the 3' end of the gene. The observation that Cdc15 could phosphorylate Ser-5, as well as Ser-2, during transcription in mitosis is in contrast to the phosphorylation marks put by the kinases in interphase (G1, S, and G2), where Cdck7/Kin28 phosphorylates Ser-5 at promoter and Bur1/Ctk1 phosphorylates Ser-2 at the 3' end of the genes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment.

    PubMed

    Janke, Abigail M; Seo, Da Hee; Rahmanian, Vahid; Conicella, Alexander E; Mathews, Kaylee L; Burke, Kathleen A; Mittal, Jeetain; Fawzi, Nicolas L

    2017-10-11

    Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. We report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.

  13. Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification.

    PubMed

    Watanabe, Yasuhiro; Yasui, Kenichi; Nakano, Toshiya; Doi, Koji; Fukada, Yasuyo; Kitayama, Michio; Ishimoto, Miho; Kurihara, Saiko; Kawashima, Mika; Fukuda, Hiroki; Adachi, Yoshiki; Inoue, Takao; Nakashima, Kenji

    2005-04-27

    Mutation of Cu/Zn superoxide dismutase (SOD1) contributes to a portion of the cases of familial amyotrophic lateral sclerosis (FALS). We previously reported on a FALS family whose members had a mutant form of SOD1 characterized by a 2-base pair (bp) deletion at codon 126 of the SOD1 gene. To investigate the cellular consequences of this mutation, we produced transgenic mice that expressed normal and mutated copies of human SOD1: wild-type SOD1 (W), wild-type SOD1 with a FLAG epitope at C-terminal (WF), mutated SOD1 with the 2-bp deletion (D), and SOD1 with the 2-bp deletion with FLAG (DF). The mice heterozygotic for the human mutated SOD1 (D and DF) showed distinct ALS-like motor symptoms, whereas the mice heterozygotic for the normal SOD1 (W and WF) mice did not. Homozygotes of D and DF lines showed the ALS symptoms at an earlier age and died earlier than the heterozygotes. By Northern blot analysis, the mRNAs for all human SOD1s were confirmed in these lines. All the human SOD1 proteins, except the D mutant, were detectable by immunoblot. The D protein was only confirmed when it was concentrated by immunoprecipitation. Neuropathologically, loss of spinal motor neurons and reactive gliosis were common features in the symptomatic lines. The remaining motor neurons in these mice also exhibited eosinophilic inclusions. The biochemical and pathological characteristics of these mice are quite similar to those of human FALS patients with same mutation. This intriguing model will provide an important source of information of the pathogenesis of FALS.

  14. Kinetic and stereochemical studies on novel inactivators of C-terminal amidation.

    PubMed Central

    Feng, J; Shi, J; Sirimanne, S R; Mounier-Lee, C E; May, S W

    2000-01-01

    C-terminal amidation, a required post-translational modification for the bioactivation of many neuropeptides, entails sequential enzymic action by peptidylglycine alpha-mono-oxygenase (PAM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PGL, EC 4.3.2.5). Here we introduce novel compounds in which an olefinic functionality is incorporated into peptide analogues as the most potent turnover-dependent inactivators of PAM. Kinetic parameters for PAM inactivation by 4-oxo-5-acetamido-6-phenyl-hex-2-enoic acid and 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid were obtained by using both the conventional dilution assay method and the more complex progress curve method. The results obtained from the progress curve method establish that these compounds exhibit the kinetic characteristics of pure competitive inactivators (i.e. no ESI complex forms during inactivation). On the basis of k(inact)/K(i) values, 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is almost two orders of magnitude more potent than benzoylacrylate, a chemically analogous olefinic inactivator that lacks the peptide moiety. Stereochemical studies established that PAM inactivation by 4-oxo-5-acetamido-6-(2-thienyl)-hex-2-enoic acid is stereospecific with respect to the moiety at the P(2) position, which is consistent with previous results with substrates and reversible inhibitors. In contrast, 2, 4-dioxo-5-acetamido-6-phenylhexanoic acid, which is a competitive inhibitor with respect to ascorbate, exhibits a low degree of stereospecificity in binding to the ascorbate sites of both PAM and dopamine-beta-hydroxylase. PMID:10947967

  15. Reduced ubiquitin C-terminal hydrolase-1 expression levels in dementia with Lewy bodies.

    PubMed

    Barrachina, Marta; Castaño, Esther; Dalfó, Esther; Maes, Tamara; Buesa, Carlos; Ferrer, Isidro

    2006-05-01

    Parkinson disease (PD) and dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal alpha-synuclein and ubiquitin in protein aggregates conforming Lewy bodies and Lewy neurites. Ubiquitin C-terminal hydrolase-1 (UCHL-1) disassembles polyubiquitin chains to increase the availability of free monomeric ubiquitin to the ubiquitin proteasome system (UPS) thus favoring protein degradation. Since mutations in the UCHL-1 gene, reducing UPS activity by 50%, have been reported in autosomal dominant PD, and UCHL-1 inhibition results in the formation of alpha-synuclein aggregates in mesencephalic cultured neurons, the present study was initiated to test UCHL-1 mRNA and protein levels in post-mortem frontal cortex (area 8) of PD and DLB cases, compared with age-matched controls. TaqMan PCR assays, and Western blots demonstrated down-regulation of UCHL-1 mRNA and UCHL-1 protein in the cerebral cortex in DLB (either in pure forms, not associated with Alzheimer disease: AD, and in common forms, with accompanying AD changes), but not in PD, when compared with age-matched controls. Interestingly, UCHL-1 mRNA and protein expressions were reduced in the medulla oblongata in the same PD cases. Moreover, UCHL-1 protein was decreased in the substantia nigra in cases with Lewy body pathology. UCHL-1 down-regulation was not associated with reduced protein levels of several proteasomal subunits, including 20SX, 20SY, 19S and 11Salpha. Yet UCHL-3 expression was reduced in the cerebral cortex of PD and DLB patients. Together, these observations show reduced UCHL-1 expression as a contributory factor in the abnormal protein aggregation in DLB, and points UCHL-1 as a putative therapeutic target in the treatment of DLB.

  16. The potential role of ubiquitin c-terminal hydrolases in oncogenesis.

    PubMed

    Fang, Ying; Fu, Da; Shen, Xi-Zhong

    2010-08-01

    Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.

  17. Interaction of CheY with the C-terminal peptide of CheZ

    SciTech Connect

    Guhaniyogi,J.; Wu, T.; Patel, S.; Stock, A.

    2008-01-01

    Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P{approx}CheY). The steady-state level of P{approx}CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P{approx}CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZC) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work, we presented high-resolution crystal structures of CheY in complex with the CheZC peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZC interaction. In addition, we present kinetic studies of the CheZC-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter.

  18. Interaction of CheY with the C-Terminal Peptide of CheZ▿ †

    PubMed Central

    Guhaniyogi, Jayita; Wu, Ti; Patel, Smita S.; Stock, Ann M.

    2008-01-01

    Chemotaxis, a means for motile bacteria to sense the environment and achieve directed swimming, is controlled by flagellar rotation. The primary output of the chemotaxis machinery is the phosphorylated form of the response regulator CheY (P∼CheY). The steady-state level of P∼CheY dictates the direction of rotation of the flagellar motor. The chemotaxis signal in the form of P∼CheY is terminated by the phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two distinct protein-protein interfaces: one involving the strongly conserved C-terminal helix of CheZ (CheZC) tethering the two proteins together and the other constituting an active site for catalytic dephosphorylation. In a previous work (J. Guhaniyogi, V. L. Robinson, and A. M. Stock, J. Mol. Biol. 359:624-645, 2006), we presented high-resolution crystal structures of CheY in complex with the CheZC peptide that revealed alternate binding modes subject to the conformational state of CheY. In this study, we report biochemical and structural data that support the alternate-binding-mode hypothesis and identify key recognition elements in the CheY-CheZC interaction. In addition, we present kinetic studies of the CheZC-associated effect on CheY phosphorylation with its physiologically relevant phosphodonor, the histidine kinase CheA. Our results indicate mechanistic differences in phosphotransfer from the kinase CheA versus that from small-molecule phosphodonors, explaining a modest twofold increase of CheY phosphorylation with the former, observed in this study, relative to a 10-fold increase previously documented with the latter. PMID:18083806

  19. A C-terminal di-leucine motif controls plasma membrane expression of PMCA4b.

    PubMed

    Antalffy, Géza; Pászty, Katalin; Varga, Karolina; Hegedűs, Luca; Enyedi, Agnes; Padányi, Rita

    2013-12-01

    Recent evidences show that the localization of different plasma membrane Ca(2+) ATPases (PMCAs) is regulated in various complex, cell type-specific ways. Here we show that in low-density epithelial and endothelial cells PMCA4b localized mostly in intracellular compartments and its plasma membrane localization was enhanced upon increasing density of cells. In good correlation with the enhanced plasma membrane localization a significantly more efficient Ca(2+) clearance was observed in confluent versus non-confluent HeLa cell cultures expressing mCherry-PMCA4b. We analyzed the subcellular localization and function of various C-terminally truncated PMCA4b variants and found that a truncated mutant PMCA4b-ct24 was mostly intracellular while another mutant, PMCA4b-ct48, localized more to the plasma membrane, indicating that a protein sequence corresponding to amino acid residues 1158-1181 contained a signal responsible for the intracellular retention of PMCA4b in non-confluent cultures. Alteration of three leucines to alanines at positions 1167-1169 resulted in enhanced cell surface expression and an appropriate Ca(2+) transport activity of both wild type and truncated pumps, suggesting that the di-leucine-like motif (1167)LLL was crucial in targeting PMCA4b. Furthermore, upon loss of cell-cell contact by extracellular Ca(2+) removal, the wild-type pump was translocated to the early endosomal compartment. Targeting PMCA4b to early endosomes was diminished by the L(1167-69)A mutation, and the mutant pump accumulated in long tubular cytosolic structures. In summary, we report a di-leucine-like internalization signal at the C-tail of PMCA4b and suggest an internalization-mediated loss of function of the pump upon low degree of cell-cell contact.

  20. N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

    PubMed Central

    Lei, Yan-Chang; Tian, Yong-Jun; Ding, Hong-Hui; Wang, Bao-Ju; Yang, Yan; Hao, You-Hua; Zhao, Xi-Ping; Lu, Meng-Ji; Gong, Fei-Li; Yang, Dong-Liang

    2006-01-01

    AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the

  1. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  2. Crystal structure of a 92-residue C-terminal fragment of TonB from Escherichia coli reveals significant conformational changes compared to structures of smaller TonB fragments.

