16 results on '"GTPase activation"'
Search Results
2. Mechanisms of decoding and peptide bond formation
- Author
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Rodnina, Marina V., Rodnina, Marina V., editor, Wintermeyer, Wolfgang, editor, and Green, Rachel, editor
- Published
- 2011
- Full Text
- View/download PDF
3. Genetic and crystallographic approaches to investigating ribosome structure and function
- Author
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Gregory, Steven T., Demirci, Hasan, Carr, Jennifer F., Belardinelli, Riccardo, Thompson, Jill R., Cameron, Dale, Rodriguez-Correa, Daniel, Murphy, Frank, Jogl, Gerwald, Dahlberg, Albert E., Rodnina, Marina V., editor, Wintermeyer, Wolfgang, editor, and Green, Rachel, editor
- Published
- 2011
- Full Text
- View/download PDF
4. High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation.
- Author
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Koraïchi, Faten, Gence, Re'mi, Bouchenot, Catherine, Grosjean, Sarah, Lajoie-Mazenc, Isabelle, Favre, Gilles, and Cabantous, Ste'phanie
- Subjects
- *
GENE expression , *CELL proliferation , *PROTEIN-protein interactions , *PREVENTION - Abstract
The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
5. The cation-dependent G-proteins: In a class of their own
- Author
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Ash, Miriam-Rose, Maher, Megan J., Mitchell Guss, J., and Jormakka, Mika
- Subjects
- *
CATIONS , *G proteins , *NUCLEOTIDES , *HYDROLYSIS , *CHEMICAL reactions , *GUANOSINE triphosphatase - Abstract
Abstract: G-proteins are some of the most important and abundant enzymes, yet their intrinsic nucleotide hydrolysis reaction is notoriously slow and must be accelerated in vivo. Recent experiments on dynamin and GTPases involved in ribosome assembly have demonstrated that their hydrolysis activities are stimulated by potassium ions. This article presents the hypothesis that cation-mediated activation of G-proteins is more common than currently realised, and that such GTPases represent a structurally and functionally unique class of G-proteins. Based on sequence analysis we provide a list of predicted cation-dependent GTPases, which encompasses almost all members of the TEES, Obg-HflX, YqeH-like and dynamin superfamilies. The results from this analysis effectively re-define the conditions under which many of these G-proteins should be studied in vitro. [Copyright &y& Elsevier]
- Published
- 2012
- Full Text
- View/download PDF
6. Insight into Catalysis of a Unique GTPase Reaction by a Combined Biochemical and FTIR Approach
- Author
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Chakrabarti, Partha P., Daumke, Oliver, Suveyzdis, Yan, Kötting, Carsten, Gerwert, Klaus, and Wittinghofer, Alfred
- Subjects
- *
INFRARED spectroscopy , *SURFACE chemistry , *FOURIER transform infrared spectroscopy , *CARRIER proteins - Abstract
Abstract: Rap1 and Rap2 are the only small guanine nucleotide-binding proteins of the Ras superfamily that do not use glutamine for GTP hydrolysis. Moreover, Rap1GAP, which stimulates the GTPase reaction of Rap1 105-fold, does not have the classical “arginine finger” like RasGAP but presumably, introduces an asparagine residue into the active site. Here, we address the requirements of this unique reaction in detail by combining various biochemical methods, such as fluorescence spectroscopy, stopped-flow and time-resolved Fourier transform infrared spectroscopy (FTIR). The fluorescence spectroscopic assay monitors primarily protein–protein interaction steps, while FTIR resolves simultaneously the elementary steps of functional groups labor-free, but it is less sensitive and needs higher concentrations. Combining both methods allows us to distinguish weather mechanistic defects caused by mutation are due to affinity or due to functionality. We show that several mutations of Asn290 block catalysis. Some of the mutants, however, still form a complex with Rap1•GDP in the presence of BeFx but not AlFx, supporting the notion that fluoride complexes are indicators of the ground versus transition state. Mutational analysis also shows that Thr61 is not required for catalysis. While replacement of Thr61 of Rap1 by Leu eliminates GTPase activation by Rap1GAP, the T61A and T61Q mutants have only a minor effect on catalysis, but change the relative rates of cleavage and (Pi −) release. While Rap1GAP(N290A) is completely inactive on wild-type Rap1, it can act on Rap1(T61Q), arguing that Asn290 in trans has a role in catalysis similar to that of the intrinsic Gln in Ras and Rho. Finally, since FTIR works at high, and thus mostly saturating, concentrations, it can clearly separate effects on affinity from purely catalytic modifications, showing that Arg388, conserved between RapGAPs and mutated in the homologous RheBGAP Tuberin, affects binding affinity severely but has no effect on the cleavage reaction itself. [Copyright &y& Elsevier]
