scholarly journals Obscurin Is a Ligand for Small Ankyrin 1 in Skeletal Muscle

2003 ◽  
Vol 14 (3) ◽  
pp. 1138-1148 ◽  
Author(s):  
Aikaterini Kontrogianni-Konstantopoulos ◽  
Ellene M. Jones ◽  
Damian B. van Rossum ◽  
Robert J. Bloch
Keyword(s):  
Striated Muscle ◽  
Direct Interaction ◽  
Kinetic Studies ◽  
Subcellular Fractionation ◽  
In Vitro Assays ◽  
Sarcomeric Protein ◽  
Rho Gef ◽  
Two Hybrid ◽  
Regular Arrays

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a ∼17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K D = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number inPseudomonas aeruginosa

2001 ◽  
Vol 183 (22) ◽  
pp. 6636-6644 ◽  
Author(s):  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Binding Motif ◽  
Yeast Two Hybrid System ◽  
Gtp Binding ◽  
C Terminus ◽  
Enhancer Binding Protein ◽  
Two Hybrid

ABSTRACT Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption offleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

scholarly journals D53 is a novel endosomal SNARE-binding protein that enhances interaction of syntaxin 1 with the synaptobrevin 2 complex in vitro

2003 ◽  
Vol 370 (1) ◽  
pp. 213-221 ◽  
Author(s):  
Véronique PROUX-GILLARDEAUX ◽  
Thierry GALLI ◽  
Isabelle CALLEBAUT ◽  
Anatoly MIKHAILIK ◽  
Georges CALOTHY ◽  
...  
Keyword(s):  
Binding Protein ◽  
Coiled Coil ◽  
Subcellular Fractionation ◽  
Snare Complex ◽  
In Vitro Assays ◽  
Hydrophobic Cluster Analysis ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Main Components

Synaptobrevin 2 (Sb2), syntaxin1 (Stx1), and synaptosomal-associated protein of 25kDa (SNAP-25) are the main components of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) complex involved in fusion of synaptic vesicles with the presynaptic plasma membrane. We report the characterization of D53, a novel SNARE-binding protein preferentially expressed in neural and neuro-endocrine cells. Its two-dimensional organization, established by the hydrophobic cluster analysis, is reminiscent of SNARE proteins. D53 contains two putative helical regions, one of which includes a large coiled-coil domain involved in the interaction with Sb2 in vitro. Following subcellular fractionation, endogenous D53 was specifically detected in the membrane-containing fraction of PC12 cells, where it co-immunoprecipitated with Sb2. Analysis by confocal microscopy showed that, in these cells, endogenous D53 co-localized partially with the transferrin receptor in early endosomes. In vitro assays revealed that binding properties of D53 to Stx1 and Sb2 are comparable with those of SNAP-25. Furthermore, D53 forms Sb2/Stx1/D53 complexes in vitro in a manner similar to SNAP-25. We propose that D53 could be involved in the assembly or disassembly of endosomal SNARE complexes by regulating Sb2/Stx interaction.

scholarly journals New Insights into the Hypotensins from Tityus serrulatus Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities

Toxins ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 846
Author(s):  
Bruno Duzzi ◽  
Cristiane Castilho Fernandes Silva ◽  
Roberto Tadashi Kodama ◽  
Daniela Cajado-Carvalho ◽  
Carla Cristina Squaiella-Baptistão ◽  
...  
Keyword(s):  
Phagocytic Activity ◽  
Clinical Manifestations ◽  
Direct Interaction ◽  
Kinetic Studies ◽  
Pressure Control ◽  
In Vivo Studies ◽  
Hypotensive Activity ◽  
Tityus Serrulatus

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 μM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice’s naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I

scholarly journals Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

2008 ◽  
Vol 19 (2) ◽  
pp. 735-744 ◽  
Author(s):  
Nai-Jung Hung ◽  
Kai-Yin Lo ◽  
Samir S. Patel ◽  
Kara Helmke ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Yeast Two Hybrid ◽  
Synthetic Lethal ◽  
Nuclear Export Receptor ◽  
Export Receptors ◽  
Two Hybrid

We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

scholarly journals Purification of the RelB and RelE Proteins ofEscherichia coli: RelE Binds to RelB and to Ribosomes

2001 ◽  
Vol 183 (8) ◽  
pp. 2700-2703 ◽  
Author(s):  
Cheryl Galvani ◽  
Jefferson Terry ◽  
Edward E. Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Direct Interaction ◽  
Binding Activity ◽  
In Vitro Assay ◽  
Ofescherichia Coli ◽  
Yeast Two Hybrid ◽  
Vitro Assay ◽  
Ribosome Binding ◽  
Two Hybrid

