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1.
Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria
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Aphasizhev R Aphasizheva I Nelson RE Gao G Simpson AM Kang X Falick AM Sbicego S Simpson L 《The EMBO journal》2003,22(4):913-924
A multiprotein, high molecular weight complex active in both U-insertion and U-deletion as judged by a pre-cleaved RNA editing assay was isolated from mitochondrial extracts of Leishmania tarentolae by the tandem affinity purification (TAP) procedure, using three different TAP-tagged proteins of the complex. This editing- or E-complex consists of at least three protein-containing components interacting via RNA: the RNA ligase-containing L-complex, a 3' TUTase (terminal uridylyltransferase) and two RNA-binding proteins, Ltp26 and Ltp28. Thirteen approximately stoichiometric components were identified by mass spectrometric analysis of the core L-complex: two RNA ligases; homologs of the four Trypanosoma brucei editing proteins; and seven novel polypeptides, among which were two with RNase III, one with an AP endo/exonuclease and one with nucleotidyltransferase motifs. Three proteins have no similarities beyond kinetoplastids. 相似文献
2.
Vernarecci S Colotti G Ornaghi P Schiebel E Chiancone E Filetici P 《Molecular microbiology》2007,65(4):1122-1138
Penta-EF-hand (PEF) proteins bind calcium and participate in a variety of calcium-dependent processes in vertebrates. In yeast, intracellular cations regulate processes like cell division and polarized growth. This study reports the identification of a unique PEF protein in Saccharomyces cerevisiae encoded by the uncharacterized open reading frame YGR058w. Pef1p has a long and unstructured N-terminal domain conserved in ascomycetes, and a highly conserved C-terminal calcium binding domain homologous to human ALG-2 and sorcin. Pef1p binds calcium and zinc and homodimerizes in vitro and in vivo like vertebrate homologues. Disruption of PEF1 induces defective growth in SDS and cation depletion conditions. Significantly, a critical substitution in the second EF hand (E218A) lowers the in vitro affinity for zinc and phenocopies growth defects. The dissection of protein-protein interactions and the cellular localization of Pef1p analogous to that of RAM pathway components controlling daughter-specific gene expression at the site of bud emergence bring out the importance of this novel protein. Our data suggest that cation homeostasis is involved in the control of polarized growth and in stress response in budding yeast. 相似文献
3.
Guanghan Gao Kestrel Rogers Feng Li Qiang Guo Daren Osato Sharleen X. Zhou Arnold M. Falick Larry Simpson 《Protist》2010,161(3):489-496
U-insertion/deletion RNA editing of mitochondrial mRNAs in trypanosome mitochondria is mediated by a core complex (RECC) containing around 16-20 proteins which is linked to several other multiprotein complexes by RNA. There are two known subcomplexes in the RECC: the REL1 subcomplex which contains the REL1 RNA ligase, the MP63 zinc finger-containing protein and the REX2 U-specific 3’-5’ exonuclease; and the REL2 subcomplex which contains the REL2 RNA ligase, the RET2 3’ TUTase and the MP81 zinc finger-containing protein. In this study we have affinity isolated recombinant TAP-tagged Leishmania major RET2 and Leishmania tarentolae MP63, REL1 and REL2 proteins after expression in baculovirus-infected insect cells. Recombinant MP63 protein was found to stimulate several in vitro activities of recombinant REL1; these activities include autoadenylation, bridged ligation and even pre-cleaved gRNA-mediated U-insertion editing with RET2 which is in the REL2 subcomplex. There was no effect of recombinant MP63 on similar REL2 ligation activities. The specificity for REL1 is consistent with MP63 being a component of the REL1 subcomplex. These results suggest that in vivo the interaction of MP63 with REL1 may play a role in regulating the overall activity of RNA editing. 相似文献
4.
The mammalian RbAp48 protein is the most extensively studied member of the conserved family of Msi1-like WD-40 repeat proteins, which are components of complexes involved in the assembly and modification of chromatin. We have isolated a plant homolog of RbAp48, AtMSI4. By metal affinity chromatography, zinc blotting and atomic absorption analysis, we demonstrate that purified recombinant RbAp48 and AtMSI4 proteins bind 3–4 metal ions per molecule of protein. Metal competition assays indicate a preference for zinc. Both N- and C-terminal halves of RbAp48 and AtMSI4 display zinc binding activity, suggesting it is an intrinsic property of the propeller structures likely to be formed by these proteins. Metal binding might mediate and/or regulate protein-protein interactions which are functionally important in chromatin metabolism. 相似文献
5.