- Published
- 2007
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- View/download PDF
7. An eIF5/eIF2 complex antagonizes guanine nucleotide exchange by eIF2B during translation initiation.
- Author
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Singh, Chingakham Ranjit, Lee, Bumjun, Udagawa, Tsuyoshi, Mohammad-Qureshi, Sarah S., Yamamoto, Yasufumi, Pavitt, Graham D., and Asano, Katsura
- Subjects
- *
G proteins , *PHYSIOLOGY , *GENETIC mutation , *TRANSFER RNA , *NUCLEOTIDES , *GENETICS - Abstract
In eukaryotic translation initiation, the eIF2·GTP/Met-tRNAiMet ternary complex (TC) binds the eIF3/eIF1/eIF5 complex to form the multifactor complex (MFC), whereas eIF2·GDP binds the pentameric factor eIF2B for guanine nucleotide exchange. eIF5 and the eIF2Bε catalytic subunit possess a conserved eIF2-binding site. Nearly half of cellular eIF2 forms a complex with eIF5 lacking Met-tRNAiMet, and here we investigate its physiological significance. eIF5 overexpression increases the abundance of both eIF2/eIF5 and TC/eIF5 complexes, thereby impeding eIF2B reaction and MFC formation, respectively. eIF2Bε mutations, but not other eIF2B mutations, enhance the ability of overexpressed eIF5 to compete for eIF2, indicating that interaction of eIF2Bε with eIF2 normally disrupts eIF2/eIF5 interaction. Overexpression of the catalytic eIF2Bε segment similarly exacerbates eIF5 mutant phenotypes, supporting the ability of eIF2Bε to compete with MFC. Moreover, we show that eIF5 overexpression does not generate aberrant MFC lacking tRNAiMet, suggesting that tRNAiMet is a vital component promoting MFC assembly. We propose that the eIF2/eIF5 complex represents a cytoplasmic reservoir for eIF2 that antagonizes eIF2B-promoted guanine nucleotide exchange, enabling coordinated regulation of translation initiation. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
8. Phosphoinositol lipids bind to phosphatidylinositol 3 (PI3)-Kinase enhancer GTPase and mediate its stimulatory effect on PI3-Kinase and Akt signalings.
- Author
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Yuanxin Hu, Zhixue Liu, and Keqiang Ye
- Subjects
- *
HYDROGEN-ion concentration , *PROTEINS , *CYTOPLASM , *HOMOLOGY (Biology) , *STEROIDS , *ENZYMES - Abstract
Phosphatidylinositol 3 (P13)-kinase enhancer (PIKE) is a nuclear GTPase that enhances P13-kinase activity in a GTP-dependent manner. Both PIKE-L and -A isoforms contain GTPase, pleckstrin homology (PH), ADP ribosylation factor-GTPase-activating protein, and two ankyrin repeats domains, and C-terminal ADP ribosylation factor-GTPase-activating protein activates its internal GTPase activity. However, whether PH domain modulates the intramolecular action and subsequently influences its downstream signalings remains elusive. Here we show that PH domain from PIKE-L robustly binds Pl(3,4,5)P3 and exclusively resides in the nucleus. By contrast, the mutant (K679,687N), unable to bind phosphoinositol lipids, translocates to the cytoplasm. This mutation substantially compromises the stimulatory effects on P13-kinase by PIKE-L. Surprisingly, PH domain from PIKE-A distributes in the cytoplasm. Similar mutation in PH domain of PIKE-A abolishes its binding to P1(3,4,5)P3 and significantly decreases its activation of Akt. More- over, amplified PIKE-A from human cancers contains mutations and highly stimulates Akt kinase activity, correlating with its GTPase activity. Thus, phosphatidylinositols regulate PIKE GTPase activity, mediating its downstream P13-kinase/Akt signaling through a feedback mechanism by binding to its PH domain. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
9. The calcium sensing receptor (CaSR) promotes Rab27B expression and activity to control secretion in breast cancer cells.