ABSTRACT The direct interaction of the Escherichia colicytotoxin RelE with its specific antidote, RelB, was demonstrated in two ways: (i) copurification of the two proteins and (ii) a positive yeast two-hybrid assay involving the relB andrelE genes. In addition, the purified RelE protein exhibited ribosome-binding activity in an in vitro assay, supporting previous observations suggesting that it is an inhibitor of translation.

scholarly journals Adenovirus E1B 55-Kilodalton Oncoprotein Binds to Daxxand Eliminates Enhancement of p53-Dependent Transcription byDaxx

2003 ◽  
Vol 77 (21) ◽  
pp. 11809-11821 ◽  
Author(s):  
Lisa Y. Zhao ◽  
April L. Colosimo ◽  
Yue Liu ◽  
Yanping Wan ◽  
Daiqing Liao
Keyword(s):  
Cell Transformation ◽  
Protein Complexes ◽  
Direct Interaction ◽  
Promyelocytic Leukemia Protein ◽  
293 Cells ◽  
Pml Bodies ◽  
Adenovirus E1b ◽  
Underlying Mechanisms ◽  
Two Hybrid

ABSTRACT The adenovirus E1B 55-kDa protein impairs the p53 pathway and enhances transformation, although the underlying mechanisms remain to be defined. We found that Daxx binds to the E1B 55-kDa protein in a yeast two-hybrid screen. The two proteins interact through their C termini. Mutation of three potential phosphorylation sites (S489/490 and T494 to alanine) within the E1B 55-kDa protein did not affect its interaction with Daxx, although such mutations were previously shown to inhibit E1B's ability to repress p53-dependent transcription and to enhance transformation. In addition to their coimmunoprecipitation in 293 extracts, purified Daxx interacted with the E1B 55-kDa protein in vitro, indicating their direct interaction. In 293 cells, Daxx colocalized with the E1B 55-kDa protein within discrete nuclear dots, where p53 was also found. Such structures were distinct from PML (promyelocytic leukemia protein) bodies, and it appeared that Daxx was displaced from PML bodies. Thus, the Daxx concentration was diminished in dots with a prominent presence of PML and vice versa. Indeed, PML overexpression led to dramatic redistribution of Daxx from p53-E1B 55-kDa protein complexes to PML bodies. Additionally, expression of the E1B 55-kDa protein in Saos2 osteosarcoma cells reduced the number of PML bodies. Our data suggest that E1B and PML compete for available Daxx in the cell. Surprisingly, Daxx significantly augmented p53-mediated transcription and the E1B 55-kDa protein eliminated this effect. Thus, it is likely that the E1B 55-kDa protein sequesters Daxx and p53 in specific nuclear locations, where p53 cannot activate transcription. One consequence of the Daxx-E1B interaction might be an alteration of normal interactions of Daxx, PML, and p53, which may contribute to cell transformation.

scholarly journals Regulation of Actin Filament Length by Muscle Isoforms of Tropomyosin and Cofilin

2020 ◽  
Vol 21 (12) ◽  
pp. 4285
Author(s):  
Katarzyna Robaszkiewicz ◽  
Małgorzata Śliwinska ◽  
Joanna Moraczewska
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Muscle Fibers ◽  
In Vitro Assays ◽  
Linear Lattice ◽  
Tropomyosin Isoforms ◽  
Slow Twitch Muscle ◽  
Actin Regulatory Proteins ◽  
Fast Twitch

In striated muscle the extent of the overlap between actin and myosin filaments contributes to the development of force. In slow twitch muscle fibers actin filaments are longer than in fast twitch fibers, but the mechanism which determines this difference is not well understood. We hypothesized that tropomyosin isoforms Tpm1.1 and Tpm3.12, the actin regulatory proteins, which are specific respectively for fast and slow muscle fibers, differently stabilize actin filaments and regulate severing of the filaments by cofilin-2. Using in vitro assays, we showed that Tpm3.12 bound to F-actin with almost 2-fold higher apparent binding constant (Kapp) than Tpm1.1. Cofilin2 reduced Kapp of both tropomyosin isoforms. In the presence of Tpm1.1 and Tpm3.12 the filaments were longer than unregulated F-actin by 25% and 40%, respectively. None of the tropomyosins affected the affinity of cofilin-2 for F-actin, but according to the linear lattice model both isoforms increased cofilin-2 binding to an isolated site and reduced binding cooperativity. The filaments decorated with Tpm1.1 and Tpm3.12 were severed by cofilin-2 more often than unregulated filaments, but depolymerization of the severed filaments was inhibited. The stabilization of the filaments by Tpm3.12 was more efficient, which can be attributed to lower dynamics of Tpm3.12 binding to actin.

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