    PubMed

    Ködding, Jiri; Killig, Frank; Polzer, Patrick; Howard, S Peter; Diederichs, Kay; Welte, Wolfram

    2005-01-28

    Uptake of siderophores and vitamin B(12) through the outer membrane of Escherichia coli is effected by an active transport system consisting of several outer membrane receptors and a protein complex of the inner membrane. The link between these is TonB, a protein associated with the cytoplasmic membrane, which forms a large periplasmic domain capable of interacting with several outer membrane receptors, e.g. FhuA, FecA, and FepA for siderophores and BtuB for vitamin B(12.) The active transport across the outer membrane is driven by the chemiosmotic gradient of the inner membrane and is mediated by the TonB protein. The receptor-binding domain of TonB appears to be formed by a highly conserved C-terminal amino acid sequence of approximately 100 residues. Crystal structures of two C-terminal TonB fragments composed of 85 (TonB-85) and 77 (TonB-77) amino acid residues, respectively, have been previously determined (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535-27540 and Koedding, J., Howard, S. P., Kaufmann, L., Polzer, P., Lustig, A., and Welte, W. (2004) J. Biol. Chem. 279, 9978-9986). In both cases the TonB fragments form dimers in solution and crystallize as dimers consisting of monomers tightly engaged with one another by the exchange of a beta-hairpin and a C-terminal beta-strand. Here we present the crystal structure of a 92-residue fragment of TonB (TonB-92), which is monomeric in solution. The structure, determined at 1.13-A resolution, shows a dimer with considerably reduced intermolecular interaction compared with the other known TonB structures, in particular lacking the beta-hairpin exchange.

  3. Generating recombinant C-terminal prion protein fragments of exact native sequence.

    PubMed

    Johanssen, V A; Barnham, K J; Masters, C L; Hill, A F; Collins, S J

    2012-02-01

    Transmissibility and distinctive neuropathology are hallmark features of prion diseases differentiating them from other neurodegenerative disorders, with pathogenesis and transmission appearing closely linked to misfolded conformers (PrP(Sc)) of the ubiquitously expressed cellular form of the prion protein (PrP(C)). Given the apparent pathogenic primacy of misfolded PrP, the utilisation of peptides based on the prion protein has formed an integral approach for providing insights into misfolding pathways and pathogenic mechanisms. In parallel with studies employing prion peptides, similar approaches in other neurodegenerative disorders such as Alzheimer Disease, have demonstrated that differential processing of parent proteins and quite minor variations in the primary sequence of cognate peptides generated from the same constitutive processing (such as Aβ1-40 versus Aβ1-42 produced from γ-secretase activity) can be associated with very different pathogenic consequences. PrP(C) also undergoes constitutive α- or β-cleavage yielding C1 (residues 112-231 human sequence) or C2 (residues 90-231), respectively, with the full cell biological significance of such processing unresolved; however, it is noteworthy that in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and murine models, the moderately extended C2 fragment predominates in the brain suggesting that the two cleavage events and the consequent C-terminal fragments may differ in their pathogenic significance. Accordingly, studies characterising biologically relevant peptides like C1 and C2, would be most valid if undertaken using peptides completely free of any inherent non-native sequence that arises as a by-product of commonly employed recombinant production techniques. To achieve this aim and thereby facilitate more representative biophysical and neurotoxicity studies, we adapted the combination of high fidelity Taq TA cloning with a SUMO-Hexa-His tag-type approach, incorporating the SUMO protease

  4. Evolution of lysine acetylation in the RNA polymerase II C-terminal domain.

    PubMed

    Simonti, Corinne N; Pollard, Katherine S; Schröder, Sebastian; He, Daniel; Bruneau, Benoit G; Ott, Melanie; Capra, John A

    2015-03-10

    RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes. To investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates. Our combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the

  5. The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation

    PubMed Central

    Hammer, Neal D.; McGuffie, Bryan A.; Zhou, Yizhou; Badtke, Matthew P.; Reinke, Ashley A.; Brännström, Kristoffer; Gestwicki, Jason E.; Olofsson, Anders; Almqvist, Fredrik; Chapman, Matthew R.

    2012-01-01

    Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1st, 2nd or 3rd repeating units were able to convert CsgA into fibers. However, mutants missing either the 4th or 5th repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units. PMID:22684146

  6. Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography.

    PubMed

    Roberts, Matthew F; Taylor, David W; Unger, Vinzenz M

    2011-06-01

    Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20Å resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100Å away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.

  7. The C-terminal domain of zDHHC2 contains distinct sorting signals that regulate intracellular localisation in neurons and neuroendocrine cells.

    PubMed

    Salaun, Christine; Ritchie, Louise; Greaves, Jennifer; Bushell, Trevor J; Chamberlain, Luke H

    2017-07-30

    The S-acyltransferase zDHHC2 mediates dynamic S-acylation of PSD95 and AKAP79/150, which impacts synaptic targeting of AMPA receptors. zDHHC2 is responsive to synaptic activity and catalyses the increased S-acylation of PSD95 that occurs following action potential blockade or application of ionotropic glutamate receptor antagonists. These treatments have been proposed to increase plasma membrane delivery of zDHHC2 via an endosomal cycling pathway, enhancing substrate accessibility. To generate an improved understanding of zDHHC2 trafficking and how this might be regulated by neuronal activity, we searched for intramolecular signals that regulate enzyme localisation. Two signals were mapped to the C-terminal tail of zDHHC2: a non-canonical dileucine motif [SxxxLL] and a downstream NP motif. Mutation of these signals enhanced plasma membrane accumulation of zDHHC2 in both neuroendocrine PC12 cells and rat hippocampal neurons, consistent with reduced endocytic retrieval. Furthermore, mutation of these signals also increased accumulation of the enzyme in neurites. Interestingly, several threonine and serine residues are adjacent to these sorting motifs and analysis of phospho-mimetic mutants highlighted a potential role for phosphorylation in regulating the efficacy of these signals. This study offers new molecular insight into the signals that determine zDHHC2 localisation and highlights a potential mechanism to regulate these trafficking signals. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

    PubMed

    Coceres, V M; Alonso, A M; Nievas, Y R; Midlej, V; Frontera, L; Benchimol, M; Johnson, P J; de Miguel, N

    2015-08-01

    The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction. © 2015 John Wiley & Sons Ltd.

  9. Crystal structure of the C-terminal domain of the RAP74 subunit of human transcription factor IIF

    SciTech Connect

    Kamada, Katsuhiko; De Angelis, Jacqueline; Roeder, Robert G.; Burley, Stephen K.

    2012-12-13

    The x-ray structure of a C-terminal fragment of the RAP74 subunit of human transcription factor (TF) IIF has been determined at 1.02-{angstrom} resolution. The {alpha}/{beta} structure is strikingly similar to the globular domain of linker histone H5 and the DNA-binding domain of hepatocyte nuclear factor 3{gamma} (HNF-3{gamma}), making it a winged-helix protein. The surface electrostatic properties of this compact domain differ significantly from those of bona fide winged-helix transcription factors (HNF-3{gamma} and RFX1) and from the winged-helix domains found within the RAP30 subunit of TFIIF and the {beta} subunit of TFIIE. RAP74 has been shown to interact with the TFIIF-associated C-terminal domain phosphatase FCP1, and a putative phosphatase binding site has been identified within the RAP74 winged-helix domain.

  10. Crystallization and preliminary X-ray analysis of the C-terminal fragment of Ski7 from Saccharomyces cerevisiae

    PubMed Central

    Lee, Ji-Young; Park, Si Hoon; Jeong, Byung-Cheon; Song, Hyun Kyu

    2014-01-01

    Ski7 (superkiller protein 7) plays a critical role in the mRNA surveillance pathway. The C-terminal fragment of Ski7 (residues 520–747) from Saccharo­myces cerevisiae was heterologously expressed in Escherichia coli and purified to homogeneity. It was successfully crystallized and preliminary X-ray data were collected to 2.0 Å resolution using synchrotron radiation. The crystal belonged to a trigonal space group, either P3121 or P3221, with unit-cell parameters a = b = 73.5, c = 83.6 Å. The asymmetric unit contains one molecule of the C-terminal fragment of Ski7 with a corresponding crystal volume per protein mass (V M) of 2.61 Å3 Da−1 and a solvent content of 52.8% by volume. The merging R factor is 6.6%. Structure determination by MAD phasing is under way. PMID:25195903

  11. Crystal Structure in the Vivo-Assembled Bacillus subtilis Spx/RNA Polymerase alpha Subunit C-Terminal Domain Complex

    SciTech Connect

    Lamour, V.; Westblade, L; Campbell, E; Darst, S

    2009-01-01

    The Bacillus subtilis Spx protein is a global transcription factor that interacts with the C-terminal domain of the RNA polymerase {alpha} subunit ({alpha}CTD) and regulates transcription of genes involved in thiol-oxidative stress, sporulation, competence, and organosulfur metabolism. Here we determined the X-ray crystal structure of the Spx/{alpha}CTD complex from an entirely new crystal form than previously reported [Newberry, K.J., Nakano, S., Zuber, P., Brennan, R.G., 2005. Crystal structure of the Bacillus subtilis anti-alpha, global transcriptional regulator, Spx, in complex with the alpha C-terminal domain of RNA polymerase. Proc. Natl. Acad. Sci. USA 102, 15839-15844]. Comparison of the previously reported sulfate-bound complex and our sulfate-free complex reveals subtle conformational changes that may be important for the role of Spx in regulating organosulfur metabolism.

  12. Solution conformation of the C-terminal domain of skeletal troponin C. Cation, trifluoperazine and troponin I binding effects.

    PubMed

    Drabikowski, W; Dalgarno, D C; Levine, B A; Gergely, J; Grabarek, Z; Leavis, P C

    1985-08-15

    Proton magnetic resonance spectroscopy has been used to study the cation (Mg2+, Ca2+)-dependent conformational states of the C-terminal domain of rabbit skeletal troponin C under a variety of solution conditions. Nuclear Overhauser data and paramagnetic probe observations provide definition of the configuration of this region of troponin C. Comparative study of homologous proteins identify common features of the tertiary structure relevant to the cation binding reaction. Complex formation with troponin I and the drug trifluoperazine is observed to adjust the solution conformation of the C-terminal domain of troponin C. The interactive conformational response to cation coordination and the binding of the drug and troponin I are discussed.