The structure of the MAPK scaffold, MP1, bound to its partner, p14. A complex with a critical role in endosomal map kinase signaling 总被引:1,自引:0,他引:1
Lunin VV Munger C Wagner J Ye Z Cygler M Sacher M 《The Journal of biological chemistry》2004,279(22):23422-23430
Scaffold proteins of the mitogen-activated protein kinase (MAPK) pathway have been proposed to form an active signaling module and enhance the specificity of the transduced signal. Here, we report a 2-A resolution structure of the MAPK scaffold protein MP1 in a complex with its partner protein, p14, that localizes the complex to late endosomes. The structures of these two proteins are remarkably similar, with a five-stranded beta-sheet flanked on either side by a total of three helices. The proteins form a heterodimer in solution and interact mainly through the edge beta-strand in each protein to generate a 10-stranded beta-sheet core. Both proteins also share structural similarity with the amino-terminal regulatory domains of the membrane trafficking proteins, sec22b and Ykt6p, as well as with sedlin (a component of a Golgi-associated membrane-trafficking complex) and the sigma2 and amino-terminal portion of the mu2 subunits of the clathrin adaptor complex AP2. Because neither MP1 nor p14 have been implicated in membrane traffic, we propose that the similar protein folds allow these relatively small proteins to be involved in multiple and simultaneous protein-protein interactions. Mapping of highly conserved, surface-exposed residues on MP1 and p14 provided insight into the potential sites of binding of the signaling kinases MEK1 and ERK1 to this complex, as well as the areas potentially involved in other protein-protein interactions. 相似文献
6.
Interactions of multiple heparin binding growth factors with neuropilin-1 and potentiation of the activity of fibroblast growth factor-2 总被引:7,自引:0,他引:7
West DC Rees CG Duchesne L Patey SJ Terry CJ Turnbull JE Delehedde M Heegaard CW Allain F Vanpouille C Ron D Fernig DG 《The Journal of biological chemistry》2005,280(14):13457-13464
The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF-binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000-1,600,000 m(-1) s(-1)). These results suggest that Npn-1 possesses a "heparin" mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins. 相似文献
7.
Daren Osato Kestrel Rogers Qiang Guo Feng Li Greg Richmond Felix Klug Larry Simpson 《RNA (New York, N.Y.)》2009,15(7):1338-1344
The RNA ligase-containing or L-complex is the core complex involved in uridine insertion/deletion RNA editing in trypanosome mitochondria. Blue native gels of glycerol gradient-separated fractions of mitochondrial lysate from cells transfected with the TAP-tagged editing protein, LC-8 (TbMP44/KREPB5), show a ∼1 MDa L-complex band and, in addition, two minor higher molecular weight REL1-containing complexes: one (L*a) co-sedimenting with the L-complex and running in the gel at around 1.2 MDa; the other (L*b) showing a continuous increase in molecular weight from 1 MDa to particles sedimenting over 70S. The L*b-complexes appear to be mainly composed of L-complex components, since polypeptide profiles of L- and L*b-complex gradient fractions were similar in composition and L*b-complex bands often degraded to L-complex bands after manipulation or freeze–thaw cycles. The L*a-complex may be artifactual since this gel shift can be produced by various experimental manipulations. However, the nature of the change and any cellular role remain to be determined. The L*b-complexes from both lysate and TAP pull-down were sensitive to RNase A digestion, suggesting that RNA is involved with the stability of the L*b-complexes. The MRP1/2 RNA binding complex is localized mainly in the L*b-complexes in substoichiometric amounts and this association is RNase sensitive. We suggest that the L*b-complexes may provide a scaffold for dynamic interaction with other editing factors during the editing process to form the active holoenzyme or “editosome.” 相似文献
8.
Systematic identification of SH3 domain-mediated human protein-protein interactions by peptide array target screening 总被引:1,自引:0,他引:1
Systematic identification of direct protein-protein interactions is often hampered by difficulties in expressing and purifying the corresponding full-length proteins. By taking advantage of the modular nature of many regulatory proteins, we attempted to simplify protein-protein interactions to the corresponding domain-ligand recognition and employed peptide arrays to identify such binding events. A group of 12 Src homology (SH) 3 domains from eight human proteins (Swiss-Prot ID: SRC, PLCG1, P85A, NCK1, GRB2, FYN, CRK) were used to screen a peptide target array composed of 1536 potential ligands, which led to the identification of 921 binary interactions between these proteins and 284 targets. To assess the efficiency of the peptide array target screening (PATS) method in identifying authentic protein-protein interactions, we examined a set of interactions mediated by the PLCgamma1 SH3 domain by coimmunoprecipitation and/or affinity pull-downs using full-length proteins and achieved a 75% success rate. Furthermore, we characterized a novel interaction between PLCgamma1 and hematopoietic progenitor kinase 1 (HPK1) identified by PATS and demonstrated that the PLCgamma1 SH3 domain negatively regulated HPK1 kinase activity. Compared to protein interactions listed in the online predicted human interaction protein database (OPHID), the majority of interactions identified by PATS are novel, suggesting that, when extended to the large number of peptide interaction domains encoded by the human genome, PATS should aid in the mapping of the human interactome. 相似文献
9.
