RESUMEN
Peroxiredoxins (Prxs) play dual roles as both thiol-peroxidases and molecular chaperones. Peroxidase activity enables various intracellular functions, however, the physiological roles of Prxs as chaperones are not well established. To study the chaperoning function of Prx, we previously sought to identify heat-induced Prx-binding proteins as the clients of a Prx chaperone. By using His-tagged Prx I as a bait, we separated ubiquitin C-terminal hydrolase-L1 (UCH-L1) as a heat-induced Prx I binding protein from rat brain crude extracts. Protein complex immunoprecipitation with HeLa cell lysates revealed that both Prx I and Prx II interact with UCH-L1. However, Prx II interacted considerably more favorably with UCH-L1 than Prx I. Prx II exhibited more effective molecular chaperone activity than Prx I when UCH-L1 was the client. Prx II interacted with UCH-L1 through its C-terminal region to protect UCH-L1 from thermal or oxidative inactivation. We found that chaperoning via interaction through C-terminal region (specific-client chaperoning) is more efficient than that involving oligomeric structural change (general-client chaperoning). Prx II binds either thermally or oxidatively unfolding early intermediates of specific clients and thereby shifted the equilibrium towards their native state. We conclude that this chaperoning mechanism provides a very effective and selective chaperoning activity.
Asunto(s)
Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Línea Celular Tumoral , Células HeLa , Calor , Humanos , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Estructura Cuaternaria de Proteína , Ratas , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/químicaRESUMEN
Peroxiredoxins (Prxs) are highly conserved antioxidant enzymes and are implicated in multiple biological processes; however, their function in oocyte meiosis has not been studied. Here we show that inhibition of Prx I and II results in spindle defects, chromosome disorganization, and impaired polarization in mouse oocytes. Prx I was specifically localized at the spindle, whereas Prx II was enriched at the oocyte cortex and chromosomes. Inhibition of Prx activity with conoidin A disturbed assembly of the microtubule organizing center (MTOC) through Aurora A regulation, leading to defects in spindle formation. Moreover, conoidin A impaired actin filament and cortical granule (CG) distribution, disrupting actin cap and CG formation, respectively. Conoidin A also increased DNA damage without significantly increasing reactive oxygen species (ROS) levels, suggesting that the effects of conoidin A on meiotic maturation are not likely associated with ROS scavenging pathways. Therefore, our data suggest that Prxs are required for spindle assembly, chromosome organization, and polarization during meiotic maturation.
Asunto(s)
Polaridad Celular/efectos de los fármacos , Cromosomas de los Mamíferos/efectos de los fármacos , Oocitos/efectos de los fármacos , Peroxirredoxinas/farmacología , Huso Acromático/efectos de los fármacos , Animales , Células Cultivadas , Cromosomas de los Mamíferos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Meiosis/efectos de los fármacos , Ratones , Oocitos/metabolismo , Peroxirredoxinas/genética , Quinoxalinas/farmacología , Huso Acromático/metabolismo , Relación Estructura-ActividadRESUMEN
AIMS/HYPOTHESIS: Insulin resistance, a major contributor to the pathogenesis of type 2 diabetes, leads to increased hepatic glucose production (HGP) owing to an impaired ability of insulin to suppress hepatic gluconeogenesis. Nuclear receptor oestrogen-related receptor γ (ERRγ) is a major transcriptional regulator of hepatic gluconeogenesis. In this study, we investigated insulin-dependent post-translational modifications (PTMs) altering the transcriptional activity of ERRγ for the regulation of hepatic gluconeogenesis. METHODS: We examined insulin-dependent phosphorylation and subcellular localisation of ERRγ in cultured cells and in the liver of C57/BL6, leptin receptor-deficient (db/db), liver-specific insulin receptor knockout (LIRKO) and protein kinase B (PKB) ß-deficient (Pkbß (-/-)) mice. To demonstrate the role of ERRγ in the inhibitory action of insulin on hepatic gluconeogenesis, we carried out an insulin tolerance test in C57/BL6 mice expressing wild-type or phosphorylation-deficient mutant ERRγ. RESULTS: We demonstrated that insulin suppressed the transcriptional activity of ERRγ by promoting PKB/Akt-mediated phosphorylation of ERRγ at S179 and by eliciting translocation of ERRγ from the nucleus to the cytoplasm through interaction with 14-3-3, impairing its ability to promote hepatic gluconeogenesis. In addition, db/db, LIRKO and Pkbß (-/-) mice displayed enhanced ERRγ transcriptional activity due to a block in PKBß-mediated ERRγ phosphorylation during refeeding. Finally, the phosphorylation-deficient mutant ERRγ S179A was resistant to the inhibitory action of insulin on HGP. CONCLUSIONS/INTERPRETATION: These results suggest that ERRγ is a major contributor to insulin action in maintaining hepatic glucose homeostasis.
