Biology subjects

Amati, F.

Publications and source records attributed to Amati, F..

5 recordsLinked to original sources

bioRxiv2026

Alternative exon splicing reveals hidden mitochondrial targeting of PLIN3

Perilipin 3 (PLIN3) is a lipid droplet-associated protein implicated in cellular lipid metabolism. We characterize a previously unrecognized alternative splicing event in human PLIN3. The generated transcript PLIN3B is detectable at the RNA level across human tissues and is modulated by antisense oligonucleotide-mediated splicing. Despite clear evidence for PLIN3B transcript expression, endogenous PLIN3B protein could not be detected using multiple complementary state-of-the-art detection strategies. When expressed exogenously, PLIN3B coding sequence encodes an unstable protein that localizes predominantly within mitochondria, in contrast to canonical PLIN3. Using super-resolution microscopy, correlative light-electron microscopy, and interaction proteomics, we show that PLIN3B accumulates intramitochondrially and engages protein quality control machinery. Mitochondrial targeting depends on residues shared by both isoforms, not altered by alternative splicing, indicating that mitochondrial targeting is normally repressed. Together, these results show that alternative exon splicing in PLIN3 reveals latent mitochondrial targeting without producing detectable endogenous protein, underscoring the need to distinguish transcript diversity from stable protein expression.

cell biology↗

bioRxiv2025

Organelle-specific lipid profiles influence/underlie metabolic health in a nutrition-dependent manner

AbstractWestern diet (WD), characterized by high energy density, saturated fat and sucrose, is a major driver of obesity and insulin resistance (IR). Although dietary fat composition influences systemic lipid metabolism and insulin sensitivity, its impact on subcellular lipid classes distribution and fatty acid (FA) incorporation in skeletal muscle remains poorly defined. We hypothesized that (1) modulating dietary FA intake remodels mitochondrial and lipid droplet (LD) lipid profiles, including phospholipids (PL) and diacylglycerol (DAG) stereoisomers implicated in lipotoxicity; and (2) organelle-specific lipid profiles relate to metabolic health. C57BL/6J mice were fed WD or control chow for 12 weeks. Whole-body metabolism, insulin sensitivity and substrate use were assessed by indirect calorimetry, glucose and insulin tolerance tests, and fasting biomarkers. Mitochondria and LDs were isolated from soleus muscle for organelle-resolved lipidomics. DAG isomers and PL classes were quantified, and FA chain length and saturation patterns were analyzed in total lysate (TL), mitochondria and LDs. Correlations were performed between lipid class abundance and metabolic parameters. WD-fed mice developed obesity, dyslipidemia and early IR. Intramyocellular lipids increased, whereas mitochondrial abundance was unchanged. Organelle-resolved lipidomics revealed distinct subcellular signatures not detectable in TL. DAG FA composition mirrored dietary FA supply across compartments, with WD increasing saturated FA (SFA) and reducing di-unsaturated FA (DiUFA) species. In contrast, PL remodeling was class- and compartment-specific, with coordinated changes in mitochondria and LDs that were masked in whole-muscle TL. Several PL classes, including ether-linked phosphatidylethanolamine (ePE), phosphatidylinositol, PE and phosphatidylcholine in TL and LD, and phosphatidylserine and phosphatidylglycerol (PG) in TL and mitochondria, were strongly associated with insulin sensitivity and substrate utilization in healthy mice, but these relationships were lost under WD. LD-associated sn1,3-DAG content was a strong predictor of metabolic health in lean mice, which related to ATGL abundance. PE and PG in LD were related to obesity markers. Dietary lipid overload induces distinct and compartment-specific remodeling of the skeletal muscle lipidome. DAG and PL classes exhibited divergent FA incorporation across mitochondria, LDs and TL, with coordinated remodeling between LDs and mitochondria that remained undetectable at the whole-muscle level. Several lipid pools, particularly LD-localized 1,3-DAG, PE and PG, were consistently related to metabolic flexibility and markers of metabolic health in healthy muscle and were disrupted in WD-fed mice. These findings identify lipid class identity, FA composition and subcellular localization as key determinants of muscle adaptation to nutritional excess and point to LD phospholipids and DAG stereoisomers as potential early molecular signatures of emerging IR.

physiology↗

bioRxiv2021

Narcolepsy with cataplexy is caused by epigenetic silencing of hypocretin neurons

Narcolepsy with cataplexy is a chronic sleep disorder characterized by hypocretin deficiency. The condition is believed to result from autoimmune destruction of hypocretin (HCRT) neurons, although direct evidence is lacking and mere Hcrt gene inactivation causes full-blown narcolepsy in mice. Here we show that the expression of another hypothalamic neuropeptide, QRFP, is lost in mouse models with HCRT cell-ablation, but tends to be even increased in Hcrt gene knockout mice, suggesting that QRFP expression can be used as a proxy for the presence or absence of HCRT neurons. Similar to Hcrt knockout mice, narcolepsy patients show intact hypothalamic QRFP expression, and cerebrospinal fluid levels of QRFP peptide are increased, suggesting their HCRT neurons are intact. We show that the human HCRT gene promoter is methylation-sensitive in vitro, and is hypermethylated in the hypothalamus of patients selectively at a putative PAX5:ETS1 binding site within the proximal HCRT promoter. