RESUMO
The whiteleg shrimp species Litopenaeus vannamei is exposed to cyclic changes of the dissolved oxygen concentration of seawater and must neutralize the adverse effects of hypoxia by using ATP as energy source. In crustaceans, the mitochondrial FOF1-ATP synthase is pivotal to the homeostasis of ATP and function prevalently as a FOF1-ATPase. Hitherto, it is unknown whether these marine invertebrates are equipped with molecules able to control the FOF1-ATPase inhibiting the ATP consumption. In this study, we report two variants of the mitochondrial FOF1-ATPase Inhibitory Factor 1 (IF1) ubiquitously expressed across tissues of the Litopenaeus vannamei transcriptome: the IF1_Lv1 and the IF1_Lv2. The IF1_Lv1, with a full-length sequence of 550 bp, encodes a 104 aa long protein and its mRNA amounts are significantly affected by hypoxia and re-oxygenation. The IF1_Lv2, with a sequence of 654 bp, encodes instead for a protein of 85 aa. Both proteins share a 69 % homology and contain a conserved minimal inhibitory sequence (IATP domain) along with a G-rich region on their N-terminus typical of the invertebrate. In light of this characterization IF1 is here discussed as an adaptive mechanism evolved by this marine species to inhibit the FOF1-ATPase activity and avoid ATP dissipation to thrive in spite of the changes in oxygen tension.
Assuntos
Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Penaeidae/genética , Penaeidae/metabolismo , Proteínas/genética , Proteínas/metabolismo , Animais , Sequência de Bases , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteína Inibidora de ATPaseRESUMO
BACKGROUND: Cancer development involves an "injury" to the respiratory machinery (Warburg effect) due to decreased or impaired mitochondrial function. This circumstance results in a down regulation of some of the ATPase subunits of the malignant tissue. The objective of this work was to assess and compare the relative expression of mRNA of mitochondrial ATPase subunits between samples of thyroid cancer and benign nodules. MATERIAL AND METHODS: Samples from 31 patients who had an operation for PTC at the General Hospital of Mexico were snap-frozen and stored at -70°C. Thirty-five patients who had an operation for benign tumors were also included in the study. mRNA expression levels of alpha, beta, gamma, and epsilon subunits of F1 and "c12" of subunit Fo were determined by real-time RT-PCR (by duplicate), in order to determine if abnormal expression of these genes could partially explain the Warburg effect in papillary thyroid cancer (PTC). RESULTS: ATP5E transcript alteration (down-expression) was highly associated to PTC diagnosis OR=11.76 (95% confidence interval, 1.245-237.98; p=0.04). CONCLUSIONS: Relative down-expression of ATP5E transcript was highly associated with PTC diagnosis. This transcript alteration may be used as a tumoral marker in papillary thyroid cancer.
Assuntos
Carcinoma/enzimologia , Carcinoma/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas/genética , RNA Mensageiro/genética , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Carcinoma/patologia , Carcinoma Papilar , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , ATPases Mitocondriais Próton-Translocadoras/biossíntese , Estudos Prospectivos , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia , Adulto Jovem , Proteína Inibidora de ATPaseRESUMO
The ATP synthase of bovine heart mitochondria possesses a regulatory subunit called the endogenous inhibitory protein (IF(1)). This subunit regulates the catalytic activity of the F(1) sector in the mitochondrial inner membrane. When DeltamuH(+) falls, IF(1) binds to the enzyme and inhibits ATP hydrolysis. On the other hand, the establishment of a DeltamuH(+) induces the release of the inhibitory action of IF(1), allowing ATP synthesis to proceed. IF(1) is also involved in the dimerization of soluble F(1). Dynamic domain analysis and normal mode analysis of the reported crystallographic structure of IF(1) revealed that it has an effective hinge formed by residues 46-52. Molecular dynamics data of a 27 residue fragment confirmed the existence of the hinge. The hinge may act as a regulatory region that links the inhibitory and anchoring domains of IF(1). The residues assigned to the hinge are conserved between mammals, but not in other species, such as yeasts. Likewise, unlike the heart inhibitor, the yeast protein does not have the residues that allow it to form stable dimers through coiled-coil interactions. Collectively, the data suggest that the hinge and the dimerization domain of the inhibitor protein from bovine heart are related to its ability to form stable dimers and to interact with other subunits of the ATP synthase.
Assuntos
Proteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Bovinos , Bases de Dados de Proteínas , Dimerização , Humanos , Modelos Teóricos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Alinhamento de Sequência , Proteína Inibidora de ATPaseRESUMO
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.