Asunto(s)
Gluconeogénesis/efectos de los fármacos , Insulina/farmacología , Hígado/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores de Estrógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Gluconeogénesis/fisiología , Hígado/metabolismo , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Receptor de Insulina/genética , Receptor de Insulina/metabolismoRESUMEN
In this study, we report the simultaneous refolding and reconstitution of the recombinant Bax inhibitor-1 (BI-1) from inclusion bodies expressed in Escherichia coli. A functional assay showed that the resulting proteoliposomes responded to acidic conditions and triggered the release of entrapped Ca(2+) from liposomes. The secondary structure of the reconstituted BI-1 was also determined using circular dichroism, which revealed an increase of alpha-helix content and a decrease of random structure when exposed to acidic solutions. These conformational changes may be responsible for the proton ion-induced Ca(2+) release of BI-1.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Cuerpos de Inclusión/química , Proteínas de la Membrana/metabolismo , Proteolípidos/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Calcio/metabolismo , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ingeniería de Proteínas , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We suggest that the cytochrome P450 2E1 (CYP2E1)-induced formation of reactive oxygen species (ROS) can be regulated by anionic phospholipids and the presence of the N-terminal region of the enzyme. When the content of cardiolipin (CL) in membranes at the expense of phosphatidylcholine matrix was increased, the ROS produced by recombinant human CYP2E1 was decreased as a function of CL concentration. On the contrary, the N-terminally truncated CYP2E1 had a decreased effect on the lipid-induced reduction of ROS formation. These results suggest that specific phospholipids can regulate the function of CYP2E1 by interaction with the enzyme including the N-terminal region(s).
Asunto(s)
Cardiolipinas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Aniones/metabolismo , Citocromo P-450 CYP2E1/química , Citocromo P-450 CYP2E1/genética , Humanos , NADP/metabolismo , Oxidación-Reducción , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Interaction of human cytochrome P450 1A2 (CYP1A2) and 2E1 (CYP2E1) with phospholipid, lysophosphatidylserine (LysoPS) in the context of a PC matrix specifically stimulated the PLD activity of both enzymes in a LysoPS concentration-dependent manner. However, other anionic lysophospholipids as well as the neutral lysophospholipids, lysophosphatidylcholine and lysophosphatidylethanolamine, had no effect. LysoPS also accompanied conformational changes in both CYPs when assayed by circular dichroism. Although the PLD activity was decreased in the presence of components required for the monooxygenase (MMO) activity, including 100% PC membranes, NADPH-cytochrome P450 reductase and NADPH, as compared to the activity in the absence of the reducing system, LysoPS recovered the PLD activity in a concentration-dependent manner. Considering that LysoPS induced a decrease in the MMO activities of both CYPs, the results suggest that the functional roles of CYP1A2 and 2E1 can be switched by interaction with a specific anionic lysophospholipid in vivo.