  13. The C-terminal dimerization motif of cyclase-associated protein is essential for actin monomer regulation.

    PubMed

    Iwase, Shohei; Ono, Shoichiro

    2016-12-01

    Cyclase-associated protein (CAP) is a conserved actin-regulatory protein that functions together with actin depolymerizing factor (ADF)/cofilin to enhance actin filament dynamics. CAP has multiple functional domains, and the function to regulate actin monomers is carried out by its C-terminal half containing a Wiskott-Aldrich Syndrome protein homology 2 (WH2) domain, a CAP and X-linked retinitis pigmentosa 2 (CARP) domain, and a dimerization motif. WH2 and CARP are implicated in binding to actin monomers and important for enhancing filament turnover. However, the role of the dimerization motif is unknown. Here, we investigated the function of the dimerization motif of CAS-2, a CAP isoform in the nematode Caenorhabditis elegans, in actin monomer regulation. CAS-2 promotes ATP-dependent recycling of ADF/cofilin-bound actin monomers for polymerization by enhancing exchange of actin-bound nucleotides. The C-terminal half of CAS-2 (CAS-2C) has nearly as strong activity as full-length CAS-2. Maltose-binding protein (MBP)-tagged CAS-2C is a dimer. However, MBP-CAS-2C with a truncation of either one or two C-terminal β-strands is monomeric. Truncations of the dimerization motif in MBP-CAS-2C nearly completely abolish its activity to sequester actin monomers from polymerization and enhance nucleotide exchange on actin monomers. As a result, these CAS-2C variants, also in the context of full-length CAS-2, fail to compete with ADF/cofilin to release actin monomers for polymerization. CAS-2C variants lacking the dimerization motif exhibit enhanced binding to actin filaments, which is mediated by WH2. Taken together, these results suggest that the evolutionarily conserved dimerization motif of CAP is essential for its C-terminal region to exert the actin monomer-specific regulatory function.

  14. C-terminal tyrosine of ferredoxin-NADP+ reductase in hydride transfer processes with NAD(P)+/H.

    PubMed

    Tejero, Jesús; Pérez-Dorado, Inmaculada; Maya, Celia; Martínez-Júlvez, Marta; Sanz-Aparicio, Julia; Gómez-Moreno, Carlos; Hermoso, Juan A; Medina, Milagros

    2005-10-18

    Ferredoxin-NADP+ reductase (FNR) catalyzes the reduction of NADP+ to NADPH in an overall reversible reaction, showing some differences in the mechanisms between cyanobacterial and higher plant FNRs. During hydride transfer it is proposed that the FNR C-terminal Tyr is displaced by the nicotinamide. Thus, this C-terminal Tyr might be involved not only in modulating the flavin redox properties, as already shown, but also in nicotinamide binding and hydride transfer. FNR variants from the cyanobacterium Anabaena in which the C-terminal Tyr has been replaced by Trp, Phe, or Ser have been produced. All FNR variants show enhanced NADP+ and NAD+ binding, especially Tyr303Ser, which correlates with a noticeable improvement of NADH-dependent reactions. Nevertheless, the Tyr303Ser variant shows a decrease in the steady-state kcat value with NADPH. Fast kinetic analysis of the hydride transfer shows that the low efficiency observed for this mutant FNR under steady-state conditions is not due to a lack of catalytic ability but rather to the strong enzyme-coenzyme interaction. Three-dimensional structures for Tyr303Ser and Tyr303Trp variants and its complexes with NADP+ show significant differences between plant and cyanobacterial FNRs. Our results suggest that modulation of coenzyme affinity is highly influenced by the strength of the C-terminus-FAD interaction and that subtle changes between plant and cyanobacterial structures are able to modify the energy of that interaction. Additionally, it is shown that the C-terminal Tyr of FNR lowers the affinity for NADP+/H to levels compatible with steady-state turnover during the catalytic cycle, but it is not involved in the hydride transfer itself.

  15. The C-Terminal Domain of the Virulence Factor MgtC Is a Divergent ACT Domain

    PubMed Central

    Yang, Yinshan; Labesse, Gilles; Carrère-Kremer, Séverine; Esteves, Kevin; Kremer, Laurent

    2012-01-01

    MgtC is a virulence factor of unknown function important for survival inside macrophages in several intracellular bacterial pathogens, including Mycobacterium tuberculosis. It is also involved in adaptation to Mg2+ deprivation, but previous work suggested that MgtC is not a Mg2+ transporter. In this study, we demonstrated that the amount of the M. tuberculosis MgtC protein is not significantly increased by Mg2+ deprivation. Members of the MgtC protein family share a conserved membrane N-terminal domain and a more divergent cytoplasmic C-terminal domain. To get insights into MgtC functional and structural organization, we have determined the nuclear magnetic resonance (NMR) structure of the C-terminal domain of M. tuberculosis MgtC. This structure is not affected by the Mg2+ concentration, indicating that it does not bind Mg2+. The structure of the C-terminal domain forms a βαββαβ fold found in small molecule binding domains called ACT domains. However, the M. tuberculosis MgtC ACT domain differs from canonical ACT domains because it appears to lack the ability to dimerize and to bind small molecules. We have shown, using a bacterial two-hybrid system, that the M. tuberculosis MgtC protein can dimerize and that the C-terminal domain somehow facilitates this dimerization. Taken together, these results indicate that M. tuberculosis MgtC does not have an intrinsic function related to Mg2+ uptake or binding but could act as a regulatory factor based on protein-protein interaction that could be facilitated by its ACT domain. PMID:22984256

  16. Structural Basis for Toughness and Flexibility in the C-terminal Passenger Domain of an Acinetobacter Trimeric Autotransporter Adhesin*

    PubMed Central

    Koiwai, Kotaro; Hartmann, Marcus D.; Linke, Dirk; Lupas, Andrei N.; Hori, Katsutoshi

    2016-01-01

    Trimeric autotransporter adhesins (TAAs) on the cell surface of Gram-negative pathogens mediate bacterial adhesion to host cells and extracellular matrix proteins. However, AtaA, a TAA in the nonpathogenic Acinetobacter sp. strain Tol 5, shows nonspecific high adhesiveness to abiotic material surfaces as well as to biotic surfaces. It consists of a passenger domain secreted by the C-terminal transmembrane anchor domain (TM), and the passenger domain contains an N-terminal head, N-terminal stalk, C-terminal head (Chead), and C-terminal stalk (Cstalk). The Chead-Cstalk-TM fragment, which is conserved in many Acinetobacter TAAs, has by itself the head-stalk-anchor architecture of a complete TAA. Here, we show the crystal structure of the Chead-Cstalk fragment, AtaA_C-terminal passenger domain (CPSD), providing the first view of several conserved TAA domains. The YadA-like head (Ylhead) of the fragment is capped by a unique structure (headCap), composed of three β-hairpins and a connector motif; it also contains a head insert motif (HIM1) before its last inner β-strand. The headCap, Ylhead, and HIM1 integrally form a stable Chead structure. Some of the major domains of the CPSD fragment are inherently flexible and provide bending sites for the fiber between segments whose toughness is ensured by topological chain exchange and hydrophobic core formation inside the trimer. Thus, although adherence assays using in-frame deletion mutants revealed that the characteristic adhesive sites of AtaA reside in its N-terminal part, the flexibility and toughness of the CPSD part provide the resilience that enables the adhesive properties of the full-length fiber across a wide range of conditions. PMID:26698633

  17. Improved synthesis of C-terminal peptide thioesters on "safety-catch" resins using LiBr/THF.

    PubMed

    Quaderer, R; Hilvert, D

    2001-10-04

    [reaction: see text] The alkanesulfonamide "safety-catch" resin has proven useful for Fmoc-based synthesis of C-terminal peptide thioesters. We now report that the yield of isolated thioester can increase significantly when the cleavage reaction is carried out in 2 M LiBr/THF rather than DMF or THF. The largest effects are seen with problematic peptides that aggregate or form secondary structures on the resin.

  18. Fertilization in C. elegans requires an intact C-terminal RING finger in sperm protein SPE-42

    PubMed Central

    2011-01-01

    Background The C. elegans sperm protein SPE-42, a membrane protein of unknown structure and molecular function, is required for fertilization. Sperm from worms with spe-42 mutations appear normal but are unable to fertilize eggs. Sequence analysis revealed the presence of 8 conserved cysteine residues in the C-terminal cytoplasmic domain of this protein suggesting these residues form a zinc-coordinating RING finger structure. Results We made an in silico structural model of the SPE-42 RING finger domain based on primary sequence analysis and previously reported RING structures. To test the model, we created spe-42 transgenes coding for mutations in each of the 8 cysteine residues predicted to coordinate Zn++ ions in the RING finger motif. Transgenes were crossed into a spe-42 null background and protein function was measured by counting progeny. We found that all 8 cysteines are required for protein function. We also showed that sequence differences between the C-terminal 29 and 30 amino acids in C. elegans and C. briggsae SPE-42 following the RING finger domain are not responsible for the failure of the C. briggsae SPE-42 homolog to rescue C. elegans spe-42 mutants. Conclusions The results suggest that a bona fide RING domain is present at the C-terminus of the SPE-42 protein and that this motif is required for sperm-egg interactions during C. elegans fertilization. Our structural model of the RING domain provides a starting point for further structure-function analysis of this critical region of the protein. The C-terminal domain swap experiment suggests that the incompatibility between the C. elegans and C. briggsae SPE-42 proteins is caused by small amino acid differences outside the C-terminal domain. PMID:21345212

  19. Dual N- and C-terminal processing of citrus chlorophyllase precursor within the plastid membranes leads to the mature enzyme.

    PubMed

    Azoulay-Shemer, Tamar; Harpaz-Saad, Smadar; Cohen-Peer, Reut; Mett, Anahit; Spicer, Victor; Lovat, Nicole; Krokhin, Oleg; Brand, Arnon; Gidoni, David; Standing, Kenneth G; Goldschmidt, Eliezer E; Eyal, Yoram

    2011-01-01

    Chl, the central player in harvesting light energy for photosynthesis, is enzymatically degraded during natural turnover, leaf senescence, fruit ripening or following biotic/abiotic stress induction. The photodynamic properties of Chl and its metabolites call for tight regulation of the catabolic pathway enzymes to avoid accumulation of intermediate breakdown products. Chlorophyllase, the Chl dephytilation enzyme, was previously demonstrated to be an initiator of Chl breakdown when transcriptionally induced to be expressed during ethylene-induced citrus fruit color break or when heterologously expressed in different plant systems. Citrus chlorophyllase was previously shown to be translated as a precursor protein, which is subsequently post-translationally processed to a mature form. We demonstrate that maturation of citrus chlorophyllase involves dual N- and C-terminal processing which appear to be rate-limiting post-translational events when chlorophyllase expression levels are high. The chlorophyllase precursor and intermediate forms were shown to be of transient nature, while the mature form accumulates over time, suggesting that processing may be involved in post-translational regulation of enzyme in vivo function. This notion is further supported by the finding that neither N- nor C-terminal processed domains are essential for chloroplast targeting of the enzyme, and that both processing events occur within the chloroplast membranes. Studies on the processing of chlorophyllase versions truncated at the N- or C-termini or mutated to abolish C-terminal processing suggest that each of the processing events is independent. Dual N- and C-terminal processing, not involving an organellar targeting signal, has rarely been documented in plants and is unique for a plastid protein.