Price HP Menon MR Panethymitaki C Goulding D McKean PG Smith DF 《The Journal of biological chemistry》2003,278(9):7206-7214
Co-translational modification of eukaryotic proteins by N-myristoylation aids subcellular targeting and protein-protein interactions. The enzyme that catalyzes this process, N-myristoyltransferase (NMT), has been characterized in the kinetoplastid protozoan parasites, Leishmania and Trypanosoma brucei. In Leishmania major, the single copy NMT gene is constitutively expressed in all parasite stages as a 48.5-kDa protein that localizes to both membrane and cytoplasmic fractions. Leishmania NMT myristoylates the target acylated Leishmania protein, HASPA, when both are co-expressed in Escherichia coli. Gene targeting experiments have shown that NMT activity is essential for viability in Leishmania. In addition, overexpression of NMT causes gross changes in parasite morphology, including the subcellular accumulation of lipids, leading to cell death. This phenotype is more extreme than that observed in Saccharomyces cerevisiae, in which overexpression of NMT activity has no obvious effects on growth kinetics or cell morphology. RNA interference assays in T. brucei have confirmed that NMT is also an essential protein in both life cycle stages of this second kinetoplastid species, suggesting that this enzyme may be an appropriate target for the development of anti-parasitic agents. 相似文献
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Kodama Y Reese ML Shimba N Ono K Kanamori E Dötsch V Noguchi S Fukunishi Y Suzuki E Shimada I Takahashi H 《Journal of structural biology》2011,174(3):434-442
Protein-protein interactions are necessary for various cellular processes, and therefore, information related to protein-protein interactions and structural information of complexes is invaluable. To identify protein-protein interfaces using NMR, resonance assignments are generally necessary to analyze the data; however, they are time consuming to collect, especially for large proteins. In this paper, we present a rapid, effective, and unbiased approach for the identification of a protein-protein interface without resonance assignments. This approach requires only a single set of 2D titration experiments of a single protein sample, labeled with a unique combination of an (15)N-labeled amino acid and several amino acids (13)C-labeled on specific atoms. To rapidly obtain high resolution data, we applied a new pulse sequence for time-shared NMR measurements that allowed simultaneous detection of a ω(1)-TROSY-type backbone (1)H-(15)N and aromatic (1)H-(13)C shift correlations together with single quantum methyl (1)H-(13)C shift correlations. We developed a structure-based computational approach, that uses our experimental data to search the protein surfaces in an unbiased manner to identify the residues involved in the protein-protein interface. Finally, we demonstrated that the obtained information of the molecular interface could be directly leveraged to support protein-protein docking studies. Such rapid construction of a complex model provides valuable information and enables more efficient biochemical characterization of a protein-protein complex, for instance, as the first step in structure-guided drug development. 相似文献
14.
The RNA interacting domain but not the protein interacting domain is highly conserved in ribosomal protein P0 总被引:9,自引:0,他引:9
Rodríguez-Gabriel MA Remacha M Ballesta JP 《The Journal of biological chemistry》2000,275(3):2130-2136
Protein P0 interacts with proteins P1alpha, P1beta, P2alpha, and P2beta, and forms the Saccharomyces cerevisiae ribosomal stalk. The capacity of RPP0 genes from Aspergillus fumigatus, Dictyostelium discoideum, Rattus norvegicus, Homo sapiens, and Leishmania infantum to complement the absence of the homologous gene has been tested. In S. cerevisiae W303dGP0, a strain containing standard amounts of the four P1/P2 protein types, all heterologous genes were functional except the one from L. infantum, some of them inducing an osmosensitive phenotype at 37 degrees C. The polymerizing activity and the elongation factor-dependent functions but not the peptide bond formation capacity is affected in the heterologous P0 containing ribosomes. The heterologous P0 proteins bind to the yeast ribosomes but the composition of the ribosomal stalk is altered. Only proteins P1alpha and P2beta are found in ribosomes carrying the A. fumigatus, R. norvegicus, and H. sapiens proteins. When the heterologous genes are expressed in a conditional null-P0 mutant whose ribosomes are totally deprived of P1/P2 proteins, none of the heterologous P0 proteins complemented the conditional phenotype. In contrast, chimeric P0 proteins made of different amino-terminal fragments from mammalian origin and the complementary carboxyl-terminal fragments from yeast allow W303dGP0 and D67dGP0 growth at restrictive conditions. These results indicate that while the P0 protein RNA-binding domain is functionally conserved in eukaryotes, the regions involved in protein-protein interactions with either the other stalk proteins or the elongation factors have notably evolved. 相似文献
15.
