The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Ronald T. Raines - One of the best experts on this subject based on the ideXlab platform.
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interaction of onconase with the human ribonuclease Inhibitor Protein
Biochemical and Biophysical Research Communications, 2008Co-Authors: Rebecca F Turcotte, Ronald T. RainesAbstract:One of the tightest known Protein–Protein interactions in biology is that between members of the ribonuclease A superfamily and the ribonuclease Inhibitor Protein (RI). Some members of this superfamily are able to kill cancer cells, and the ability to evade RI is a major determinant of whether a ribonuclease will be cytotoxic. The archetypal cytotoxic ribonuclease, onconase (ONC), is in late-stage clinical trials for the treatment of malignant mesothelioma. We present here the first measurement of the inhibition of the ribonucleolytic activity of ONC by RI. This inhibition occurs with Ki = 0.15 μM in a solution of low salt concentration.
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Inhibition of human pancreatic ribonuclease by the human ribonuclease Inhibitor Protein.
Journal of molecular biology, 2007Co-Authors: R. Jeremy Johnson, J G Mccoy, G N Phillips, Craig A. Bingman, Ronald T. RainesAbstract:The ribonuclease Inhibitor Protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI x RNase 1 complex was determined at a resolution of 1.95 A, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI.RNase 1 complex was found to be extremely stable (t(1/2)=81 days; K(d)=2.9 x 10(-16) M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10(3)-fold lower association rate for complex formation and 5.9 x 10(9)-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important Protein-Protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.
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inhibition of human pancreatic ribonuclease by the human ribonuclease Inhibitor Protein
Journal of Molecular Biology, 2005Co-Authors: Jeremy R Johnson, J G Mccoy, G N Phillips, Craig A. Bingman, Ronald T. RainesAbstract:The ribonuclease Inhibitor Protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI {center_dot} RNase 1 complex was determined at a resolution of 1.95 {angstrom}, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only nine such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI {center_dot} RNase 1 complex was found to be extremely stable (t{sub 1/2} = 81 days; K{sub d} = 2.9 x 10{sup -16}). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1, Arg39 and Arg91, that are especially important for both the formation and stability of the complex, and are thus termed 'electrostatic targeting residues'. Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1more » that maintained ribonucleolytic activity and conformational stability but had a 2.8 x 10{sup 3}-fold lower association rate for complex formation and 5.9 x 10{sup 9}-fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important Protein-Protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.« less
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fluorescence assay for the binding of ribonuclease a to the ribonuclease Inhibitor Protein
Analytical Biochemistry, 2002Co-Authors: Richele L Abel, Chiwook Park, Marcia C Haigis, Ronald T. RainesAbstract:Abstract Ribonuclease A (RNase A) and the ribonuclease Inhibitor Protein (RI) form one of the tightest known Protein–Protein complexes. RNase A variants and homologues, such as G88R RNase A, that retain ribonucleolytic activity in the presence of RI are toxic to cancer cells. Herein, a new and facile assay is described for measuring the equilibrium dissociation constant (Kd) and dissociation rate constant (kd) for complexes of RI and RNase A. This assay is based on the decrease in fluorescence intensity that occurs when a fluorescein-labeled RNase A binds to RI. To allow time for equilibration, the assay is most readily applied to those complexes with Kd values in the nanomolar range or higher. Using this assay, the value of Kd for the complex of RI with fluorescein-labeled G88R RNase A was determined to be 0.55 ± 0.03 nM. In addition, the value of Kd was determined for the complex of RI with unlabeled G88R RNase A to be 0.57 ± 0.05 nM by using a competition assay with fluorescein-labeled G88R RNase A. Finally, the value of kd for the complex of RI with fluorescein-labeled G88R RNase A was determined to be (7.5 ± 0.4) × 10−3 s−1 by monitoring the increase in fluorescence intensity upon dissociation. This assay can be used to characterize complexes of RI with a wide variety of RNase A variants and homologues, including those with cytotoxic activity.
Armando Gómez-puyou - One of the best experts on this subject based on the ideXlab platform.
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Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without Inhibitor Protein.
Archives of biochemistry and biophysics, 2005Co-Authors: M. Tuena De Gómez-puyou, Lenin Domínguez-ramírez, Gerardo Pérez-hernández, Armando Gómez-puyouAbstract:Abstract The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the Inhibitor Protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500–650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the Inhibitor Protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of Inhibitor Protein, indicating that the Protein stabilizes the global structure of particulate F1.