- Author
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Zavala-Barrera, Cesar, del-Río-Robles, Jorge Eduardo, García-Jiménez, Irving, Egusquiza-Alvarez, Carlos Alejandro, Hernández-Maldonado, Jennifer Paulina, Vázquez-Prado, José, and Reyes-Cruz, Guadalupe
- Subjects
- *
CANCER cells , *GUANINE nucleotide exchange factors , *SECRETORY granules , *BREAST cancer , *CHEMOTACTIC factors , *HELA cells - Abstract
Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1β, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment. [Display omitted] • CaSR stimulates Rab27B expression and activity. • CaSR activates Rab27B via MADD GEF/Rab3GEP. • PKA and PI3K/mTOR participate in the activation of Rab27B by CaSR. • CaSR-dependent secretion of cytokines and chemotactic factors involves Rab27B. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
10. High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation
- Author
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Faten Koraïchi, Gilles Favre, Sarah Grosjean, Isabelle Lajoie-Mazenc, Stéphanie Cabantous, Rémi Gence, and Catherine Bouchenot
- Subjects
0301 basic medicine ,RHOA ,RHOB ,Genetic Vectors ,Green Fluorescent Proteins ,GTPase ,GTPase activation ,03 medical and health sciences ,0302 clinical medicine ,Protein Interaction Mapping ,Guanine Nucleotide Exchange Factors ,Humans ,Small GTPase ,GEF ,rhoB GTP-Binding Protein ,biology ,Cell-based assays ,Cell Biology ,High-Throughput Screening Assays ,Tools and Resources ,Cell biology ,Protein–protein interaction ,HEK293 Cells ,030104 developmental biology ,High-content analysis ,030220 oncology & carcinogenesis ,High-content screening ,biology.protein ,Split-GFP ,GTP Phosphohydrolase Activators ,Guanine nucleotide exchange factor ,Cellular model ,Ras superfamily ,rhoA GTP-Binding Protein ,Protein Binding - Abstract
The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains., Summary: The development of a fluorescent reporter of GTPase activation based on tripartite split-GFP that enables the evaluation of GEF activity and the effect of modulators of GTPase activation in a high-content analysis.
- Published
- 2017
11. The cation-dependent G-proteins: In a class of their own
- Author
-
Miriam-Rose Ash, Megan J. Maher, Mika Jormakka, and J. Mitchell Guss
- Subjects
Dynamins ,Protein Conformation ,G protein ,Molecular Sequence Data ,Molecular Conformation ,Biophysics ,GTPase ,GTPase activation ,Biology ,Models, Biological ,Biochemistry ,Ribosome ,GTP Phosphohydrolases ,Ribosome assembly ,03 medical and health sciences ,GTP-binding protein regulators ,Protein structure ,GTP-Binding Proteins ,Structural Biology ,Cations ,Genetics ,Humans ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,030304 developmental biology ,Dynamin ,0303 health sciences ,Binding Sites ,Sequence Homology, Amino Acid ,Hydrolysis ,030302 biochemistry & molecular biology ,Potassium stimulation ,Cell Biology ,Dynamin-like GTPase ,3. Good health ,Cell biology ,Ribosome-assembly GTPase ,Cation-dependent GTPase ,Potassium ,G-protein classification ,Ribosomes - Abstract
G-proteins are some of the most important and abundant enzymes, yet their intrinsic nucleotide hydrolysis reaction is notoriously slow and must be accelerated in vivo. Recent experiments on dynamin and GTPases involved in ribosome assembly have demonstrated that their hydrolysis activities are stimulated by potassium ions. This article presents the hypothesis that cation-mediated activation of G-proteins is more common than currently realised, and that such GTPases represent a structurally and functionally unique class of G-proteins. Based on sequence analysis we provide a list of predicted cation-dependent GTPases, which encompasses almost all members of the TEES, Obg-HflX, YqeH-like and dynamin superfamilies. The results from this analysis effectively re-define the conditions under which many of these G-proteins should be studied in vitro.
- Published
- 2012
12. The Escherichia coli SRP Receptor Forms a Homodimer at the Membrane.
- Author
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Kempf, Georg, Stjepanovic, Goran, Sloan, Jeremy, Hendricks, Astrid, Lapouge, Karine, and Sinning, Irmgard
- Subjects
- *
ESCHERICHIA coli , *SIGNAL recognition particle receptor , *HOMODIMERS , *GUANOSINE triphosphate , *HYDROLYSIS - Abstract
Summary The Escherichia coli signal recognition particle (SRP) receptor, FtsY, plays a fundamental role in co-translational targeting of membrane proteins via the SRP pathway. Efficient targeting relies on membrane interaction of FtsY and heterodimerization with the SRP protein Ffh, which is driven by detachment of α helix (αN1) in FtsY. Here we show that apart from the heterodimer, FtsY forms a nucleotide-dependent homodimer on the membrane, and upon αN1 removal also in solution. Homodimerization triggers reciprocal stimulation of GTP hydrolysis and occurs in vivo. Biochemical characterization together with integrative modeling suggests that the homodimer employs the same interface as the heterodimer. Structure determination of FtsY NG+1 with GMPPNP shows that a dimerization-induced conformational switch of the γ-phosphate is conserved in Escherichia coli , filling an important gap in SRP GTPase activation. Our findings add to the current understanding of SRP GTPases and may challenge previous studies that did not consider homodimerization of FtsY. Graphical Abstract Highlights • The SRP receptor FtsY forms a homodimer at the membrane in vivo and in vitro • Homodimer formation relies on lipid-triggered detachment of helix αN1 • Homodimer formation triggers stimulation of GTP hydrolysis • The homodimer uses the same interface as the SRP/SR GTPase heterodimer Efficient protein targeting requires heterodimerization and activation of the GTPases present in the SRP receptor FtsY and the SRP protein Ffh. We now show that FtsY forms a homodimer at the membrane using the same interaction surface as the heterodimer. Homodimerization adds to the complex interaction landscape of protein targeting. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
