The Experts below are selected from a list of 1044 Experts worldwide ranked by ideXlab platform
Kwan Yong Choi - One of the best experts on this subject based on the ideXlab platform.
-
maintenance of alpha helical structures by phenyl rings in the active site tyrosine triad contributes to catalysis and stability of ketosteroid isomerase from pseudomonas putida biotype b
Biochemistry, 2001Co-Authors: Gyu Hyun Nam, Do Soo Jang, Sunshin Cha, Taehee Lee, Do Hyung Kim, Bee Hak Hong, Young Sung Yun, Kwan Yong ChoiAbstract:Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic rearrangement of Δ5-3-Ketosteroids at rates comparable with the diffusion-controlled limit. The tyrosine triad (Tyr14···Tyr55···Tyr30) forming a hydrogen-bond network in the apolar active site of KSI has been characterized in an effort to identify the roles of the phenyl rings in catalysis, stability, and unfolding of the enzyme. The replacement of Tyr14, a catalytic residue, with serine resulted in a 33-fold decrease of kcat, while the replacements of Tyr30 and Tyr55 with serine decreased kcat by 4- and 51-fold, respectively. The large decrease of kcat for Y55S could be due to the structural perturbation of α-helix A3, which results in the reorientation of the active-site residues as judged by the crystal structure of Y55S determined at 2.2 A resolution. Consistent with the analysis of the Y55S crystal structure, the far-UV circular dichroism spectra of Y14S, Y30S, and Y55S indicated that the elimi...
-
pseudoreversion of the catalytic activity of y14f by the additional substitution s of tyrosine with phenylalanine in the hydrogen bond network of delta 5 3 ketosteroid isomerase from pseudomonas putida biotype b
Biochemistry, 2001Co-Authors: Gildon Choi, Bee Hak Hong, Min Sung Kim, Kwan Yong ChoiAbstract:Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Δ5-3-Ketosteroids to their conjugated Δ4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Oδ2···O of Wat504···Tyr-14 Oη···Tyr-55 Oη···Tyr-30 Oη in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26−51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Oδ2 and C3−O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type e...
Chelain R Goodman - One of the best experts on this subject based on the ideXlab platform.
-
abstract p3 05 10 glucocorticoid and aldosterone mimic progestin induction of a therapy resistant cytokeratin 5 positive cell population in estrogen receptor positive breast cancer through a bcl6 dependent mechanism
Cancer Research, 2015Co-Authors: Takahiro Sato, Amy R Peck, Melanie A Girondo, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith P Mitchell, Terry Hyslop, Chelain R GoodmanAbstract:Resistance to anti-estrogen therapy remains a significant problem in patients diagnosed with estrogen receptor-α (ERα) positive breast cancer. Recent progress has defined a "lumino-basal" subclass of ERα-positive breast cancer characterized by mosaic presence of a minor population of ERα-negative cells expressing the basal cytokeratin-5 (CK5). The CK5-positive cells are therapy-resistant and have increased tumorigenic potential. Initial studies have suggested that progestins but not other steroids expand this CK5+ cell population. Unexpectedly, we discovered that at least two 3-Ketosteroids other than progestins, glucocorticoids and mineralocorticoids, are capable of inducing the CK5+/ERα- cell population. CK5+ cells induced by glucocorticoid or aldosterone showed increased clonogenicity in soft agar, expressed the stem cell marker CD44, showed loss of ERα and PR expression, and demonstrated therapy-resistance with reduced apoptosis in response to chemotherapy, and were further enriched following adjuvant antiestrogen or chemotherapies. Induction of CK5+ cells by 3-Ketosteroids was consistently preceded by induction of Bcl6, a transcriptional repressor implicated in breast cancer progression. Suppression of Bcl6 by shRNA or the Bcl6 suppressor, prolactin, abolished 3-ketosteroid-induction of CK5+ cells. Prolactin also blocked 3-ketosteroid-induced colony formation in vitro and suppressed progestin-induction of the CK5+ cell population in T47D xenograft tumors in vivo. Survival analyses with recursive partitioning revealed that CK5 and Bcl6 transcripts or protein levels in ERα+ breast cancer identify patients at high or low risk for tumor in two independent cohorts. The observations provide a mechanism by which stress-related or pharmacologic elevation of glucocorticoids may adversely affect patients with ERα+/CK5+ breast cancer, and may justify further exploring of inhibitors to 3-ketosteroid receptors or Bcl6 for therapeutic benefit. Citation Format: Hallgeir Rui, Takahiro Sato, Amy Peck, Melanie A Girondo, Chengbao Liu, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith Mitchell, Terry Hyslop, Chelain Goodman. Glucocorticoid and aldosterone mimic progestin-induction of a therapy-resistant cytokeratin-5 positive cell population in estrogen receptor-positive breast cancer through a Bcl6-dependent mechanism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-10.
Paul Talalay - One of the best experts on this subject based on the ideXlab platform.