  20. XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair.

    PubMed

    Gabel, Scott A; DeRose, Eugene F; London, Robert E

    2013-12-01

    The function of X-ray cross complementing group 1 protein (XRCC1), a scaffold that binds to DNA repair enzymes involved in single-strand break and base excision repair, requires that it be recruited to sites of damaged DNA. However, structural insights into this recruitment are currently limited. Sequence analysis of the first unstructured linker domain of XRCC1 identifies a segment consistent with a possible REV1 interacting region (X1RIR) motif. The X1RIR motif is present in translesion polymerases that can be recruited to the pol /REV1 DNA repair complex via a specific interaction with the REV1 C-terminal domain. NMR and fluorescence titration studies were performed on XRCC1-derived peptides containing this putative RIR motif in order to evaluate the binding affinity for the REV1 C-terminal domain. These studies demonstrate an interaction of the XRCC1-derived peptide with the human REV1 C-terminal domain characterized by dissociation constants in the low micromolar range. Ligand competition studies comparing the XRCC1 RIR peptide with previously studied RIR peptides were found to be inconsistent with the NMR based Kd values. These discrepancies were resolved using a fluorescence assay for which the RIR–REV1 system is particularly well suited. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol κ and pol η RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. These studies suggest the possible involvement of XRCC1 and its associated repair factors in post replication repair.

  1. XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair

    PubMed Central

    Gabel, Scott A.; DeRose, Eugene F.; London, Robert E.

    2014-01-01

    The function of X-ray cross complementing group 1 protein (XRCC1), a scaffold that binds to DNA repair enzymes involved in single-strand break and base excision repair, requires that it be recruited to sites of damaged DNA. However, structural insights into this recruitment are currently limited. Sequence analysis of the first unstructured linker domain of XRCC1 identifies a segment consistent with a possible REV1 interacting region (RIR) motif. The RIR motif is present in translesion polymerases that can be recruited to the pol ζ/REV1 DNA repair complex via a specific interaction with the REV1 C-terminal domain. NMR and fluorescence titration studies were performed on XRCC1-derived peptides containing this putative RIR motif in order to evaluate the binding affinity for the REV1 C-terminal domain. These studies demonstrate an interaction of the XRCC1-derived peptide with the human REV1 C-terminal domain characterized by dissociation constants in the low micromolar range. Ligand competition studies comparing the X1 RIR peptide with previously studied RIR peptides were found to be inconsistent with the NMR based Kd values. These discrepancies were resolved using a fluorescence assay for which the RIR – REV1 system is particularly well suited. The structure of a REV1-XRCC1 peptide complex was determined by using NOE restraints to dock the unlabeled XRCC1 peptide with a labeled REV1 C-terminal domain. The structure is generally homologous with previously determined complexes with the pol κ and pol η RIR peptides, although the helical segment in XRCC1 is shorter than was observed in these cases. These studies suggest the possible involvement of XRCC1 and its associated repair factors in post replication repair. PMID:24409475

  2. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase.

    PubMed

    Wray, Lewis V; Fisher, Susan H

    2008-04-01

    The Bacillus subtilis GlnR transcription factor regulates gene expression in response to changes in nitrogen availability. Glutamine synthetase transmits the nitrogen regulatory signal to GlnR. The DNA-binding activity of GlnR is activated by a transient protein-protein interaction with feedback-inhibited glutamine synthetase that stabilizes GlnR-DNA complexes. This signal transduction mechanism was analysed by creating mutant GlnR proteins with partial or complete truncations of their C-terminal domains. The truncated GlnR proteins were found to constitutively repress gene expression in vivo. This constitutive repression did not require glutamine synthetase. Purified mutant GlnR proteins bound DNA in vitro more tightly than wild-type GlnR protein and this binding was not activated by feedback-inhibited glutamine synthetase. While full-length GlnR is monomeric, the truncated GlnR proteins contained significant levels of dimers. These results indicate that the C-terminal region of GlnR acts as an autoinhibitory domain that prevents GlnR dimerization and thus impedes DNA binding. The GlnR C-terminal domain is also required for the interaction between GlnR and feedback-inhibited glutamine synthetase. Compared with the full-length GlnR protein, the truncated GlnR proteins were defective in their interaction with feedback-inhibited glutamine synthetase in cross-linking experiments.

  3. Structural aspects and chaperone activity of human HspB3: role of the "C-terminal extension".

    PubMed

    Asthana, Abhishek; Raman, Bakthisaran; Ramakrishna, Tangirala; Rao, Ch Mohan

    2012-09-01

    HspB3, an as yet uncharacterized sHsp, is present in muscle, brain, heart, and in fetal tissues. A point mutation correlates with the development of axonal motor neuropathy. We purified recombinant human HspB3. Circular dichroism studies indicate that it exhibits β-sheet structure. Gel filtration and sedimentation velocity experiments show that HspB3 exhibits polydisperse populations with predominantly trimeric species. HspB3 exhibits molecular chaperone-like activity in preventing the heat-induced aggregation of alcohol dehydrogenase (ADH). It exhibits moderate chaperone-like activity towards heat-induced aggregation of citrate synthase. However, it does not prevent the DTT-induced aggregation of insulin, indicating that it exhibits target protein-dependent molecular chaperone-like activity. Unlike other sHsps, it has a very short C-terminal extension. Fusion of the C-terminal extension of αB-crystallin results in altered tertiary and quaternary structure, and increase in polydispersity of the chimeric protein, HspB3αB-CT. The chimeric protein shows comparable chaperone-like activity towards heat-induced aggregation of ADH and citrate synthase. However, it shows enhanced activity towards DTT-induced aggregation of insulin. Our study, for the first time, provides the structural and chaperone functional characterization of HspB3 and also sheds light on the role of the C-terminal extension of sHsps.

  4. The Contribution of the C-Terminal Tails of Microtubules in Altering the Force Production Specifications of Multiple Kinesin-1.

    PubMed

    Feizabadi, Mitra Shojania

    2016-09-01

    The extent to which beta tubulin isotypes contribute to the function of microtubules and the microtubule-driven transport of molecular motors is poorly understood. The major differences in these isotypes are associated with the structure of their C-terminal tails. Recent studies have revealed a few aspects of the C-terminal tails' regulatory role on the activities of some of the motor proteins on a single-molecule level. However, little attention is given to the degree to which the function of a team of motor proteins can be altered by the microtubule's tail. In a set of parallel experiments, we investigated this open question by studying the force production of several kinesin-1 (kinesin) molecular motors along two groups of microtubules: regular ones and those microtubules whose C-terminals are cleaved by subtilisin digestion. The results indicate that the difference between the average of the force production of motors along two types of microtubules is statistically significant. The underlying mechanism of such production is substantially different as well. As compared to untreated microtubules, the magnitude of the binding time of several kinesin-1 is almost three times greater along subtilisin-treated microtubules. Also, the velocity of the group of kinesin molecules shows a higher sensitivity to external loads and reduces significantly under higher loads along subtilisin-treated microtubules. Together, this work shows the capacity of the tails in fine-tuning the force production characteristics of several kinesin molecules.

  5. Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block.

    PubMed

    Kawakami, Toru; Yoshikawa, Ryo; Fujiyoshi, Yuki; Mishima, Yuichi; Hojo, Hironobu; Tajima, Shoji; Suetake, Isao

    2015-11-01

    The post-translational modification of histones plays an important role in gene expression. We report herein on a method for synthesizing such modified histones by ligating chemically prepared N-terminal peptides and C-terminal recombinant peptide building blocks. Based on their chemical synthesis, core histones can be categorized as two types; histones H2A, H2B and H4 which contain no Cys residues, and histone H3 which contains a Cys residue(s) in the C-terminal region. A combination of native chemical ligation and desulphurization can be simply used to prepare histones without Cys residues. For the synthesis of histone H3, the endogenous Cys residue(s) must be selectively protected, while keeping the N-terminal Cys residue of the C-terminal building block that is introduced for purposes of chemical ligation unprotected. To this end, a phenacyl group was successfully utilized to protect endogenous Cys residue(s), and the recombinant peptide was ligated with a peptide containing a Cys-Pro ester (CPE) sequence as a thioester precursor. Using this approach it was possible to prepare all of the core histones H2A, H2B, H3 and H4 with any modifications. The resulting proteins could then be used to prepare a core histone library of proteins that have been post-translationally modified. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  6. Enhanced sampling molecular dynamics identifies PrP(Sc) structures harboring a C-terminal β-core.

    PubMed

    Baillod, Pascal; Garrec, Julian; Colombo, Maria-Carola; Tavernelli, Ivano; Rothlisberger, Ursula

    2012-12-11

    We perform a replica exchange molecular dynamics simulation corresponding to a 2.8 μs total time for the extensive enhanced sampling of the conformational space of the C-terminal part (residues 124-226) of the mouse prion protein (PrP); 1.3% of the conformations sampled display a high level of β-structure (≥19 residues), allowing the assessment of β-propensities along the sequence and highlighting the most structurally labile hot spots. A clustering algorithm is applied to sort the structures of this pool according to their fold. Ten β-rich folds are thus defined and analyzed with regard to their topology, accumulation temperatures, and structural characteristics. In contrast to the so-called spiral and β-helix models suggesting that the β-rich core of the scrapie isoform (PrP(Sc)) comprises the N-terminal tail and part of the C-terminal domain up to helix 1 (H1), we present putative structural models for monomeric precursors of PrP(Sc) and PrP β-oligomers that are characterized by a C-terminal β-rich core, in agreement with the suggestions of a series of recent experiments.

  7. Stepwise assembly of functional C-terminal REST/NRSF transcriptional repressor complexes as a drug target.

    PubMed

    Inui, Ken; Zhao, Zongpei; Yuan, Juan; Jayaprakash, Sakthidasan; Le, Le T M; Drakulic, Srdja; Sander, Bjoern; Golas, Monika M

    2017-02-20

    In human cells, thousands of predominantly neuronal genes are regulated by the repressor element 1 (RE1)-silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF). REST/NRSF represses transcription of these genes in stem cells and non-neuronal cells by tethering corepressor complexes. Aberrant REST/NRSF expression and intracellular localization are associated with cancer and neurodegeneration in humans. To date, detailed molecular analyses of REST/NRSF and its C-terminal repressor complex have been hampered largely by the lack of sufficient amounts of purified REST/NRSF and its complexes. Therefore, the aim of this study was to express and purify human REST/NRSF and its C-terminal interactors in a baculovirus multiprotein expression system as individual proteins and coexpressed complexes. All proteins were enriched in the nucleus, and REST/NRSF was isolated as a slower migrating form, characteristic of nuclear REST/NRSF in mammalian cells. Both REST/NRSF alone and its C-terminal repressor complex were functionally active in histone deacetylation and histone demethylation and bound to RE1/neuron-restrictive silencer element (NRSE) sites. Additionally, the mechanisms of inhibition of the small-molecule drugs 4SC-202 and SP2509 were analyzed. These drugs interfered with the viability of medulloblastoma cells, where REST/NRSF has been implicated in cancer pathogenesis. Thus, a resource for molecular REST/NRSF studies and drug development has been established.