The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bisphosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P less than 0.002 and P less than 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding site is observed (P less than 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P less than 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network. 相似文献
16.
Kawahashi Y Doi N Takashima H Tsuda C Oishi Y Oyama R Yonezawa M Miyamoto-Sato E Yanagawa H 《Proteomics》2003,3(7):1236-1243
Protein microarrays or proteome chips are potentially powerful tools for comprehensive analysis of protein-protein interactions. In interaction analysis, a set of immobilized proteins is arrayed on slides and each slide is probed with a set of fluorescently labeled proteins. Here we have developed and tested an in vitro protein microarray, in which both arraying and probing proteins were prepared by cell-free translation. The in vitro synthesis of fluorescently labeled proteins was accomplished by a new method: a fluorophore-puromycin conjugate was incorporated into a protein at the C-terminus on the ribosome. The resulting fluorescently labeled proteins were confirmed to be useful for probing protein-protein interactions on protein microarrays in model experiments. Since the in vitro protein microarrays can easily be extended to a high-throughput format and also combined with in vitro display technologies such as the streptavidin-biotin linkage in emulsions method (Doi and Yanagawa, FEBS Lett. 1999, 457, 227-230), our method should be useful for large-scale analysis of protein-protein interactions. 相似文献
17.
The interactions between proteins allow the cell's life. A number of experimental, genome-wide, high-throughput studies have been devoted to the determination of protein-protein interactions and the consequent interaction networks. Here, the bioinformatics methods dealing with protein-protein interactions and interaction network are overviewed. 1. Interaction databases developed to collect and annotate this immense amount of data; 2. Automated data mining techniques developed to extract information about interactions from the published literature; 3. Computational methods to assess the experimental results developed as a consequence of the finding that the results of high-throughput methods are rather inaccurate; 4. Exploitation of the information provided by protein interaction networks in order to predict functional features of the proteins; and 5. Prediction of protein-protein interactions. 相似文献
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The stability of the human erythrocyte membrane skeletal network is reported to be dependent on the state of aggregation of spectrin and decreased or increased by polyphosphate anions or the polyamine, spermine, respectively. We have employed polyacrylamide gel electrophoresis and electron spin resonance (ESR) utilizing spin labels specific for membrane proteins, bilayer lipids, or cell-surface sialic acid in order to gain insight into these observations and into the reliability of the ESR spectra of the protein-specific spin label used to correctly report the interactions of the skeletal protein network. The major findings are: (1) We confirm previous reports that the preferred state of spectrin aggregation in the skeletal network is tetrameric and that spectrin can be reversibly transformed to dimeric spectrin and back to tetrameric spectrin on the membrane. (2) The ESR spectra of the protein specific maleimide spin label employed accurately reflect the state of aggregation of spectrin. (3) As dimeric spectrin is increased on the membrane or when 2,3-bis-phosphoglycerate was added to spin-labeled membranes, increased segmental motion of protein spin label binding sites reflecting decreased protein-protein interactions in the skeletal network is observed (P < 0.002 and P < 0.005, respectively). (4) Conversely, as protein-protein interactions between skeletal proteins or between skeletal proteins and the bilayer are increased by spermine (reflected in the total inability to extract spectrin from the membrane in contrast to control membranes), highly decreased segmental motion of the protein specific spin label binding sites is observed (P < 0.005). (5) The dimeric-tetrameric state of spectrin aggregation on the membrane does not have influence on the order or motion of bilayer lipids nor on the rotational rate of spin-labeled, cell-surface sialic acid, a result also observed when protein-protein interactions were decreased by 2,3-bisphosphoglycerate. In contrast, increased protein-protein interactions by addition of spermine produced a small, but significant, increase in order and decrease in motion of bilayer lipids near the membrane surface as well as a nearly 40% decrease in the apparent rotational correlation time of spin labeled, cell surface sialic acid (P < 0.002). These latter observations are discussed with reference to possible associations of phospholipids and the major, transmembrane sialoglycoprotein with the skeletal protein network. 相似文献
20.
Liew CK Kowalski K Fox AH Newton A Sharpe BK Crossley M Mackay JP 《Structure (London, England : 1993)》2000,8(11):1157-1166
BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function. 相似文献