-
ultraviolet spectroscopic evidence for decreased motion of the active site tyrosine residue of delta 5 3 ketosteroid isomerase by steroid binding
Biochemistry, 1995Co-Authors: Qinjian Zhao, Albert S Mildvan, Paul TalalayAbstract:Delta(5)-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the highly efficient conversion of Delta(5)-3-Ketosteroids to Delta(4)-3-Ketosteroids by a stereoselective and intramolecular transfer of the 4 beta-proton to the 6 beta-position. Tyr-14 is the critical general acid and Asp-38 is the general base involved in catalysis. The UV absorption bandwidths of Tyr-14 were much narrower than those of the other two tyrosines (Tyr-55 and Tyr-88) of isomerase or of the N-acetyltyrosine ethyl ester in aqueous solution, suggesting that Tyr-14 is restricted in its mobility. Further immobilization of this residue occurs upon steroid binding. Thus, 5 alpha-estrane-3,17-dione, an A-ring saturated steroid, induces significant narrowing of the tyrosine absorption bands (pi --> pi*) of the main peak (279.5 nm) and the shoulder (285.5 nm) of Tyr-14, with no significant changes in lambda(max). No effects of steroid binding were found on the absorption bandwidths of Tyr-55, Tyr-88, or the phenylalanine residues. The ratio of the absorbance (A(max)) at the absorption maximum (lambda(max)) to that at lambda(max) plus 4 nm (A(max+4)) was used as a measure of peak sharpness. Specifically, the ratios of A(279.5)/A(283.5) (main peak) and A(285.5)/A(289.5) (shoulder) of Tyr-14 of the free enzyme at 25 degrees C were 1.25 and 1.89, respectively, and they increased to 1.41 and 2.70, respectively, in the complex. A more precise measurement of the band narrowing from 4.2 to 3.1 nm between the inflection points was obtained from the derivative spectra. The absorption bands of free and steroid-bound isomerase were narrowed significantly by lowering the temperature and were broadened by denaturation, suggesting that the unusual peak-sharpening effects induced by steroid binding arise from the restricted motion of Tyr-lit, as well as from more directional hydrogen bonding resulting from the displacement of water molecules from the active site and decreased flexibility of the protein. Larger enthalpy of the sharpening effects was observed for the steroid-bound enzyme (-0.527 +/- 0.016 kcal/mol) than for the free enzyme (-0.250 +/- 0.018 kcal/mol) by lowering the temperature, indicating that interactions of Tyr-14 with its environment, which restrain its motion, are stronger in the steroid-bound enzyme than in the free enzyme. Hydrogen-bonding modes of Tyr-14, mobility of the active site, and protein flexibility are the environmental factors determining the absorption bandwidths of the critical Tyr-14 residue.
-
enzymatic and nonenzymatic polarizations of alpha beta unsaturated Ketosteroids and phenolic steroids implications for the roles of hydrogen bonding in the catalytic mechanism of delta 5 3 ketosteroid isomerase
Biochemistry, 1995Co-Authors: Qinjian Zhao, Albert S Mildvan, Paul TalalayAbstract:Ketosteroids (e.g., 19-nortestosterone) and phenolic steroids (e.g., 17 beta-estradiol and 17 beta-dihydroequilenin), which are potent competitive inhibitors of delta 5-3-ketosteroid isomerase (isomerase, EC 5.3.3.1) of Pseudomonas testosteroni, undergo significant polarization upon binding to the active site of the enzyme. The 10 nm red shift of the UV absorption maximum of the enone chromophore of 19-nortestosterone, which occurs in the enzyme-steroid complex, resembles that observed when this steroid is exposed to strong acid. The UV and fluorescence spectral changes of 17 beta-estradiol and 17 beta-dihydroequilenin in the enzyme-bound complex resemble the spectra of ionized phenolate species in aqueous basic solutions. Since most enzymes bind their substrates and competitive inhibitors in a solvent-inaccessible hydrophobic environment, and the generation of charges in such nonpolar environments is unfavorable, we investigated the possibility that the spectral perturbations of the steroids might arise from strong hydrogen bonding in nonpolar environments. For this purpose, the spectral properties of model compounds capable of forming intramolecular hydrogen bonds were studied in nonpolar solvents. Thus, 4-hydroxyandrost-4-ene-3,17-dione, in which the 4-hydroxyl group is intramolecularly hydrogen-bonded to the 3-carbonyl group through a five-membered ring, exhibits a lambda max of 276.0 nm, while the corresponding 4-methyl ether, 4-methoxyandrost-4-ene-3,17-dione, which cannot form an internal hydrogen bond, shows a lambda max of 258.5 nm in aqueous solution.(ABSTRACT TRUNCATED AT 250 WORDS)
Sachin A. Gupte - One of the best experts on this subject based on the ideXlab platform.