  8. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

    PubMed Central

    Bewley, Kathryn D.; Bennallack, Philip R.; Burlingame, Mark A.; Robison, Richard A.; Griffitts, Joel S.

    2016-01-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts. PMID:27791142

  9. The AtMYB12 activation domain maps to a short C-terminal region of the transcription factor.

    PubMed

    Stracke, Ralf; Turgut-Kara, Neslihan; Weisshaar, Bernd

    2017-03-11

    The Arabidopsis thaliana R2R3-MYB transcription factor MYB12 is a light-inducible, flavonol-specific activator of flavonoid biosynthesis. The transactivation activity of the AtMYB12 protein was analyzed using a C-terminal deletion series in a transient A. thaliana protoplast assay with the goal of mapping the activation domain (AD). Although the deletion of the last 46 C-terminal amino acids did not affect the activation capacity, the deletion of the last 98 amino acids almost totally abolished transactivation of two different target promoters. A domain swap experiment using the yeast GAL4 DNA-binding domain revealed that the region from positions 282 to 328 of AtMYB12 was sufficient for transactivation. In contrast to the R2R3-MYB ADs known thus far, that of AtMYB12 is not located at the rearmost C-terminal end of the protein. The AtMYB12 AD is conserved in other experimentally proven R2R3-MYB flavonol regulators from different species.

  10. The C-terminal domain is the primary determinant of histone H1 binding to chromatin in vivo.

    PubMed

    Hendzel, Michael J; Lever, Melody A; Crawford, Ellen; Th'ng, John P H

    2004-05-07

    We have used a combination of kinetic measurements and targeted mutations to show that the C-terminal domain is required for high-affinity binding of histone H1 to chromatin, and phosphorylations can disrupt binding by affecting the secondary structure of the C terminus. By measuring the fluorescence recovery after photo-bleaching profiles of green fluorescent protein-histone H1 proteins in living cells, we find that the deletion of the N terminus only modestly reduces binding affinity. Deletion of the C terminus, however, almost completely eliminates histone H1.1 binding. Specific mutations of the C-terminal domain identified Thr-152 and Ser-183 as novel regulatory switches that control the binding of histone H1.1 in vivo. It is remarkable that the single amino acid substitution of Thr-152 with glutamic acid was almost as effective as the truncation of the C terminus to amino acid 151 in destabilizing histone H1.1 binding in vivo. We found that modifications to the C terminus can affect histone H1 binding dramatically but have little or no influence on the charge distribution or the overall net charge of this domain. A comparison of individual point mutations and deletion mutants, when reviewed collectively, cannot be reconciled with simple charge-dependent mechanisms of C-terminal domain function of linker histones.

  11. Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation.

    PubMed

    Bewley, Kathryn D; Bennallack, Philip R; Burlingame, Mark A; Robison, Richard A; Griffitts, Joel S; Miller, Susan M

    2016-11-01

    Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.

  12. Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity*

    PubMed Central

    Galian, Carmen; Björkholm, Patrik; Bulleid, Neil; von Heijne, Gunnar

    2012-01-01

    Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is “marginal”: less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane. PMID:22431723

  13. Alternatively spliced C-terminal domains regulate the surface expression of large conductance calcium-activated potassium (BKCa) channels

    PubMed Central

    Kim, Eun Young; Ridgway, Lon D.; Zou, Shengwei; Chiu, Yu-Hsin; Dryer, Stuart E.

    2007-01-01

    The Slo1 gene, also known as KCNMA1, encodes the pore-forming subunits of large-conductance Ca2+-activated K+ (BKCa) channels. Products of this gene are widely expressed in vertebrate tissues, and occur in a large number (≥ 20) of alternatively spliced variants that vary in their gating properties, susceptibility to modulation, and trafficking to the plasma membrane. Motifs in the large cytoplasmic C-terminal are especially important in determining the functional properties of BKCa channels. Here we report that chick ciliary ganglion neurons express transcripts and proteins of two Slo1 splice variants that differ at the extreme C-terminal. We refer to these variants as VEDEC and QEDRL (or QEERL for the orthologous mammalian versions), after the five terminal amino acid residues in each isoform. Individual ciliary ganglion neurons preferentially express these variants in different subcellular compartments. Moreover, QEERL channels show markedly higher levels of constitutive expression on the plasma membrane than VEDEC channels in HEK293T and NG108-15 cells. However, growth factor treatment can stimulate surface expression of VEDEC channels to levels comparable to those seen with QEERL. In addition, we show that co-expression of a soluble protein comprised of VEDEC C-terminal tail residues markedly increases cell surface expression of full-length VEDEC channels, suggesting that this region binds to proteins that cause retention of the these channels in intracellular stores. PMID:17478049

  14. Cholesterol dependent conformational exchange of the C-terminal domain of the influenza A M2 protein

    PubMed Central

    Kim, Sangwoo S.; Upshur, Mary Alice; Saotome, Kei; Sahu, Indra D.; McCarrick, Robert M.; Feix, Jimmy B.; Lorigan, Gary A.; Howard, Kathleen P.

    2016-01-01

    The C-terminal amphipathic helix of the influenza A M2 protein plays a critical cholesterol dependent role in viral budding. To provide atomic-level detail on the impact cholesterol has on the conformation of M2 protein, we spin-labeled sites right before and within the C-terminal amphipathic helix of the M2 protein. We studied the spin-labeled M2 proteins in membranes both with and without cholesterol. We used a multipronged site-directed spin-label electron paramagnetic resonance (SDSL-EPR) approach and collected data on line shapes, relaxation rates, accessibility of sites to the membrane, and distances between symmetry related sites within the tetrameric protein. We demonstrate that the C-terminal amphipathic helix of M2 populates at least two conformations in POPC/POPG 4:1 bilayers. Furthermore, we show that the conformational state that becomes more populated in the presence of cholesterol is less dynamic, less membrane buried, and more tightly packed than the other state. Cholesterol dependent changes in M2 could be attributed to the changes cholesterol induces in bilayer properties and/or direct binding of cholesterol to the protein. We propose a model consistent with all our experimental data that suggests that the predominant conformation we observe in the presence of cholesterol is relevant for the understanding of viral budding. PMID:26569023

  15. Influence of C-terminal tail deletion on structure and stability of hyperthermophile Sulfolobus tokodaii RNase HI.

    PubMed

    Chen, Lin; Zhang, Ji-Long; Zheng, Qing-Chuan; Chu, Wen-Ting; Xue, Qiao; Zhang, Hong-Xing; Sun, Chia-Chung

    2013-06-01

    The C-terminus tail (G144-T149) of the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) plays an important role in this protein's hyperstabilization and may therefore be a good protein stability tag. Detailed understanding of the structural and dynamic effects of C-terminus tail deletion is required for gaining insights into the thermal stability mechanism of Sto-RNase HI. Focused on Sulfolobus tokodaii RNase HI (Sto-RNase HI) and its derivative lacking the C-terminal tail (ΔC6 Sto-RNase HI) (PDB codes: 2EHG and 3ALY), we applied molecular dynamics (MD) simulations at four different temperatures (300, 375, 475, and 500 K) to examine the effect of the C-terminal tail on the hyperstabilization of Sto-RNase HI and to investigate the unfolding process of Sto-RNase HI and ΔC6 Sto-RNase HI. The simulations suggest that the C-terminal tail has significant impact in hyperstabilization of Sto-RNase HI and the unfolding of these two proteins evolves along dissimilar pathways. Essential dynamics analysis indicates that the essential subspaces of the two proteins at different temperatures are non-overlapping within the trajectories and they exhibit different directions of motion. Our work can give important information to understand the three-state folding mechanism of Sto-RNase HI and to offer alternative strategies to improve the protein stability.

  16. Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope.

    PubMed

    Seybold, Christian; Elserafy, Menattallah; Rüthnick, Diana; Ozboyaci, Musa; Neuner, Annett; Flottmann, Benjamin; Heilemann, Mike; Wade, Rebecca C; Schiebel, Elmar

    2015-06-22

    The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1's function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31-Cdc31 interactions between Sfi1-Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.

  17. NMR assignment and secondary structure of coiled coil domain of C-terminal myosin binding subunit of myosin phosphatase.

    PubMed

    Sharma, Alok K; Rigby, Alan C

    2014-07-01

    Protein-protein interactions between the C-terminal domain of Myosin Binding Subunit (MBS) of MLC Phosphatase (MBS(CT180); C-terminal 180 aa) and the N-terminal coiled coil (CC) leucine zipper (LZ) domain of PKGIα, PKG-Iα(1-159) play an important role in the process of Smooth Muscle Cell relaxation. The paucity of three-dimensional structural information for MBS(CT180) prevents an atomic level understanding of the MBS-PKG contractile complex. MBS(CT180) is comprised of three structurally different sub-domains including a non-canonical CC, a CC, and a LZ. Recently we reported polypeptide purification and biophysical characterization of the CC domain and the LZ domain of MBS(CT180) (Sharma et al, Prot Expr Purif 2012). Here we report (1)H, (13)C, (15)N chemical shift assignments of homodimeric CC MBS domain encompassing amino acid residues Asp931-Leu980 using 2D and 3D heteronuclear NMR spectroscopy. Secondary structure analyses deduced from these NMR chemical shift data have identified a contiguous stretch of 36 residues from Phe932 to Ala967 that is involved in the formation of coiled coil α-helical region within CC MBS domain. The N-terminal residue Asp931 and the C-terminally positioned residues Thr968-Ala975, Arg977, and Ser978 adopt nonhelical loop conformations.

  18. The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease

    SciTech Connect

    Lin, J.J.; Sancar, A. )

    1991-08-01

    The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3{prime} end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5{prime} and the fourth phosphodiester bond 3{prime} to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease.

  19. Crystal Structure of the C-terminal Domain of Splicing Factor Prp8 Carrying Retinitis Pigmentosa Mutants

    SciTech Connect

    Zhang,L.; Shen, J.; Guarnieri, M.; Heroux, A.; Yang, K.; Zhao, R.

    2007-01-01

    Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an / structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn{sup 2+} coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn{sup 2+}-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

  20. Collision-Induced Dissociation Fragmentation Inside Disulfide C-Terminal Loops of Natural Non-Tryptic Peptides

    NASA Astrophysics Data System (ADS)

    Samgina, Tatiana Y.; Vorontsov, Egor A.; Gorshkov, Vladimir A.; Artemenko, Konstantin A.; Zubarev, Roman A.; Ytterberg, Jimmy A.; Lebedev, Albert T.

