RESUMO
Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes' lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization-high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography-ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.
Assuntos
GTP Fosfo-Hidrolases , Lipidômica , Mitocôndrias , Fosfolipídeos , Animais , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/deficiência , Camundongos , Mitocôndrias/metabolismo , Fosfolipídeos/metabolismo , Camundongos Knockout , Fibroblastos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genéticaRESUMO
É£-aminobutyric acid (GABA) is a fourcarbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown.
RESUMO
The occurrence of methyl carbamates of phosphatidylethanolamines and phosphatidylserines in the lipid extract of mitochondria obtained from mouse embryonic fibroblasts was ascertained by hydrophilic interaction liquid chromatography with electrospray ionization single and multi-stage mass spectrometry, performed using sinergically a high resolution (quadrupole-Orbitrap) and a low resolution (linear ion trap) spectrometer. Two possible routes to the synthesis of methyl carbamates of phospholipids were postulated and evaluated: (i) a chemical transformation involving phosgene, occurring as a photooxidation by-product in the chloroform used for lipid extraction, and methanol, also used for the latter; (ii) an enzymatic methoxycarbonylation reaction due to an accidental bacterial contamination, that was unveiled subsequently on the murine mitochondrial sample. A specific lipid extraction performed on a couple of standard phosphatidyl-ethanolamines/-serines, based on purposely photo-oxidized chloroform and deuterated methanol, indicated route (i) as negligible in the specific case, thus highlighting the enzymatic route related to bacterial contamination as the most likely source of methyl carbamates. The unambiguous recognition of the latter might represent the starting point toward a better understanding of their generation in biological systems and a minimization of their occurrence when an artefactual formation is ascertained.