Biology subjects

Prado, F. M.

Publications and source records attributed to Prado, F. M..

3 recordsLinked to original sources

bioRxiv2026

PARG activity is required for cell death by parthanatos

Cell death by a non-apoptotic pathway termed parthanatos is induced by hyperactivation of the DNA damage sensor PARP1, which uses NAD+ as a substrate to catalyse the poly-ADP-ribosylation (PARylation) of proteins. Parthanatos has been implicated in several pathological conditions such as ischaemia-reperfusion injury and neurodegenerative processes, including Alzheime[r]s and Parkinso[n]s diseases, as well as rare genetic disorders characterized by PARP1 hyperactivation. However, the precise sequence of molecular events by which excessive PARP1 activity causes cell death is currently unclear. Here we show that, in addition to PARP1-dependent PARylation, the execution of parthanatos also requires the hydrolysis of poly-ADP-ribose (PAR) chains by the glycohydrolase PARG. While complete inhibition of PARG activity prevents parthanatos, low levels of residual PARG activity are sufficient to support cell death by this pathway. Due to a phenotypic discrepancy between CRISPR/Cas9-generated PARG KO cells and PARG inhibitor-treated cells, we uncovered a new PARG splice variant predicted to encode an isoform of 53 kDa, termed PARG53, and provide evidence for the incorrect annotation of the previously reported isoforms PARG55 and PARG60. PARP1 hyperactivation during parthanatos leads to rapid and profound depletion of NAD+ and ATP pools, but full PARG inhibition only prevents the depletion of ATP, indicating that the depletion of these core metabolites can be uncoupled, and that ATP depletion is more closely correlated with cell death. This work sheds light on the initial steps of parthanatos induction, demonstrating that PAR formation and PAR hydrolysis are both required for this type of cell death.

biochemistry↗

bioRxiv2025

Simultaneous detection and quantification of adenine nucleotides in mammalian cultured cells by HPLC

Adenine nucleotides, including ATP, ADP, ADP-ribose, AMP, NAD+ and NADH, play central roles in cellular homeostasis and involved in multiple metabolic and signaling pathways. Owing to their broad functional relevance in cell biology, the accurate quantification of these metabolites is essential for diverse research areas such as bioenergetics, cell signaling and cancer biology. Several analytical methods have been described for the measurement of adenine nucleotides, ranging from enzymatic assays to mass spectrometry-based approaches. In this study, we developed a reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV-Vis detection that enables the simultaneous quantification of ATP, ADP, ADP-ribose, AMP, NAD+ and NADH. This method is simple, sensitive within the physiological concentration range of all analytes, and capable of detecting biologically relevant changes in ATP, AMP and NAD+ levels induced by pharmacological treatments. Therefore, it presents an accessible and reliable alternative for quantification of these nucleotides in biological samples.

biochemistry↗

bioRxiv2024

Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species

Plasmalogens are glycerophospholipids with a vinyl-ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species (ROS), our study reveals the generation reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) we demonstrate that singlet molecular oxygen [O2 (1{Delta}g)] reacts with the vinyl ether bond, producing hydroperoxyl acetal as major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates lead to the formation of excited triplet carbonyls, O2 (1{Delta}g), and electrophilic phospholipid and fatty aldehyde species, as secondary reactive products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoantracene as a triplet enhancer. Moreover, O2 (1{Delta}g) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phopspholipid and fatty aldehydes by LC-MS analysis using two probes that specifically reacts with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen oxidized products. Significance StatementPlasmalogens, the most abundant subclass of ether lipids in mammalian cells, have traditionally been regarded as antioxidants. However, our study reveals a new perspective, shedding light on the generation of chemiexcited and reactive lipid species during plasmalogen photooxidation. We provide direct evidence revealing the production of excited triplet carbonyls and singlet molecular oxygen as secondary reactive products originating from dioxetane and hydroperoxyacetal intermediates. Importantly, we also demonstrate the generation of electrophilic alpha-beta unsaturated phospholipids and fatty aldehydes through plasmalogen oxidation. These findings highlight the production of excited states and reactive lipid species resulting from plasmalogen oxidation, which can potentially induce oxidative modifications in biological systems.

biochemistry↗