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  1. Structure and regulatory role of the C-terminal winged helix domain of the archaeal minichromosome maintenance complex

    PubMed Central

    Wiedemann, Christoph; Szambowska, Anna; Häfner, Sabine; Ohlenschläger, Oliver; Gührs, Karl-Heinz; Görlach, Matthias

    2015-01-01

    The minichromosome maintenance complex (MCM) represents the replicative DNA helicase both in eukaryotes and archaea. Here, we describe the solution structure of the C-terminal domains of the archaeal MCMs of Sulfolobus solfataricus (Sso) and Methanothermobacter thermautotrophicus (Mth). Those domains consist of a structurally conserved truncated winged helix (WH) domain lacking the two typical ‘wings’ of canonical WH domains. A less conserved N-terminal extension links this WH module to the MCM AAA+ domain forming the ATPase center. In the Sso MCM this linker contains a short α-helical element. Using Sso MCM mutants, including chimeric constructs containing Mth C-terminal domain elements, we show that the ATPase and helicase activity of the Sso MCM is significantly modulated by the short α-helical linker element and by N-terminal residues of the first α-helix of the truncated WH module. Finally, based on our structural and functional data, we present a docking-derived model of the Sso MCM, which implies an allosteric control of the ATPase center by the C-terminal domain. PMID:25712103

  2. The C-terminal part of microcin B is crucial for DNA gyrase inhibition and antibiotic uptake by sensitive cells.

    PubMed

    Shkundina, Irina; Serebryakova, Marina; Severinov, Konstantin

    2014-05-01

    Microcin B (McB) is a ribosomally synthesized antibacterial peptide. It contains up to nine oxazole and thiazole heterocycles that are introduced posttranslationally and are required for activity. McB inhibits the DNA gyrase, a validated drug target. Previous structure-activity analyses indicated that two fused heterocycles located in the central part of McB are important for antibacterial action and gyrase inhibition. Here, we used site-specific mutagenesis of the McB precursor gene to assess the functional significance of the C-terminal part of McB that is located past the second fused heterocycle and contains two single heterocycles as well as an unmodified four-amino-acid C-terminal tail. We found that removal of unmodified C-terminal amino acids of McB, while having no effect on fused heterocycles, has a very strong negative effect on activity in vivo and in vitro. In fact, even nonconservative point substitutions in the last McB amino acid have a very strong effect by simultaneously decreasing uptake and ability to inhibit the gyrase. The results highlight the importance of unmodified McB amino acids for function and open the way for creation of recombinant McB derivatives with an altered or expanded spectrum of antibacterial action.

  3. Collision-induced dissociation fragmentation inside disulfide C-terminal loops of natural non-tryptic peptides.

    PubMed

    Samgina, Tatiana Y; Vorontsov, Egor A; Gorshkov, Vladimir A; Artemenko, Konstantin A; Zubarev, Roman A; Ytterberg, Jimmy A; Lebedev, Albert T

    2013-07-01

    Collision-induced dissociation (CID) spectra of long non-tryptic peptides are usually quite complicated and rather difficult to interpret. Disulfide bond formed by two cysteine residues at C-terminus of frog skin peptides precludes one to determine sequence inside the forming loop. Thereby, chemical modification of S-S bonds is often used in "bottom up" sequencing approach. However, low-energy CID spectra of natural non-tryptic peptides with C-terminal disulfide cycle demonstrate an unusual fragmentation route, which may be used to elucidate the "hidden" C-terminal sequence. Low charge state protonated molecules experience peptide bond cleavage at the N-terminus of C-terminal cysteine. The forming isomeric acyclic ions serve as precursors for a series of b-type ions revealing sequence inside former disulfide cycle. The reaction is preferable for peptides with basic lysine residues inside the cycle. It may also be activated by acidic protons of Asp and Glu residues neighboring the loop. The observed cleavages may be quite competitive, revealing the sequence inside disulfide cycle, although S-S bond rupture does not occur in this case.

  4. Functions of the C-terminal domains of apoptosis-related proteins of the Bcl-2 family.

    PubMed

    Gómez-Fernández, Juan C

    2014-10-01

    Bcl-2 family proteins are involved in cell homeostasis, where they regulate cell death. Some of these proteins are pro-apoptotic and others pro-survival. Moreover, many of them share a similar domain composition with several of the so-called BH domains, although some only have a BH3 domain. A C-terminal domain is present in all the multi-BH domain proteins and in some of the BH3-only ones. This C-terminal domain is hydrophobic or amphipathic, for which reason it was thought when they were discovered that they were membrane anchors. Although this is indeed one of their functions, it has since been observed that they may also serve as regulators of the function of some members of this family, such as Bax. They may also serve to recognize the target membrane of some of these proteins, which only after an apoptotic signal, are incorporated into a membrane. It has been shown that peptides that imitate the sequence of C-terminal domains can form pores and may serve as a model to design cytotoxic molecules.

  5. Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

    PubMed Central

    Seybold, Christian; Elserafy, Menattallah; Rüthnick, Diana; Ozboyaci, Musa; Neuner, Annett; Flottmann, Benjamin; Heilemann, Mike; Wade, Rebecca C.

    2015-01-01

    The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation. PMID:26076691

  6. Ubiquitin C-Terminal Hydrolase L1 (UCH-L1) Promotes Hippocampus-Dependent Memory via Its Deubiquitinating Effect on TrkB.

    PubMed

    Guo, Yun-Yun; Lu, Yi; Zheng, Yuan; Chen, Xiao-Rong; Dong, Jun-Lu; Yuan, Rong-Rong; Huang, Shu-Hong; Yu, Hui; Wang, Yue; Chen, Zhe-Yu; Su, Bo

    2017-06-21

    Multiple studies have established that brain-derived neurotrophic factor (BDNF) plays a critical role in the regulation of synaptic plasticity via its receptor, TrkB. In addition to being phosphorylated, TrkB has also been demonstrated to be ubiquitinated. However, the mechanisms of TrkB ubiquitination and its biological functions remain poorly understood. In this study, we demonstrate that ubiquitin C-terminal hydrolase L1 (UCH-L1) promotes contextual fear conditioning learning and memory via the regulation of ubiquitination of TrkB. We provide evidence that UCH-L1 can deubiquitinate TrkB directly. K460 in the juxtamembane domain of TrkB is the primary ubiquitination site and is regulated by UCH-L1. By using a peptide that competitively inhibits the association between UCH-L1 and TrkB, we show that the blockade of UCH-L1-regulated TrkB deubiquitination leads to increased BDNF-induced TrkB internalization and consequently directs the internalized TrkB to the degradation pathway, resulting in increased degradation of surface TrkB and attenuation of TrkB activation and its downstream signaling pathways. Moreover, injection of the peptide into the DG region of mice impairs hippocampus-dependent memory. Together, our results suggest that the ubiquitination of TrkB is a mechanism that controls its downstream signaling pathways via the regulation of its endocytosis and postendocytic trafficking and that UCH-L1 mediates the deubiquitination of TrkB and could be a potential target for the modulation of hippocampus-dependent memory.SIGNIFICANCE STATEMENT Ubiquitin C-terminal hydrolase L1 (UCH-L1) has been demonstrated to play important roles in the regulation of synaptic plasticity and learning and memory. TrkB, the receptor for brain-derived neurotrophic factor, has also been shown to be a potent regulator of synaptic plasticity. In this study, we demonstrate that UCH-L1 functions as a deubiquitinase for TrkB. The blockage of UCH-L1-regulated deubiquitination of Trk

  7. Conformational states of the full-length glucagon receptor

    PubMed Central

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-01-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism. PMID:26227798

  8. Conformational states of the full-length glucagon receptor

    NASA Astrophysics Data System (ADS)

    Yang, Linlin; Yang, Dehua; de Graaf, Chris; Moeller, Arne; West, Graham M.; Dharmarajan, Venkatasubramanian; Wang, Chong; Siu, Fai Y.; Song, Gaojie; Reedtz-Runge, Steffen; Pascal, Bruce D.; Wu, Beili; Potter, Clinton S.; Zhou, Hu; Griffin, Patrick R.; Carragher, Bridget; Yang, Huaiyu; Wang, Ming-Wei; Stevens, Raymond C.; Jiang, Hualiang

    2015-07-01

    Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

  9. Effects of the HN gene c-terminal extensions on the Newcastle disease virus virulence

    USDA-ARS?s Scientific Manuscript database

    The hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is a multifunctional protein that has receptor recognition, neuraminidase and fusion promotion activities. Sequence analysis revealed that the HN gene of many extremely low virulence NDV strains encodes a larger open reading frame...

  10. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions.

    PubMed

    Vedovato, Natascia; Gadsby, David C

    2010-07-01

    Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K-adenosine triphosphatase (ATPase) alpha subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis alpha1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain-sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 microM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump-induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the

  11. Nox5 Stability and Superoxide Production is Regulated by C-terminal Binding of Hsp90 and Co-Chaperones

    PubMed Central

    Chen, Feng; Haigh, Steven; Yu, Yanfang; Benson, Tyler; Wang, Yusi; Li, Xueyi; Dou, Huijuan; Bagi, Zsolt; Verin, Alexander D.; Stepp, David W.; Csanyi, Gabor; Chadli, Ahmed; Weintraub, Neal L.; Smith, Susan M. E.; Fulton, David J.R.

    2015-01-01

    Heat shock protein 90 (Hsp90) is a molecular chaperone that orchestrates the folding and stability of proteins that regulate cellular signaling, proliferation and inflammation. We have previously shown that Hsp90 controls the production of reactive oxygen species by modulating the activity of Noxes1-3 and 5, but not Nox4. The goal of the current study was to define the regions on Nox5 that bind Hsp90 and determine how Hsp90 regulates enzyme activity. In isolated enzyme activity assays, we found that Hsp90 inhibitors selectively decrease superoxide, but not hydrogen peroxide, production. The addition of Hsp90 alone only modestly increases Nox5 enzyme activity but in combination with the co-chaperones, Hsp70, HOP, Hsp40, and p23 it robustly stimulated superoxide, but not hydrogen peroxide, production. Proximity ligation assays reveal that Nox5 and Hsp90 interact in intact cells. In cell lysates using a co-IP approach, Hsp90 binds to Nox5 but not Nox4, and the degree of binding can be influenced by calcium-dependent stimuli. Inhibition of Hsp90 induced the degradation of full length, catalytically inactive and a C-terminal fragment (aa398–719) of Nox5. In contrast, inhibition of Hsp90 did not affect the expression levels of N-terminal fragments (aa1–550) suggesting that Hsp90 binding maintains the stability of C-terminal regions. In Co-IP assays, Hsp90 was bound only to the C-terminal region of Nox5. Further refinement using deletion analysis revealed that the region between aa490–550 mediates Hsp90 binding. Converse mapping experiments show that the C-terminal region of Nox5 bound to the M domain of Hsp90 (aa310–529). In addition to Hsp90, Nox5 bound other components of the foldosome including co-chaperones Hsp70, HOP, p23 and Hsp40. Silencing of HOP, Hsp40 and p23 reduced Nox5-dependent superoxide. In contrast, increased expression of Hsp70 decreased Nox5 activity whereas a mutant of Hsp70 failed to do so. Inhibition of Hsp90 results in the loss of higher

  12. The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions

    PubMed Central

    Vedovato, Natascia

    2010-01-01

    Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the

  13. Isolation and characterization of nucleotide-binding site and C-terminal leucine-rich repeat-resistance gene candidates in bananas.

