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1.
J Bioenerg Biomembr ; 50(1): 59-69, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29313294

RESUMO

The plasma membrane Ca2+-ATPase (PMCA) removes Ca2+ from the cytosol into the extracellular space. Its catalytic activity can be stimulated by calmodulin (CaM) or by limited proteolysis. We evaluated the effect of chlorpromazine (CPZ) and dimethyl sulfoxide (DMSO) over the hydrolytic activity of PMCA. Activity was monitored in three different forms: native, CaM-activated and proteolyzed by trypsin. CPZ appears to inhibit PMCA without directly interfering with the C-terminal site, since it is affected by CaM and proteolysis. Although the treatment of PMCA with trypsin and CaM produces an activation, it also produces an enzymatic form that is more sensitive to inhibition by CPZ. The same case was observed in the DMSO inhibition experiments. In the absence of CPZ, DMSO produces a progressive loss of activity, but in the presence of CPZ the profile of activity against DMSO changes and produces a recovery of activity, indicating a possible partition of CPZ by the solvent. Increasing Ca2+ concentrations indicated that CPZ interacts with PMCA rather than with CaM. This observation is supported by docking analysis that suggests that the CPZ-PMCA interaction is non-competitive. We propose that CPZ interacts with the state of lower affinity for Ca2 +.


Assuntos
Clorpromazina/farmacologia , Dimetil Sulfóxido/farmacologia , Membrana Eritrocítica/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Biocatálise/efeitos dos fármacos , Calmodulina/farmacologia , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Humanos , Simulação de Acoplamento Molecular , Tripsina/farmacologia
2.
Int J Mol Sci ; 17(8)2016 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-27556455

RESUMO

Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.


Assuntos
Esterases/química , Esterases/metabolismo , Doença de Parkinson/enzimologia , Esterases/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Simulação de Acoplamento Molecular , Mutação , Nitrofenóis/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/genética , Proteína Desglicase DJ-1/química , Proteína Desglicase DJ-1/genética , Proteína Desglicase DJ-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo
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