Assuntos
Trifosfato de Adenosina/química , Guanidina/química , Mitocôndrias Cardíacas/enzimologia , Proteínas/química , ATPases Translocadoras de Prótons/química , Partículas Submitocôndricas/enzimologia , Animais , Aurovertinas/química , Bovinos , Ativação Enzimática , Hidrólise , Desnaturação Proteica , Desacopladores/química , Proteína Inibidora de ATPaseRESUMO
The ATPase inhibitor protein (IP) of mitochondria was detected in the plasma membrane of living endothelial cells by flow cytometry, competition assays, and confocal microscopy of cells exposed to IP antibodies. The plasma membranes of endothelial cells also possess beta-subunits of the mitochondrial ATPase. Plasma membranes have the capacity to bind exogenous IP. TNF-alpha decreases the level of beta-subunits and increases the amount of IP, indicating that the ratio of IP to beta-subunit exhibits significant variations. Therefore, it is probable that the function of IP in the plasma membrane of endothelial cells is not limited to regulation of catalysis.
Assuntos
Membrana Celular/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , Proteínas/metabolismo , Anticorpos/imunologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Humanos , Microscopia Confocal , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Proteínas/análise , Proteínas/imunologia , Solubilidade , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Proteína Inibidora de ATPaseRESUMO
The F1-inhibitor protein complex (F1-IP) was purified from heart submitochondrial particles. Size exclusion chromatography of the endogenous complex showed that it contains dimers (D) and monomers (M) of F1-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 F1, the M species accumulates through a process that is faster than the release of IP from F1. These findings indicate that the activation of F1-IP involves the transformation of D into M, which subsequently loses IP. The spectroscopic characteristics of D, M, and free F1 show that the binding of IP and dimerization modifies the fluorescence intensity of tyrosine residues and that of the single tryptophan of F1 which is far from the IP binding site.
Assuntos
Mitocôndrias Cardíacas/enzimologia , Proteínas/química , Proteínas/metabolismo , Animais , Bovinos , Cromatografia em Gel , Dimerização , Ativação Enzimática/fisiologia , Fluorescência , Conformação Proteica , Proteína Inibidora de ATPaseRESUMO
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.
Assuntos
Mitocôndrias Cardíacas/enzimologia , Proteínas/química , Proteínas/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Partículas Submitocôndricas/enzimologia , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Cinética , Pressão , Proteína Inibidora de ATPaseRESUMO
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.
Assuntos
Mitocôndrias Cardíacas/enzimologia , Proteínas/metabolismo , ATPases Translocadoras de Prótons/química , Partículas Submitocôndricas/enzimologia , Trifosfato de Adenosina/farmacologia , Animais , Bovinos , Ativação Enzimática , Estabilidade Enzimática , Temperatura Alta , Cinética , Proteínas/isolamento & purificação , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , Termodinâmica , Proteína Inibidora de ATPaseRESUMO
The binding of ATP radiolabeled in the adenine ring or in the gamma- or alpha-phosphate to F1-ATPase in complex with the endogenous inhibitor protein was measured in bovine heart submitochondrial particles by filtration in Sephadex centrifuge columns or by Millipore filtration techniques. These particles had 0.44 +/- 0.05 nmol of F1 mg-1 as determined by the method of Ferguson et al. [(1976) Biochem. J. 153, 347]. By incubation of the particles with 50 microM ATP, and low magnesium concentrations (less than 0.1 microM MgATP), it was possible to observe that 3.5 mol of [gamma-32P]ATP was tightly bound per mole of F1 before the completion of one catalytic cycle. With [gamma-32P]ITP, only one tight binding site was detected. Half-maximal binding of adenine nucleotides took place with about 10 microM. All the bound radioactive nucleotides were released from the enzyme after a chase with cold ATP or ADP; 1.5 sites exchanged with a rate constant of 2.8 s-1 and 2 with a rate constant of 0.45 s-1. Only one of the tightly bound adenine nucleotides was released by 1 mM ITP; the rate constant was 3.2 s-1. It was also observed that two of the bound [gamma-32P]ATP were slowly hydrolyzed after removal of medium ATP; when the same experiment was repeated with [alpha-32P]ATP, all the label remained bound to F1, suggesting that ADP remained bound after completion of ATP hydrolysis. Particles in which the natural ATPase inhibitor protein had been released bound tightly only one adenine nucleotide per enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Nucleotídeos de Adenina/metabolismo , Mitocôndrias Cardíacas/metabolismo , Proteínas/metabolismo , Partículas Submitocôndricas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Inosina Trifosfato/metabolismo , Cinética , Mitocôndrias Cardíacas/ultraestrutura , Proteína Inibidora de ATPaseRESUMO
An inhibitor of Crithidia fasciculata and Trypanosoma cruzi H+ -ATP synthase (ATPase) was isolated from these organims mitochondrial particles, either by (a) ammonium sulfate-cholate extraction followed by heat treatment and ethanol precipitation, or (b) gel-filtration on Sephadex G-50, followed by a similar purification procedure. Inactivation by trypsin supported the inhibitor peptide structure. Removal of the peptide inhibitor increased about three-fold the specific activity of the protozoan ATPases. The isolated peptides and a highly purified bovine heart ATPase inhibitor inhibited C. fasciculata ATPase as a function of the peptide concentration.