13. Multi-functional regulator MapZ controls both positioning and timing of FtsZ polymerization.
- Author
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Feng Z, Zhang J, Xu D, Jiang YL, Zhou CZ, and Chen Y
- Subjects
- Hydrolysis, Protein Domains, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Cytoskeletal Proteins chemistry, Cytoskeletal Proteins metabolism, Guanosine Triphosphate chemistry, Guanosine Triphosphate metabolism, Protein Multimerization, Streptococcus pneumoniae chemistry, Streptococcus pneumoniae metabolism
- Abstract
The tubulin-like GTPase protein FtsZ, which forms a discontinuous cytokinetic ring at mid-cell, is a central player to recruit the division machinery to orchestrate cell division. To guarantee the production of two identical daughter cells, the assembly of FtsZ, namely Z-ring, and its precise positioning should be finely regulated. In Streptococcus pneumoniae , the positioning of Z-ring at the division site is mediated by a bitopic membrane protein MapZ (mid-cell-anchored protein Z) through direct interactions between the intracellular domain (termed MapZ-N (the intracellular domain of MapZ)) and FtsZ. Using nuclear magnetic resonance titration experiments, we clearly assigned the key residues involved in the interactions. In the presence of MapZ-N, FtsZ gains a shortened activation delay, a lower critical concentration for polymerization and a higher cooperativity towards GTP hydrolysis. On the other hand, MapZ-N antagonizes the lateral interactions of single-stranded filaments of FtsZ, thus slows down the formation of highly bundled FtsZ polymers and eventually maintains FtsZ at a dynamic state. Altogether, we conclude that MapZ is not only an accelerator to trigger the polymerization of FtsZ, but also a brake to tune the velocity to form the end-product, FtsZ bundles. These findings suggest that MapZ is a multi-functional regulator towards FtsZ that controls both the precise positioning and proper timing of FtsZ polymerization., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2019
- Full Text
- View/download PDF
14. Expression and Localisation of Wave Isoforms in Human Schwann and Schwannoma Cells
- Author
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Schmid, M.
- Subjects
Westernblot ,Schwann ,Wave isoforms ,Schwannoma ,NF2 ,otorhinolaryngologic diseases ,cytoskeleton ,Cell ,tumours ,GTPase activation ,merlin ,Immunocytochemistry ,Human - Abstract
Schwannoma, the main tumours associated with Neurofibromatosis Type II, are caused by loss of function mutations in the NF2 gene, encoding for the tumour suppressor Merlin. Schwannoma cells display protrusive structures, due to deregulation of the cytoskeleton. The cytoskeleton is controlled by small Rho GTPases, such as Rac1 and Cdc42, known to be activated continuously in merlin-deficient human schwannoma cells (NF2-/-). Downstream Rac signalling leads to the activaton of Wave signalling complexes, which have been shown to bind and activate the Arp2/3 complex, a crucial regulator of the actin cytoskeleton that is involved in formation of protrusive structures. This investigation used an established NF2 model of primary human Schwann and schwannoma cells to investigate the expression and localisation of Pan-Wave and Wave-2. Using commercially available antibodies, immunocytochemistry (ICC) labelling for Pan-Wave showed an atypical distribution in both human Schwann and schwannoma cells. Western blotting for Pan-Wave in NF2-/- (schwannoma cells) also showed multiple non-specific bands. ICC for Wave2 in human schwannoma showed atypical distribution of this isoform. Western blotting for Wave2 using whole Schwann cell lysate, schwannoma lysate and HL60 cell lysate confirmed the non-specificity of the antibody. Therefore the specificity regarding commercially available antibodies is discussed.
- Published
- 2008
15. tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes
- Author
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Cheng, Kimberley, Ivanova, Natalia, Scheres, Sjores, Pavlov, Michael Y, Maria Carazo, Jose, Hebert, Hans, Ehrenberg, Måns, Lindahl, Martin, Cheng, Kimberley, Ivanova, Natalia, Scheres, Sjores, Pavlov, Michael Y, Maria Carazo, Jose, Hebert, Hans, Ehrenberg, Måns, and Lindahl, Martin
- Abstract
Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation. (C) 2009 Elsevier Inc. All rights reserved., QC 20100708
- Published
- 2010
- Full Text
- View/download PDF
16. Phosphoinositol Lipids Bind to Phosphatidylinositol 3 (PI3)-Kinase Enhancer GTPase and Mediate Its Stimulatory Effect on PI3-Kinase and Akt Signalings
- Author
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Hu, Yuanxin and Ye, Keqiang
- Published
- 2005
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