Asunto(s)
Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Lisofosfolípidos/metabolismo , Fosfolipasa D/metabolismo , Animales , Citocromo P-450 CYP1A2/química , Inhibidores del Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2E1/química , Inhibidores del Citocromo P-450 CYP2E1 , Humanos , Lisofosfolípidos/química , Lisofosfolípidos/farmacología , Fosfolipasa D/química , Conformación Proteica , RatasRESUMEN
Radiotherapy is one of the major treatment modalities for lung cancer. Cell killing by ionizing radiation is mediated primarily through the reactive oxygen species (ROS) and ROS-driven oxidative stress. Prx1, a peroxiredoxin family member, was shown to be frequently elevated in lung cancer cells and tissues. Although the antioxidant function of Prx1 is expected to affect the radiotherapy response of lung cancer, the physiologic significance of its peroxidase activity in irradiated cells is unclear because the catalytic Cys52 is easily inactivated by ROS due to its overoxidation to sulfinic or sulfonic acid. In this study, we investigated the role of Prx1 in radiation sensitivity of human lung cancer cells, with special emphasis on the redox status of the catalytic Cys52. We found that overexpression of Prx1 enhances the clonogenic survival of irradiated cells and suppresses ionizing radiation-induced c-Jun NH2-terminal kinase (JNK) activation and apoptosis. The peroxidase activity of Prx1, however, is not essential for inhibiting JNK activation. The latter effect is mediated through its association with the glutathione S-transferase pi (GSTpi)-JNK complex, thereby preventing JNK release from the complex. Reduced JNK activation is observed when the peroxidase activity of Prx1 is compromised by Cys52 overoxidation or in the presence of the Cys52 to Ser52 mutant (Prx1C52S) lacking peroxidase activity. We show that both Prx1 and Prx1C52S interact with the GSTpi-JNK complex and suppress the release of JNK from the complex. Our study provides new insight into the antiapoptotic function of Prx1 in modulating radiosensitivity and provides the impetus to monitor the influence of Prx1 levels in the management of lung cancer.
Asunto(s)
Gutatión-S-Transferasa pi/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/radioterapia , Peroxirredoxinas/metabolismo , Antioxidantes/metabolismo , Apoptosis/fisiología , Sitios de Unión , Cisteína/metabolismo , Activación Enzimática/efectos de la radiación , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Neoplasias Pulmonares/metabolismo , Oxidación-Reducción , Peroxirredoxinas/biosíntesis , Tolerancia a Radiación/fisiología , Transducción de Señal/efectos de la radiaciónRESUMEN
The lateral segregation of anionic phospholipids phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS) was detected after addition of cytochrome P450 2B1 (CYP2B1). The tendency of lipid clustering was highly dependent on the type of anionic phospholipids examined. PA was the most highly clustered while PI and PS clustered to a lesser degree. Moreover, liposomes containing anionic phospholipids form anionic phospholipid-rich microdomains in the presence of CYP2B1. Anionic phospholipids (mostly notably PA) also increased the ability of CYP2B1 to bind to lipid monolayers. In addition to the ability of CYP2B1 to modulate the physical properties of the membrane, the membrane itself can have reciprocal effects on the activity and conformation of CYP2B1. The catalytic activity of CYP2B1 increased as a function of anionic phospholipid concentration and in the presence of 10 mol% PA, the activity increased by 85%. These results suggest a bi-directional coupling between the CYP2B1 and anionic phospholipids.
Asunto(s)
Citocromo P-450 CYP2B1/química , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Proteínas de la Membrana/química , Fosfolípidos/química , Aniones , Sitios de Unión , Citocromo P-450 CYP2B1/ultraestructura , Unión ProteicaRESUMEN
The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.
Asunto(s)
Proteínas del Choque Térmico HSP40/farmacología , Chaperonas Moleculares , Tiorredoxinas/metabolismo , Zinc/metabolismo , Cisteína/química , Cisteína/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP40/genética , Humanos , Peróxido de Hidrógeno/farmacología , Luciferasas/metabolismo , Oxidantes/farmacología , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos Híbridos , Dedos de ZincRESUMEN