    PubMed

    Lu, Y; Xu, W H; Xie, Y X; Zhang, X; Pu, J J; Qi, Y X; Li, H P

    2011-12-15

    Commercial banana varieties are highly susceptible to fungal pathogens, as well as bacterial pathogens, nematodes, viruses, and insect pests. The largest known family of plant resistance genes encodes proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for the isolation of candidate genes in banana that may be involved in plant defense. Six degenerate PCR primers were designed to target NBS and additional domains were tested on commercial banana species Musa acuminata subsp malaccensis and the Musa AAB Group propagated in vitro and plants maintained in a greenhouse. Total DNA was isolated by a modified CTAB extraction technique. Four resistance gene analogs were amplified and deposited in GenBank and assigned numbers HQ199833-HQ199836. The predicted amino acid sequences compared to the amino acid sequences of known resistance genes (MRGL1, MRGL2, MRGL3, and MRGL4) revealed significant sequence similarity. The presence of consensus domains, namely kinase-1a, kinase-2 and hydrophobic domain, provided evidence that the cloned sequences belong to the typical non-Toll/interleukin-1 receptor-like domain NBS-LRR gene family.

  14. β-Amyloid (Aβ) Oligomers Impair Brain-derived Neurotrophic Factor Retrograde Trafficking by Down-regulating Ubiquitin C-terminal Hydrolase, UCH-L1*

    PubMed Central

    Poon, Wayne W.; Carlos, Anthony J.; Aguilar, Brittany L.; Berchtold, Nicole C.; Kawano, Crystal K.; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W.

    2013-01-01

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival. PMID:23599427

  15. β-Amyloid (Aβ) oligomers impair brain-derived neurotrophic factor retrograde trafficking by down-regulating ubiquitin C-terminal hydrolase, UCH-L1.

    PubMed

    Poon, Wayne W; Carlos, Anthony J; Aguilar, Brittany L; Berchtold, Nicole C; Kawano, Crystal K; Zograbyan, Vahe; Yaopruke, Tim; Shelanski, Michael; Cotman, Carl W

    2013-06-07

    We previously found that BDNF-dependent retrograde trafficking is impaired in AD transgenic mouse neurons. Utilizing a novel microfluidic culture chamber, we demonstrate that Aβ oligomers compromise BDNF-mediated retrograde transport by impairing endosomal vesicle velocities, resulting in impaired downstream signaling driven by BDNF/TrkB, including ERK5 activation, and CREB-dependent gene regulation. Our data suggest that a key mechanism mediating the deficit involves ubiquitin C-terminal hydrolase L1 (UCH-L1), a deubiquitinating enzyme that functions to regulate cellular ubiquitin. Aβ-induced deficits in BDNF trafficking and signaling are mimicked by LDN (an inhibitor of UCH-L1) and can be reversed by increasing cellular UCH-L1 levels, demonstrated here using a transducible TAT-UCH-L1 strategy. Finally, our data reveal that UCH-L1 mRNA levels are decreased in the hippocampi of AD brains. Taken together, our data implicate that UCH-L1 is important for regulating neurotrophin receptor sorting to signaling endosomes and supporting retrograde transport. Further, our results support the idea that in AD, Aβ may down-regulate UCH-L1 in the AD brain, which in turn impairs BDNF/TrkB-mediated retrograde signaling, compromising synaptic plasticity and neuronal survival.

  16. Crystal structure of TRIM20 C-terminal coiled-coil/B30.2 fragment: implications for the recognition of higher order oligomers.

    PubMed

    Weinert, Christopher; Morger, Damien; Djekic, Aleksandra; Grütter, Markus G; Mittl, Peer R E

    2015-06-04

    Many tripartite motif-containing (TRIM) proteins, comprising RING-finger, B-Box, and coiled-coil domains, carry additional B30.2 domains on the C-terminus of the TRIM motif and are considered to be pattern recognition receptors involved in the detection of higher order oligomers (e.g. viral capsid proteins). To investigate the spatial architecture of domains in TRIM proteins we determined the crystal structure of the TRIM20Δ413 fragment at 2.4 Å resolution. This structure comprises the central helical scaffold (CHS) and C-terminal B30.2 domains and reveals an anti-parallel arrangement of CHS domains placing the B-box domains 170 Å apart from each other. Small-angle X-ray scattering confirmed that the linker between CHS and B30.2 domains is flexible in solution. The crystal structure suggests an interaction between the B30.2 domain and an extended stretch in the CHS domain, which involves residues that are mutated in the inherited disease Familial Mediterranean Fever. Dimerization of B30.2 domains by means of the CHS domain is crucial for TRIM20 to bind pro-IL-1β in vitro. To exemplify how TRIM proteins could be involved in binding higher order oligomers we discuss three possible models for the TRIM5α/HIV-1 capsid interaction assuming different conformations of B30.2 domains.

  17. Mild Clinical Features and Histopathologically Atypical Cores in Two Korean Families with Central Core Disease Harboring RYR1 Mutations at the C-Terminal Region

    PubMed Central

    Jung, Na-Yeon; Park, Yeong-Eun; Shin, Jin-Hong; Lee, Chang Hun; Jung, Dae-Soo

    2015-01-01

    Background Central core disease (CCD) is a congenital myopathy characterized by distinctive cores in muscle fibers. Mutations in the gene encoding ryanodine receptor 1 (RYR1) have been identified in most CCD patients. Case Report Two unrelated patients presented with slowly progressive or nonprogressive proximal muscle weakness since childhood. Their family history revealed some members with the same clinical problem. Histological analysis of muscle biopsy samples revealed numerous peripheral cores in the muscle fibers. RYR1 sequence analysis disclosed a novel mutation in exon 101 (c.14590T>C) and confirmed a previously reported mutation in exon 102 (c.14678G>A). Conclusions We report herein two families with CCD in whom missense mutations at the C-terminal of RYR1 were identified. Although it has been accepted that such mutations are usually associated with a severe clinical phenotype and clearly demarcated central cores, our patients exhibited a mild clinical phenotype without facial muscle involvement and skeletal deformities, and atypical cores in their muscle biopsy specimens. PMID:25628744

  18. The C-terminal domain of the nuclear factor I-B2 isoform is glycosylated and transactivates the WAP gene in the JEG-3 cells

    SciTech Connect

    Mukhopadhyay, Sudit S. . E-mail: suditmukhopadhy@yahoo.com; Rosen, Jeffrey M. . E-mail: jrosen@bcm.tmc.edu

    2007-07-06

    The transcription factor nuclear factor I (NFI) has been shown previously both in vivo and in vitro to be involved in the cooperative regulation of whey acidic protein (WAP) gene transcription along with the glucocorticoid receptor and STAT5. In addition, one of the specific NFI isoforms, NFI-B2, was demonstrated in transient co-transfection experiments in JEG cells, which lack endogenous NFI, to be preferentially involved in the cooperative regulation of WAP gene expression. A comparison of the DNA-binding specificities of the different NFI isoforms only partially explained their differential ability to activate the WAP gene transcription. Here, we analyzed the transactivation regions of two NFI isoforms by making chimeric proteins between the NFI-A and B isoforms. Though, their DNA-binding specificities were not altered as compared to the corresponding wild-type transcription factors, the C-terminal region of the NFI-B isoform was shown to preferentially activate WAP gene transcription in cooperation with GR and STAT5 in transient co-transfection assays in JEG-3 cells. Furthermore, determination of serine and threonine-specific glycosylation (O-linked N-acetylglucosamine) of the C-terminus of the NFI-B isoform suggested that the secondary modification by O-GlcNAc might play a role in the cooperative regulation of WAP gene transcription by NFI-B2 and STAT5.

  19. Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains*

    PubMed Central

    Wielandt, Alex Green; Pedersen, Jesper Torbøl; Falhof, Janus; Kemmer, Gerdi Christine; Lund, Anette; Ekberg, Kira; Fuglsang, Anja Thoe; Pomorski, Thomas Günther; Buch-Pedersen, Morten Jeppe; Palmgren, Michael

    2015-01-01

    Eukaryotic P-type plasma membrane H+-ATPases are primary active transport systems that are regulated at the post-translation level by cis-acting autoinhibitory domains, which can be relieved by protein kinase-mediated phosphorylation or binding of specific lipid species. Here we show that lysophospholipids specifically activate a plant plasma membrane H+-ATPase (Arabidopsis thaliana AHA2) by a mechanism that involves both cytoplasmic terminal domains of AHA2, whereas they have no effect on the fungal counterpart (Saccharomyces cerevisiae Pma1p). The activation was dependent on the glycerol backbone of the lysophospholipid and increased with acyl chain length, whereas the headgroup had little effect on activation. Activation of the plant pump by lysophospholipids did not involve the penultimate residue, Thr-947, which is known to be phosphorylated as part of a binding site for activating 14-3-3 protein, but was critically dependent on a single autoinhibitory residue (Leu-919) upstream of the C-terminal cytoplasmic domain in AHA2. A corresponding residue is absent in the fungal counterpart. These data indicate that plant plasma membrane H+-ATPases evolved as specific receptors for lysophospholipids and support the hypothesis that lysophospholipids are important plant signaling molecules. PMID:25971968

  20. Mild Clinical Features and Histopathologically Atypical Cores in Two Korean Families with Central Core Disease Harboring RYR1 Mutations at the C-Terminal Region.

    PubMed

    Jung, Na-Yeon; Park, Yeong-Eun; Shin, Jin-Hong; Lee, Chang Hun; Jung, Dae-Soo; Kim, Dae-Seong

    2015-01-01

    Central core disease (CCD) is a congenital myopathy characterized by distinctive cores in muscle fibers. Mutations in the gene encoding ryanodine receptor 1 (RYR1) have been identified in most CCD patients. Two unrelated patients presented with slowly progressive or nonprogressive proximal muscle weakness since childhood. Their family history revealed some members with the same clinical problem. Histological analysis of muscle biopsy samples revealed numerous peripheral cores in the muscle fibers. RYR1 sequence analysis disclosed a novel mutation in exon 101 (c.14590T>C) and confirmed a previously reported mutation in exon 102 (c.14678G>A). We report herein two families with CCD in whom missense mutations at the C-terminal of RYR1 were identified. Although it has been accepted that such mutations are usually associated with a severe clinical phenotype and clearly demarcated central cores, our patients exhibited a mild clinical phenotype without facial muscle involvement and skeletal deformities, and atypical cores in their muscle biopsy specimens.

