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1.
ACS Chem Biol ; 19(8): 1786-1793, 2024 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-39037001

RESUMO

The oxidation of the cellular membrane through lipid peroxidation (LPO) is linked to aging and disease. Despite the physiological importance, the chemical mechanisms underlying LPO and oxidative reactions in membranes in general remain incompletely understood, and challenges exist in translating LPO inhibitor efficacies from in vitro to in vivo. The complexity of LPO, including multiple oxidation reactions in complex membrane environments and the difficulty in quantifying reaction kinetics, underlies these difficulties. In this work, we developed a robust and straightforward method for quantifying the oxidation rate kinetics of fluorescent molecules and determined the oxidation kinetics of widely fluorophores used as indicators of membrane LPO, diphenylhexatriene (DPH), BODIPY-C11, and Liperfluo. The measurement is initiated by lipoxygenase, which provides chemical specificity and enables a straightforward interpretation of oxidation kinetics. Our results reveal that the membrane composition significantly impacts the observed kinetics oxidation in DPH and BODIPY-C11 but not Liperfluo. Reaction mechanisms for their lipid peroxide-induced oxidation are proposed. This work provides a foundation for the quantitative analysis of LPO with fluorescence and extricating the complexity of oxidation reactions within membranes.


Assuntos
Compostos de Boro , Corantes Fluorescentes , Peroxidação de Lipídeos , Oxirredução , Cinética , Corantes Fluorescentes/química , Compostos de Boro/química , Membrana Celular/metabolismo , Difenilexatrieno/química , Difenilexatrieno/análogos & derivados , Lipídeos de Membrana/metabolismo , Lipídeos de Membrana/química , Humanos , Lipoxigenase/metabolismo
2.
Langmuir ; 40(14): 7456-7462, 2024 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-38546877

RESUMO

The primary constituents of honeybee venom, melittin and phospholipase A2 (PLA2), display toxin synergism in which the PLA2 activity is significantly enhanced by the presence of melittin. It has been shown previously that this is accomplished by the disruption in lipid packing, which allows PLA2 to become processive on the membrane surface. In this work, we show that melittin is capable of driving miscibility phase transition in giant unilamellar vesicles (GUVs) and that it raises the miscibility transition temperature (Tmisc) in a concentration-dependent manner. The induced phase separation enhances the processivity of PLA2, particularly at its boundaries, where a substantial difference in domain thickness creates a membrane discontinuity. The catalytic action of PLA2, in response, induces changes in the membrane, rendering it more conducive to melittin binding. This, in turn, facilitates further lipid phase separation and eventual vesicle lysis. Overall, our results show that melittin has powerful membrane-altering capabilities that activate PLA2 in various membrane contexts. More broadly, they exemplify how this biochemical system actively modulates and capitalizes on the spatial distribution of membrane lipids to efficiently achieve its objectives.


Assuntos
Venenos de Abelha , Meliteno , Meliteno/farmacologia , Lipossomas Unilamelares , Fosfolipases A2 , Lipídeos de Membrana
3.
bioRxiv ; 2024 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-38187697

RESUMO

Desmosterol and cholesterol are essential lipid components of the sperm plasma membrane. Cholesterol efflux is required for capacitation, a process through which sperm acquire fertilizing ability. In this study, using a transgenic mouse model overexpressing 24-dehydrocholesterol reductase (DHCR24), an enzyme in the sterol biosynthesis pathway responsible for the conversion of desmosterol to cholesterol, we show that disruption of sterol homeostasis during spermatogenesis led to defective sperm morphology characterized by incomplete mitochondrial packing in the midpiece, reduced sperm count and motility, and a decline in male fertility with increasing paternal age, without changes in body fat composition. Sperm depleted of desmosterol exhibit inefficiency in the acrosome reaction, metabolic dysfunction, and an inability to fertilize the egg. These findings provide molecular insights into sterol homeostasis for sperm capacitation and its impact on male fertility.

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