Thioredoxin (Trx) is a cellular redox enzyme that plays multiple roles in regulating cell growth and apoptosis. Jun activation domain-binding protein 1 (Jab1) was originally identified as a coactivator of activator protein 1 (AP-1) transcription and was also shown to promote degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Recently, Jab1 expression was associated with the progression and poor prognosis of pituitary, epithelial ovarian, and breast cancers, suggesting that it plays a role in oncogenesis. Here, we report that Trx specifically interacts with and modulates the function of Jab1. Fluorescence resonance energy transfer and co-immunoprecipitation studies revealed that Trx and Jab1 colocalize and directly interact with each other. Further, Trx negatively regulates two important Jab1-controlled signaling pathways, activation of AP-1 transcription and degradation of p27Kip1, probably through a direct interaction between Trx and C-terminal of Jab1. The negative effect of Trx on AP-1 activity is Jab1-dependent, as it disappears when Jab1 levels are suppressed by an antisense approach. In addition, Trx competes with p27Kip1 for Jab1 binding. Taken together, our results suggest that Trx may regulate cell cycle and growth through a novel modulation of Jab1-mediated proliferation signals, further indicating that Trx may have the ability to control tumor progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Tiorredoxinas/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Sitios de Unión , Complejo del Señalosoma COP9 , Línea Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Cisteína/química , ADN Complementario/metabolismo , Progresión de la Enfermedad , Disulfuros , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica , Genes Reporteros , Glutatión Transferasa/metabolismo , Células HeLa , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Mutación , Neoplasias/metabolismo , Oligonucleótidos Antisentido/química , Oxidación-Reducción , Péptido Hidrolasas , Pronóstico , Unión Proteica , Proteínas Recombinantes/química , Transducción de Señal , Factores de Tiempo , Activación Transcripcional , Técnicas del Sistema de Dos HíbridosRESUMEN
The observation that purified yeast glutamine synthetase is rapidly inactivated in a thiol-containing buffer yet retains activity in crude extracts containing the same thiol led to our discovery of an enzyme that protects against oxidation in a thiol-containing system. This novel antioxidant enzyme was shown to reduce hydroperoxides and, more recently, peroxynitrite with the use of electrons provided by a physiological thiol like thioredoxin. It defined a family of proteins, present in organisms from all kingdoms, that was named peroxiredoxin (Prx). All Prx enzymes contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of Prx (Prx I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of Cys residues that participate in catalysis. The relative abundance of Prx enzymes in mammalian cells appears to protect cellular components by removing the low levels of peroxides produced as a result of normal cellular metabolism. During catalysis, the active site cysteine is occasionally overoxidized to cysteine sulfinic acid. Contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells, studies on the fate of the overoxidized Prx species revealed a mechanism by which the catalytically active thiol form is recovered. This sulfinic reduction is a slow, ATP-dependent process that is specific to 2-Cys Prx isoforms. This reversible overoxidation may represent an adaptation unique to eukaryotic cells that accommodates the intracellular messenger function of H(2)O(2), but experimental validation of such speculation is yet to come.
Asunto(s)
Antioxidantes/fisiología , Peroxidasas/fisiología , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Proteína Quinasa CDC2/metabolismo , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Humanos , Isoenzimas/metabolismo , Oxidorreductasas/metabolismo , Peroxidasas/antagonistas & inhibidores , Peroxiredoxina VI , Peroxirredoxinas , Fosforilación , Compuestos de Sulfhidrilo/metabolismo , Tiorredoxinas/metabolismoRESUMEN
In eukaryotic cells, peroxiredoxins are both antioxidants and regulators of H(2)O(2)-mediated signaling. We previously found that peroxiredoxin-I (Prx-I) was overexpressed in non-small cell lung cancer (NSCLC) tissue. Since overexpressed protein can induce a humoral immune response, we examined whether serum from NSCLC patients exhibited immunoreactivity against Prx-I using Western blotting. We found that 25 (47%) of 53 NSCLC patients tested had autoantibodies against Prx-I in their sera, whereas such activity was detected in 4 (8%) sera from 50 healthy subjects. Prx-I itself was detected in the sera from 18 (34%) of 53 NSCLC patients but in only 1 (2%) serum from 50 controls. Moreover, 17% of NSCLC sera were positive to both Prx-I antibody and antigen but none in control sera. The data indicate both Prx-I autoantibody and circulating antigen are potential biomarkers for use in serological diagnosis of NSCLC. Interestingly enough, we found that Prx-I was secreted by lung adenocarcinoma cells (A549) but not by non-cancer lung cells (BEAS 2B) or breast cancer cells (MCF7). This cell culture study suggests the possibility of Prx-I secretion from NSCLC tumor tissues.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/inmunología , Proteínas de Choque Térmico/fisiología , Neoplasias Pulmonares/inmunología , Peroxidasas/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Electroforesis en Gel de Poliacrilamida , Femenino , Proteínas de Choque Térmico/inmunología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Peroxidasas/inmunología , Peroxirredoxinas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products.
Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa , Rajidae/genética , Animales , Cartilla de ADN , Sondas de ADN , ADN Mitocondrial/genética , Complejo IV de Transporte de Electrones/genética , Análisis de Secuencia de ADN , Especificidad de la Especie , Polimerasa TaqRESUMEN
SIGNIFICANCE: Reversible protein glutathionylation plays an important role in cellular regulation, signaling transduction, and antioxidant defense. This redox-sensitive mechanism is involved in regulating the functions of peroxiredoxins (Prxs), a family of ubiquitously expressed thiol-specific peroxidase enzymes. Glutathionylation of certain Prxs at their active-site cysteines not only provides reducing equivalents to support their peroxidase activity but also protects Prxs from irreversible hyperoxidation. Typical 2-Cys Prx also functions as a molecular chaperone when it exists as a decamer and/or higher molecular weight complexes. The hyperoxidized sulfinic derivative of 2-Cys Prx is reactivated by sulfiredoxin (Srx). In this review, the roles of glutathionylation in the regulation of Prxs are discussed with respect to their molecular structure and functions as antioxidants, molecular chaperones, and signal modulators. RECENT ADVANCES: Recent findings reveal that glutathionylation regulates the quaternary structure of Prx. Glutathionylation of Prx I at Cys(83) converts the decameric Prx to its dimers with the loss of chaperone activity. The findings that dimer/oligomer structure specific Prx I binding proteins, e.g., phosphatase and tensin homolog (PTEN) and mammalian Ste20-like kinase-1 (MST1), regulate cell cycle and apoptosis, respectively, suggest a possible link between glutathionylation and those signaling pathways. CRITICAL ISSUES: Knowing how glutathionylation affects the interaction between Prx I and its nearly 20 known interacting proteins, e.g., PTEN and MST1 kinase, would reveal new insights on the physiological functions of Prx. FUTURE DIRECTIONS: In vitro studies reveal that Prx oligomerization is linked to its functional changes. However, in vivo dynamics, including the effect by glutathionylation, and its physiological significance remain to be investigated.
Asunto(s)
Antioxidantes/metabolismo , Glutatión/metabolismo , Chaperonas Moleculares/metabolismo , Peroxirredoxinas/metabolismo , Transducción de Señal , Animales , HumanosRESUMEN
Reactive oxygen species are involved in ovulation. The aim of this study was to examine gonadotropin regulation of antioxidant enzyme sulfiredoxin (Srx) and peroxiredoxin 2 (PRDX2) expressions and modification during the ovulatory process in rats. Administration of antioxidants in vivo reduced ovulation rate and cumulus expansion. LH treatment increased H(2)O(2) levels within 15 min, which, in turn, induced Srx gene expression in cultured preovulatory follicles. Treatment of preovulatory follicles with catalase suppressed the stimulatory effect of LH on Akt phosphorylation. LH- or H(2)O(2)-stimulated Srx mRNA levels were suppressed by inhibitors of antioxidant agents and MAPK kinase. An in vivo injection of equine chorionic gonadotropin-human chorionic gonadotropin (hCG) stimulated Srx mRNA within 1 h in granulosa but not thecal cells of preovulatory follicles. Srx protein levels were stimulated from 3 h post-hCG injection. Immunofluorescence analysis revealed that oocytes expressed the Srx protein. Furthermore, hCG treatment increased Srx expression in mural granulosa, theca and cumulus cells, but the Srx protein was not detected in corpora lutea. Gene expression of PRDX2, identified as an Srx-dependent modified enzyme, was stimulated by gonadotropins. In situ hybridization analysis demonstrated that PRDX2 mRNA was detected in oocytes and theca cells as well as granulosa cells of some antral and preovulatory follicles. High levels of PRDX2 mRNA were detected in corpora lutea. Total levels of PRDX2 protein were not changed by gonadotropins. However, levels of hyperoxidized PRDX2 increased within 2-3 h after the hCG injection. Taken together, gonadotropin stimulation of Srx expression and PRDX2 modification in the ovary suggest the existence of an antioxidant system to maintain H(2)O(2) production and elimination during the periovulatory period.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Hormona Luteinizante/farmacología , Ovario/metabolismo , Ovulación/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Peroxirredoxinas/metabolismo , Animales , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ovario/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Peroxirredoxinas/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismoRESUMEN
An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site.
RESUMEN
To investigate the differences in the functional roles of peroxiredoxins (Prxs) and glutathione peroxidase (GPx) of Schizosaccharomyces pombe, we examined the peroxidase and molecular chaperone properties of the recombinant proteins. TPx (thioredoxin peroxidase) exhibited a capacity for peroxide reduction with the thioredoxin system. GPx also showed thioreoxin-dependent peroxidase activity rather than GPx activity. The peroxidase activity of BCP (bacterioferritin comigratory protein) was similar to that of TPx. However, peroxidase activity was not observed for PMP20 (peroxisomal membrane protein 20). TPx, PMP20, and GPx inhibited thermal aggregation of citrate synthase at 43(o)C, but BCP failed to inhibit the aggregation. The chaperone activities of PMP20 and GPx were weaker than that of TPx. The peroxidase and chaperone properties of TPx, BCP, and GPx of the fission yeast are similar to those of Saccharomyces cerevisiae. The fission yeast PMP20 without thioredoxin-dependent peroxidase activity may act as a molecular chaperone.