  1. Synthesis of gastrin antagonists, analogues of the C-terminal tetrapeptide of gastrin, by introduction of a beta-homo residue.

    PubMed

    Rodriguez, M; Fulcrand, P; Laur, J; Aumelas, A; Bali, J P; Martinez, J

    1989-03-01

    A series of analogues of Boc-Trp-Leu-Asp-Phe-NH2, a potent gastrin agonist, were synthesized by introducing a beta-homo residue in the sequence. These compounds were tested in vivo on acid secretion, in the anesthetized rat, and for their ability to inhibit binding of labeled gastrin to its receptors on gastric mucosal cells. These analogues behaved as gastrin antagonists. The most potent compounds in this series were Boc-Trp-Leu-beta-homo-Asp-NHCH2C6H5 (10) (IC50 = 1 microM, ED50 = 0.2 mg/kg), Boc-Trp-Leu-beta-homo-Asp-NHCH2CH2C6H5 (11) (IC50 = 0.75 microM, ED50 = 0.5 mg/kg), Boc-Trp-Leu-beta-homo-Asp-Phe-NH2 (12) (IC50 = 1.5 microM, ED50 = 0.1 mg/kg), and Boc-Trp-Leu-beta-homo-Asp-D-Phe-NH2 (13) (IC50 = 2 microM, ED50 = 0.1 mg/kg). We could demonstrate the importance of the region of the peptide bond between leucine and aspartic acid and of the structure of the C-terminal dipeptide Asp-Phe-NH2, for exhibiting biological activity on acid secretion.

  2. C-terminal phosphorylation regulates the kinetics of a subset of melanopsin-mediated behaviors in mice.

    PubMed

    Somasundaram, Preethi; Wyrick, Glenn R; Fernandez, Diego Carlos; Ghahari, Alireza; Pinhal, Cindy M; Simmonds Richardson, Melissa; Rupp, Alan C; Cui, Lihong; Wu, Zhijian; Brown, R Lane; Badea, Tudor Constantin; Hattar, Samer; Robinson, Phyllis R

    2017-03-07

    Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and mediate several non-image-forming visual functions, including circadian photoentrainment and the pupillary light reflex (PLR). ipRGCs act as autonomous photoreceptors via the intrinsic melanopsin-based phototransduction pathway and as a relay for rod/cone input via synaptically driven responses. Under low light intensities, where only synaptically driven rod/cone input activates ipRGCs, the duration of the ipRGC response will be determined by the termination kinetics of the rod/cone circuits. Little is known, however, about the termination kinetics of the intrinsic melanopsin-based phototransduction pathway and its contribution to several melanopsin-mediated behaviors. Here, we show that C-terminal phosphorylation of melanopsin determines the recovery kinetics of the intrinsic melanopsin-based photoresponse in ipRGCs, the duration of the PLR, and the speed of reentrainment. In contrast, circadian phase alignment and direct effects of light on activity (masking) are not influenced by C-terminal phosphorylation of melanopsin. Electrophysiological measurements demonstrate that expression of a virally encoded melanopsin lacking all C-terminal phosphorylation sites (C terminus phosphonull) leads to a prolonged intrinsic light response. In addition, mice expressing the C terminus phosphonull in ipRGCs reentrain faster to a delayed light/dark cycle compared with mice expressing virally encoded WT melanopsin; however, the phase angle of entrainment and masking were indistinguishable. Importantly, a sustained PLR in the phosphonull animals is only observed at brighter light intensities that activate melanopsin phototransduction, but not at dimmer light intensities that activate only the rod/cone pathway. Taken together, our results highlight how the kinetics of the melanopsin photoresponse differentially regulate distinct light-mediated behaviors.

  3. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    DOE PAGES

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; ...

    2014-11-22

    The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. We expressed short, long and extralong MnPs heterologously and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. Furthermore, the tail, which is anchored by numerous contacts, not only affectsmore » the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.« less

  4. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    SciTech Connect

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T.

    2014-11-22

    The genome ofCeriporiopsis subvermisporaincludes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. We expressed short, long and extralong MnPs heterologously and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. Furthermore, the tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of anin silicoshortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

  5. C-terminal region of DNA ligase IV drives XRCC4/DNA ligase IV complex to chromatin

    SciTech Connect

    Liu, Sicheng; Liu, Xunyue; Kamdar, Radhika Pankaj; Wanotayan, Rujira; Sharma, Mukesh Kumar; Adachi, Noritaka; Matsumoto, Yoshihisa

    2013-09-20

    Highlights: •Chromatin binding of XRCC4 is dependent on the presence of DNA ligase IV. •C-terminal region of DNA ligase IV alone can recruit itself and XRCC4 to chromatin. •Two BRCT domains of DNA ligase IV are essential for the chromatin binding of XRCC4. -- Abstract: DNA ligase IV (LIG4) and XRCC4 form a complex to ligate two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). It is not fully understood how these proteins are recruited to DSBs. We recently demonstrated radiation-induced chromatin binding of XRCC4 by biochemical fractionation using detergent Nonidet P-40. In the present study, we examined the role of LIG4 in the recruitment of XRCC4/LIG4 complex to chromatin. The chromatin binding of XRCC4 was dependent on the presence of LIG4. The mutations in two BRCT domains (W725R and W893R, respectively) of LIG4 reduced the chromatin binding of LIG4 and XRCC4. The C-terminal fragment of LIG4 (LIG4-CT) without N-terminal catalytic domains could bind to chromatin with XRCC4. LIG4-CT with W725R or W893R mutation could bind to chromatin but could not support the chromatin binding of XRCC4. The ability of C-terminal region of LIG4 to interact with chromatin might provide us with an insight into the mechanisms of DSB repair through NHEJ.

  6. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi.

    PubMed

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T

    2014-12-01

    The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn(2+)-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn(2+) oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd(2+) binds at the Mn(2+)-oxidation site and competitively inhibits oxidation of both Mn(2+) and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

  7. Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication

    PubMed Central

    Hansmann, Britta; Schröder, Jens-Michael; Gerstel, Ulrich

    2015-01-01

    Soil- and waterborne bacteria such as Pseudomonas aeruginosa are constantly challenging body surfaces. Since infections of healthy skin are unexpectedly rare, we hypothesized that the outermost epidermis, the stratum corneum, and sweat glands directly control the growth of P. aeruginosa by surface-provided antimicrobials. Due to its high abundance in the upper epidermis and eccrine sweat glands, filaggrin-2 (FLG2), a water-insoluble 248 kDa S100 fused-type protein, might possess these innate effector functions. Indeed, recombinant FLG2 C-terminal protein fragments display potent antimicrobial activity against P. aeruginosa and other Pseudomonads. Moreover, upon cultivation on stratum corneum, P. aeruginosa release FLG2 C-terminus-containing FLG2 fragments from insoluble material, indicating liberation of antimicrobially active FLG2 fragments by the bacteria themselves. Analyses of the underlying antimicrobial mechanism reveal that FLG2 C-terminal fragments do not induce pore formation, as known for many other antimicrobial peptides, but membrane blebbing, suggesting an alternative mode of action. The association of the FLG2 fragment with the inner membrane of treated bacteria and its DNA-binding implicated an interference with the bacterial replication that was confirmed by in vitro and in vivo replication assays. Probably through in situ-activation by soil- and waterborne bacteria such as Pseudomonads, FLG2 interferes with the bacterial replication, terminates their growth on skin surface and thus may contributes to the skin’s antimicrobial defense shield. The apparent absence of FLG2 at certain body surfaces, as in the lung or of burned skin, would explain their higher susceptibility towards Pseudomonas infections and make FLG2 C-terminal fragments and their derivatives candidates for new Pseudomonas-targeting antimicrobials. PMID:26371476

  8. Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

    SciTech Connect

    Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

    2008-09-30

    Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS.

  9. Contribution of N- and C-terminal Kv4.2 channel domains to KChIP interaction [corrected].

    PubMed

    Callsen, Britta; Isbrandt, Dirk; Sauter, Kathrin; Hartmann, L Sven; Pongs, Olaf; Bähring, Robert

    2005-10-15

    Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.

  10. Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes.

    PubMed

    Laurin, Yoann; Eyer, Joel; Robert, Charles H; Prevost, Chantal; Sacquin-Mora, Sophie

    2017-03-28

    Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

  11. Endomorphin-2 analogs with C-terminal esterification produce potent systemic antinociception with reduced tolerance and gastrointestinal side effects.

    PubMed

    Wang, Chang-Lin; Qiu, Ting-Ting; Yang, Dai-Jun; Yuan, Bi-Yu; Han, Feng-Tong; Li, Li; Gu, Ning

    2017-04-01

    C-terminal esterification of opioid peptides may change their opioid activities due to the modified physicochemical properties. In the present study, the pharmacological activities of C-terminal esterified endomorphin-2 (EM-2) analogs 1-3 were characterized by in vitro metabolic stability and octanol/buffer distribution assays. Also, the antinociceptive profiles in the radiant heat paw withdrawal test and related side effects of these analogs were determined. Our results showed that all three analogs significantly increased the metabolic stability and lipophilicity. Moreover, analogs 1-3 displayed potent antinociceptive activities after intracerebroventricular (i.c.v.) administration. Analogs 1 and 3 exhibited about 2-fold higher antinociception than EM-2, and differential opioid mechanisms were involved. In addition, EM-2 at 50 μmol/kg failed to produce any significant antinociceptive activity after subcutaneous (s.c.) administration, whereas equimolar dose of analogs 1-3 produced significant analgesic effects. Analog 3 showed the highest antinociceptive activity after systemic administration, which was consistent with its in vitro stability and lipophilicity. We further evaluated the antinociceptive tolerance of analogs 1-3. In acute tolerance test, analogs 1-3 shifted the dose-response curves rightward by only 1.4-3.2 fold as determined by tolerance ratio, whereas EM-2 by 5.6-fold, demonstrating reduced antinociceptive tolerance. Also, analogs 1 and 2 decreased chronic antinociceptive tolerance by central and peripheral administration of drugs. In particular, analogs 3 displayed insignificant chronic antinociceptive tolerance. Furthermore, analogs 1-3 were less prone to induce gastrointestinal side effects at analgesic doses. The present investigation gave the evidence that C-terminal esterified modifications of EM-2 will facilitate the development of novel opioid analgesics with reduced side effects.

  12. Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi

    PubMed Central

    Fernández-Fueyo, Elena; Acebes, Sandra; Ruiz-Dueñas, Francisco J.; Martínez, María Jesús; Romero, Antonio; Medrano, Francisco Javier; Guallar, Victor; Martínez, Angel T.

    2014-01-01

    The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences. PMID:25478843

  13. Role of the C-Terminal Region of Vervet Monkey Polyomavirus 1 VP1 in Virion Formation

    PubMed Central

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