Biology subjects

Goni, F. M.

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

4 recordsLinked to original sources

bioRxiv2023

A progesterone derivative linked to a stable phospholipid activates breast cancer cell response without leaving the cell membrane

In hormone-responsive breast cancer cells, progesterone (P4) has been shown to act via its nuclear receptor (PR), a ligand-activated transcription factor. A small fraction of PR is palmitoylated and anchored to the cell membrane (mbPR) forming a complex with estrogen receptor alpha (ER). Upon hormone exposure, either directly or via interaction with ER mbPR activates the SRC/RAS/ERK kinase pathway leading to phosphorylation of PR by ERK. Kinase activation is essential for P4 gene regulation, as the ERK and MSK1 kinases are recruited by the PR to its genomic binding sites and trigger chromatin remodeling. An interesting open question is whether activation of mbPR can result in gene regulation in the absence of ligand binding to intracellular PR. This matter has been investigated in the past using P4 attached to serum albumin, but the attachment is leaky and albumin can be endocytosed and degraded, liberating P4. Here we propose a more stringent approach to address this issue by ensuring attachment of P4 to the cell membrane via covalent binding to a stable phospholipid. This strategy identifies the actions of P4 independent of hormone binding to intracellular PR. We found that a membrane-attached progestin can activate mbPR, the ERK signalling pathway leading to PR phosphorylation, initial gene regulation and entry into the cell cycle, in the absence of detectable intracellular progestin.

genomics↗

bioRxiv2022

Phase behaviour of C18-N-acyl sphingolipids, the prevalent species in human brain

Lipidomic analysis of the N-acyl components of sphingolipids in different mammalian tissues had revealed that brain tissue differed from all the other samples in that SM contained mainly C18:0 and C24:1 N-acyl chains, and that the most abundant Cer species was C18:0. Only in the nervous system was C18:0 found in sizable proportions. The high levels of C18:0 and C16:0, respectively in brain and non-brain SM, were important because SM is by far the most abundant sphingolipid in the plasma membrane. In view of these observations, the present paper is devoted to a comparative study of the properties of C16:0 and C18:0 sphingolipids (SM and Cer) pure and in mixtures of increasing complexities, using differential scanning calorimetry, confocal microscopy of giant unilamellar vesicles, and correlative fluorescence microscopy and atomic force microscopy of supported lipid bilayers. Membrane rigidity was measured by force spectroscopy. It was found that in mixtures containing dioleoyl phosphatidylcholine, sphingomyelin and cholesterol, i.e. representing the lipids predominant in the outer monolayer of cell membranes, lateral inhomogeneities occurred, with the formation of rigid domains within a continuous fluid phase. Inclusion of saturated Cer in the system was always found to increase the rigidity of the segregated domains. C18:0-based sphingolipids exhibit hydrocarbon chain-length asymmetry, and some singularities observed with this N-acyl chain, e.g. complex calorimetric endotherms, could be attributed to this property. Moreover, C18:0-based sphingolipids, that are typical of the excitable cells, were less miscible with the fluid phase than their C16:0 counterparts. The results could be interpreted as suggesting that the predominance of C18:0 Cer in the nervous system would contribute to the tightness of its plasma membranes, thus facilitating maintenance of the ion gradients.

biophysics↗

bioRxiv2020

LC3 subfamily in cardiolipin-mediated mitophagy: A comparison of the LC3A, LC3B and LC3C homologs

Among the described indicators of mitochondrial damage, externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to trigger mitophagy, acting as a signal for binding the autophagy protein LC3B. However, the behavior of the other LC3 subfamily members has not been explored yet. In the present contribution, a comparative analysis of the interaction of LC3B, LC3A and LC3C with CL-containing model membranes, as well as their ability to translocate to mitochondria was assessed. Binding of LC3A to CL was higher than that of LC3B, and both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization by rotenone in HeLa-NDPK-D or SH-SY5Y cells. Two residues located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a higher binding than LC3B or LC3A to CL, binding was less specific, and colocalization of LC3C with mitochondria was not rotenonedependent. These results suggest that, at variance with LC3A, LC3C does not participate in the rotenone-CL mitophagy mechanism. The data support the notion that the various LC3/GABARAP family members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy.

biophysics↗

bioRxiv2020

CHO/LY-B cell growth under limiting sphingolipid supply: correlation between lipid composition and biophysical properties of sphingolipid-restricted cell membranes

ABSTRACTSphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO line in which serine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In the present study LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, i.e. 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 h. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of control LY-B cell growth rates. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led to a 6-fold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Concomitant changes were detected in glycerophospholipids under SL-restriction conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