Estradiol-17beta stimulates specific receptor and endogenous nitric oxide-dependent dynamic endothelial protein S-nitrosylation: analysis of endothelial nitrosyl-proteome.
Covalent adduction of a nitrosyl group to cysteines [S-nitrosylation (S-NO)] is emerging as a key route for nitric oxide (NO) to directly modulate protein functions. Here, we studied the effects of estrogens on endothelial protein S-NO and analyzed the nitrosyl-proteomes by biotin/CyDye switch technique combined with two-dimensional fluorescence difference gel electrophoresis and identified nitrosoproteins by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Estradiol-17beta (E2) rapidly stimulated protein S-NO in human umbilical vein endothelial cells, maximizing within 10- to 30-min post-E2 (10 nm) exposure. E2-BSA also rapidly stimulated protein S-NO. Both E2 and E2-BSA-induced protein S-NO was blocked by ICI 182,780 and N-nitro-l-arginine-methylester. Human umbilical vein endothelial cells expressed estrogen receptor (ER)alpha and ERbeta; both seemed to be required for E2 stimulation of protein S-NO because: 1) neither ERalpha or ERbeta agonist alone, but their combination, stimulated protein S-NO; and 2) either ERalpha or ERbeta antagonist blocked E2-induced protein S-NO. Numerous nitrosoproteins (spots) were observed on two-dimensional fluorescence difference gel. One hundred spots of interest were picked up; 58 were identified and, of which 15 were novel nitrosoproteins, 28 were up-regulated, 11 were decreased, and the rest were unchanged by E2. Pathway analysis suggested that nitrosoproteins are involved in regulating various endothelial functions, including apoptosis, cell structure and metabolism, redox homeostasis, etc. Thus, estrogens stimulate dynamic endothelial protein S-NO via mechanisms linked to specific ERs possibly on the plasma membrane and endogenous NO. These findings signify a critical next step for the understanding of the biological targets of enhanced NO production by estrogens.
Department of Obstetrics and Gynecology, University of California Irvine, Irvine, California 92697, USA.
Hong-Hai
HH
Zhang
Lin
L
Feng
Itamar
I
Livnat
Jeong-Kyu
JK
Hoh
Jae-Yoon
JY
Shim
Wu-Xiang
WX
Liao
Dong-Bao
DB
Chen
D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology*; D004965:Estrogen Antagonists / Q000494:pharmacology; D000077267:Fulvestrant; D006801:Humans; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D015538:Nitrosation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D011960:Receptors, Estrogen / Q000819:agonists* / Q000378:metabolism / Q000502:physiology; D013379:Substrate Specificity / Q000187:drug effects
D004965:Estrogen Antagonists; D009566:Nitrates; D011506:Proteins; D020543:Proteome; D011960:Receptors, Estrogen; D000077267:Fulvestrant; D009569:Nitric Oxide; D004958:Estradiol