RESUMO
Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.
Assuntos
Cisteína/genética , Oxirredução/efeitos dos fármacos , Tiorredoxina Redutase 1/genética , Tiorredoxinas/genética , Animais , Cisteína/química , Fator de Crescimento Epidérmico/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , PTEN Fosfo-Hidrolase/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Selênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfetos/metabolismo , Sulfetos/farmacologia , Tiorredoxina Redutase 1/química , Tiorredoxinas/químicaRESUMO
Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that (226)RIVRG(230) and (160)IKNP(163) regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues (226)RIVRG(230) and (160)IKNP(163) are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly(165)-Ile(190) and Ser(200)-Glu(225) are adjacent to each other in the membrane.
Assuntos
Bacteriófagos/genética , Grupo dos Citocromos b/química , Mapeamento de Epitopos , NADPH Oxidases , Neutrófilos/enzimologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/imunologia , Dimerização , Ensaio de Imunoadsorção Enzimática , Humanos , Testes de PrecipitinaRESUMO
The bacteriophage P1 Cre/loxP system has become a powerful tool for in vivo manipulation of the genomes of transgenic mice. Although in vitro studies have shown that Cre can catalyze recombination between cryptic "pseudo-loxP" sites in mammalian genomes, to date there have been no reports of loxP-site infidelity in transgenic animals. We produced lines of transgenic mice that use the mouse Protamine 1 (Prm1) gene promoter to express Cre recombinase in postmeiotic spermatids. All male founders and all Cre-bearing male descendents of female founders were sterile; females were unaffected. Sperm counts, sperm motility, and sperm morphology were normal, as was the mating behavior of the transgenic males and the production of two-celled embryos after mating. Mice that expressed similar levels of a derivative transgene that carries an inactive Cre exhibited normal male fertility. Analyses of embryos from matings between sterile Cre-expressing males and wild-type females indicated that Cre-catalyzed chromosome rearrangements in the spermatids that lead to abortive pregnancies with 100% penetrance. Similar Cre-mediated, but loxP-independent, genomic alterations may also occur in somatic tissues that express Cre, but, because of the greater difficulty of assessing deleterious effects of somatic mutations, these may go undetected. This study indicates that, following the use of the Cre/loxP site-specific recombination systems in vivo, it is prudent to eliminate or inactivate the Cre recombinase gene as rapidly as possible.