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Interconversion Between Dimers and Monomers of Endogenous Mitochondrial F_1-Inhibitor Protein Complexes and the Release of the Inhibitor Protein. Spectroscopic Characteristics of the Complexes
Journal of Bioenergetics and Biomembranes, 2004Co-Authors: Lenin Domínguez-ramírez, Armando Gómez-puyou, Georgina Garza-ramos, Hugo Najera, Guillermo Mendoza-hernández, M. Tuena De Gómez-puyouAbstract:The F_1-Inhibitor Protein complex (F_1-IP) was purified from heart submitochondrial particles. Size exclusion chromatography of the endogenous complex showed that it contains dimers (D) and monomers (M) of F_1-IP. Further chromatographic analysis showed that D and M interconvert. At high Protein concentrations, the interconversion reaction is shifted toward the D species. The release of the inhibiting action of IP is faster at low than at high Protein concentrations. During activation of F_1, the M species accumulates through a process that is faster than the release of IP from F_1. These findings indicate that the activation of F_1-IP involves the transformation of D into M, which subsequently loses IP. The spectroscopic characteristics of D, M, and free F_1 show that the binding of IP and dimerization modifies the fluorescence intensity of tyrosine residues and that of the single tryptophan of F_1 which is far from the IP binding site.
M. Tuena De Gómez-puyou - One of the best experts on this subject based on the ideXlab platform.
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Effect of denaturants on multisite and unisite ATP hydrolysis by bovine heart submitochondrial particles with and without Inhibitor Protein.
Archives of biochemistry and biophysics, 2005Co-Authors: M. Tuena De Gómez-puyou, Lenin Domínguez-ramírez, Gerardo Pérez-hernández, Armando Gómez-puyouAbstract:Abstract The effect of guanidinium hydrochloride (GdnHCl) on multisite and unisite ATPase activity by F0F1 of submitochondrial particles from bovine hearts was studied. In particles without control by the Inhibitor Protein, 50 mM GdnHCl inhibited multisite hydrolysis by about 85%; full inhibition required around 500 mM. In the range of 500–650 mM, GdnHCl enhanced the rate of unisite catalysis by promoting product release; it also increased the rate of hydrolysis of ATP bound to the catalytic site without GdnHCl. GdnHCl diminished the affinity of the enzyme for aurovertin. The effects of GdnHCl were irreversible. The results suggest that disruption of intersubunit contacts in F0F1 abolishes multisite hydrolysis and stimulates of unisite hydrolysis. Particles under control by the Inhibitor Protein were insensitive to concentrations of GdnHCl that induce the aforementioned alterations of F0F1 free of Inhibitor Protein, indicating that the Protein stabilizes the global structure of particulate F1.
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Interconversion Between Dimers and Monomers of Endogenous Mitochondrial F_1-Inhibitor Protein Complexes and the Release of the Inhibitor Protein. Spectroscopic Characteristics of the Complexes
Journal of Bioenergetics and Biomembranes, 2004Co-Authors: Lenin Domínguez-ramírez, Armando Gómez-puyou, Georgina Garza-ramos, Hugo Najera, Guillermo Mendoza-hernández, M. Tuena De Gómez-puyouAbstract:The F_1-Inhibitor Protein complex (F_1-IP) was purified from heart submitochondrial particles. Size exclusion chromatography of the endogenous complex showed that it contains dimers (D) and monomers (M) of F_1-IP. Further chromatographic analysis showed that D and M interconvert. At high Protein concentrations, the interconversion reaction is shifted toward the D species. The release of the inhibiting action of IP is faster at low than at high Protein concentrations. During activation of F_1, the M species accumulates through a process that is faster than the release of IP from F_1. These findings indicate that the activation of F_1-IP involves the transformation of D into M, which subsequently loses IP. The spectroscopic characteristics of D, M, and free F_1 show that the binding of IP and dimerization modifies the fluorescence intensity of tyrosine residues and that of the single tryptophan of F_1 which is far from the IP binding site.
Richard E Morton - One of the best experts on this subject based on the ideXlab platform.
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cetp and lipid transfer Inhibitor Protein are uniquely affected by the negative charge density of the lipid and Protein domains of ldl
Journal of Lipid Research, 2003Co-Authors: Richard E Morton, Diane J GreeneAbstract:LipoProtein surface charge influences choles- teryl ester transfer Protein (CETP) activity and its associa- tion with lipoProteins; however, the relationship between these events is not clear. Additionally, although CETP and its regulator, lipid transfer Inhibitor Protein (LTIP), bind to lipoProteins, it is not known how the charge density of lipo- Protein Protein and lipid domains influences these factors. Here, the electronegativity of the Protein (by acetylation) and surface lipid (oleate addition) domains of LDL were modified. LDL-only lipid transfer assays measured changes in CETP and LTIP activities. CETP activity was stimulated by � 10 � M oleate but completely suppressed by � 20 � M. The same electronegative potential induced by acetylation mildly stimulated CETP. Modification-induced enhanced binding of CETP did not correlate with CETP activity. LTIP activity was completely blocked by � 10 � M oleate but only mildly suppressed by acetylation. LTIP binding to LDL was not decreased by oleate. Thus, the negative charge of LDL surface lipids, but not Protein, is an important regula- tor of CETP and LTIP activity. Altered binding could not explain changes in CETP activity, suggesting that the extent of CETP binding is not normally rate limiting to its activity. Physiologic and pathophysiologic conditions that modify the negative charge of lipoProtein surface lipids will sup- press LTIP activity first, followed by CETP. —Morton, R. E., and D. J. Greene. CETP and lipid transfer Inhibitor Protein are uniquely affected by the negative charge density of the lipid and Protein domains of LDL. J. Lipid Res. 2003. 44: 2287-2296.
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Cholesteryl ester transfer Protein and its plasma regulator: lipid transfer Inhibitor Protein.
Current opinion in lipidology, 1999Co-Authors: Richard E MortonAbstract:The interconnections between cholesteryl ester transfer Protein (CETP) expression and lipid metabolism, and the possible roles of CETP in atherogenesis are examined. The importance of lipid transfer Inhibitor Protein in modulating CETP activity is detailed, and the consequences of this Inhibitory activity on CETP-mediated events are proposed.
Jerson L. Silva - One of the best experts on this subject based on the ideXlab platform.
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Pressure Effects on the Interaction between Natural Inhibitor Protein and Mitochondrial F1–ATPase☆☆☆
Archives of biochemistry and biophysics, 1998Co-Authors: Luz Alba M. G. Fornells, Horacio Guimarães-motta, Jorge Saad Nehme, Orlando B. Martins, Jerson L. SilvaAbstract:Abstract Pressure stability of the complex formed between F1-ATPase and the Inhibitor Protein (IP) was studied in the membrane-bound and soluble, purified forms of beef-heart mitochondrial enzymes. A latent preparation of submitochondrial particles (SMP-MgATP) initially exhibits low hydrolytic activity. Dissociation of IP increases the activity about 10-fold. This increase occurs in parallel with an increase in sensitivity to pressure inactivation. The membrane-bound, latent IP–F1-ATPase complex is activated 2.5-fold when incubated at a pressure of 1.7 kbar, suggesting dissociation of IP. A fully active preparation of submitochondrial particles depleted of IP (AS-particles) is highly pressure labile when compared with the latent form. In the absence of IP, soluble purified F1-ATPase is also inactivated by pressure. In contrast, the soluble IP–F1-ATPase complex is very resistant to pressure, as evidenced by enzymatic and fluorescence studies. Based on the pressure–titration experiments, binding of IP stabilizes the F1-ATPase complex by 1.54 kcal per mole of complex. The substrate MgATP confers additional protection on both preparations only in the presence of IP. Glycerol appears to prevent dissociation of IP and therefore protects SMP-MgATP from pressure inactivation. Our results demonstrate that in addition to its regulatory role in catalysis, IP stabilizes the structure of the F1-ATPase complex. The pressure-induced dissociation of IP from F1-ATPase and its prevention by glycerol suggest that nonpolar in addition to electrostatic interactions are important for the binding of IP to the regulatory site.
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A contribution of the mitochondrial adenosinetriphosphatase Inhibitor Protein to the thermal stability of the F0F1-ATPase complex.
Zeitschrift fur Naturforschung. C Journal of biosciences, 1997Co-Authors: Jorge Saad-nehme, André Luis Bezerra, Luz Alba M. G. Fornells, Jerson L. Silva, José Roberto Meyer-fernandesAbstract:A complete inactivation is observed after a 3 min pre-incubation at 70 degrees C with mitochondrial F0F1-ATPase complex depleted of the ATPase natural Inhibitor Protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial particles (particles that after a 4 h-pre-incubation at 42 degrees C released the endogenous Inhibitor Protein). However, latent MgATP-submitochondrial particles (particles containing the Inhibitor Protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 micrograms/ml of purified ATPase Inhibitor Protein there is an increase of 15-fold in the half-time for thermal inactivation (t0.5), showing that the Inhibitor Protein protects the mitochondrial F0F1-ATPase complex against thermal inactivation.