Asunto(s)
Glutatión Peroxidasa/metabolismo , Peroxirredoxinas/metabolismo , Schizosaccharomyces/enzimología , Citrato (si)-Sintasa/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/fisiología , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiología , Peroxirredoxinas/genética , Peroxirredoxinas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
We investigated the functional activity of recombinant Bax inhibitor-1 reconstituted into liposomes. When proteoliposomes were suspended in acidic solutions, encapsulated Ca(2+) was released from the membranes, as previously suggested [Kim HR, Lee GH, Ha KC, Ahn T, Moon JY, Lee BJ, Cho SG, Kim S, Seo YR, Shin YJ et al. (2008) J Biol Chem283, 15946-15955]. Concomitantly, proton ions were internalized when assayed using the time-dependent change in the fluorescence of the pH-sensitive dye oxonol V entrapped in the proteoliposomes. The influx of proton ions was confirmed by observing tritium accumulation in the membranes. However, the external acidity of the membranes per se did not induce proton ion influx without internalized Ca(2+). These results suggest that reconstituted Bax inhibitor-1 has a Ca(2+)/H(+) antiporter-like activity.
Asunto(s)
Antiportadores/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Transporte de Catión/metabolismo , Liposomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Calcio/metabolismo , Humanos , Indoles/metabolismo , Proteínas de la Membrana/genéticaRESUMEN
To screen chaperone proteins from Schizosaccharomyce pombe (S. pombe), we prepared recombinant citrate synthase of the fission yeast as a substrate of anti-aggregation assay. Purified recombinant citrate synthase showed citrate synthase activity and was suitable for the substrate of chaperone assay. Several heat stable proteins including aspartyl aminopeptidase (AAP) for candidates of chaperone were screened from the supernatant fraction of heat-treated crude extract of S. pombe. The purified AAP migrated as a single band of 47 kDa on SDS-polyacrylamide gel electrophoresis. The native size of AAP was estimated as 200 kDa by a HPLC gel permeation chromatography. This enzyme can remove the aspartyl residue at N-terminus of angiotensin I. In addition, AAP showed the heat stability and protected the aggregation of citrate synthase caused by thermal denaturation. This study showed that S. pombe AAP is a moonlight protein that has aspartyl aminopeptidase and chaperone activities.
Asunto(s)
Aminopeptidasas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/enzimología , Aminopeptidasas/química , Aminopeptidasas/aislamiento & purificación , Activación Enzimática , Estabilidad de Enzimas , Calor , Chaperonas Moleculares/química , Chaperonas Moleculares/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Schizosaccharomyces/metabolismo , Factores de TiempoRESUMEN
Steroidogenesis decreases with aging in the testis, whereas the levels of reactive oxygen species (ROS) increase. In addition, ROS have been reported to inhibit testicular steroidogenesis. Here, we investigated the effects of ROS on the transcriptional activity of Nur77, one of the major transcription factors that regulate the expression of steroidogenic enzyme genes. ROS signaling inhibited Nur77 transactivation, which was diminished by either treatment with c-Jun N-terminal kinase (JNK) inhibitor or the expression of a dominant negative form of JNK. This suggests the involvement of JNK signaling, which elevates the expression of c-Jun as well as its phosphorylation in Leydig cells. In transient transfection assays, c-Jun suppressed Nur77 transactivation in a dose-dependent manner. Further studies using c-Jun mutants revealed that the protein level of c-Jun, but not phosphorylation itself, was important for the suppression of Nur77 transactivation. Nur77 directly interacted with c-Jun in vivo, which blocked the DNA binding activity of Nur77. Together, these results suggest that ROS signaling-mediated c-Jun upregulation suppresses the expression of steroidogenic enzyme genes by inhibiting Nur77 transactivation, resulting in the reduction of testicular steroidogenesis. These findings may provide a mechanistic explanation for the age-related decline in testicular steroid hormone production.