-
Glucose-6-Phosphate Dehydrogenase and NADPH Redox Regulates Cardiac Myocyte L-Type Calcium Channel Activity and Myocardial Contractile Function
2016Co-Authors: Dhwajbahadur K. Rawat, Peter Hecker, Makino Watanabe, Sukrutha Chettimada, Richard J. Levy, Takao Okada, John G. Edwards, Sachin A. GupteAbstract:We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca2+ currents (ICa-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-Ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit ICa-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca2+ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and ICa-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of 280 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO2 and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak ICa-L amplitudes, and decreased left ventricular developed pressure and 6dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of ICa-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads t
-
glucose 6 phosphate dehydrogenase and nadph redox regulates cardiac myocyte l type calcium channel activity and myocardial contractile function
PLOS ONE, 2012Co-Authors: Dhwajbahadur K. Rawat, Makino Watanabe, Sukrutha Chettimada, Richard J. Levy, Takao Okada, John G. Edwards, Peter A Hecker, Sachin A. GupteAbstract:We recently demonstrated that a 17-ketosteroid, epiandrosterone, attenuates L-type Ca2+ currents (ICa-L) in cardiac myocytes and inhibits myocardial contractility. Because 17-Ketosteroids are known to inhibit glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, and to reduce intracellular NADPH levels, we hypothesized that inhibition of G6PD could be a novel signaling mechanism which inhibit ICa-L and, therefore, cardiac contractile function. We tested this idea by examining myocardial function in isolated hearts and Ca2+ channel activity in isolated cardiac myocytes. Myocardial function was tested in Langendorff perfused hearts and ICa-L were recorded in the whole-cell patch configuration by applying double pulses from a holding potential of −80 mV and then normalized to the peak amplitudes of control currents. 6-Aminonicotinamide, a competitive inhibitor of G6PD, increased pCO2 and decreased pH. Additionally, 6-aminonicotinamide inhibited G6PD activity, reduced NADPH levels, attenuated peak ICa-L amplitudes, and decreased left ventricular developed pressure and ±dp/dt. Finally, dialyzing NADPH into cells from the patch pipette solution attenuated the suppression of ICa-L by 6-aminonicotinamide. Likewise, in G6PD-deficient mice, G6PD insufficiency in the heart decreased GSH-to-GSSG ratio, superoxide, cholesterol and acetyl CoA. In these mice, M-mode echocardiographic findings showed increased diastolic volume and end-diastolic diameter without changes in the fraction shortening. Taken together, these findings suggest that inhibiting G6PD activity and reducing NADPH levels alters metabolism and leads to inhibition of L-type Ca2+ channel activity. Notably, this pathway may be involved in modulating myocardial contractility under physiological and pathophysiological conditions during which the pentose phosphate pathway-derived NADPH redox is modulated (e.g., ischemia-reperfusion and heart failure).
Takahiro Sato - One of the best experts on this subject based on the ideXlab platform.
-
abstract p3 05 10 glucocorticoid and aldosterone mimic progestin induction of a therapy resistant cytokeratin 5 positive cell population in estrogen receptor positive breast cancer through a bcl6 dependent mechanism
Cancer Research, 2015Co-Authors: Takahiro Sato, Amy R Peck, Melanie A Girondo, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith P Mitchell, Terry Hyslop, Chelain R GoodmanAbstract:Resistance to anti-estrogen therapy remains a significant problem in patients diagnosed with estrogen receptor-α (ERα) positive breast cancer. Recent progress has defined a "lumino-basal" subclass of ERα-positive breast cancer characterized by mosaic presence of a minor population of ERα-negative cells expressing the basal cytokeratin-5 (CK5). The CK5-positive cells are therapy-resistant and have increased tumorigenic potential. Initial studies have suggested that progestins but not other steroids expand this CK5+ cell population. Unexpectedly, we discovered that at least two 3-Ketosteroids other than progestins, glucocorticoids and mineralocorticoids, are capable of inducing the CK5+/ERα- cell population. CK5+ cells induced by glucocorticoid or aldosterone showed increased clonogenicity in soft agar, expressed the stem cell marker CD44, showed loss of ERα and PR expression, and demonstrated therapy-resistance with reduced apoptosis in response to chemotherapy, and were further enriched following adjuvant antiestrogen or chemotherapies. Induction of CK5+ cells by 3-Ketosteroids was consistently preceded by induction of Bcl6, a transcriptional repressor implicated in breast cancer progression. Suppression of Bcl6 by shRNA or the Bcl6 suppressor, prolactin, abolished 3-ketosteroid-induction of CK5+ cells. Prolactin also blocked 3-ketosteroid-induced colony formation in vitro and suppressed progestin-induction of the CK5+ cell population in T47D xenograft tumors in vivo. Survival analyses with recursive partitioning revealed that CK5 and Bcl6 transcripts or protein levels in ERα+ breast cancer identify patients at high or low risk for tumor in two independent cohorts. The observations provide a mechanism by which stress-related or pharmacologic elevation of glucocorticoids may adversely affect patients with ERα+/CK5+ breast cancer, and may justify further exploring of inhibitors to 3-ketosteroid receptors or Bcl6 for therapeutic benefit. Citation Format: Hallgeir Rui, Takahiro Sato, Amy Peck, Melanie A Girondo, Chengbao Liu, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith Mitchell, Terry Hyslop, Chelain Goodman. Glucocorticoid and aldosterone mimic progestin-induction of a therapy-resistant cytokeratin-5 positive cell population in estrogen receptor-positive breast cancer through a Bcl6-dependent mechanism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-10.
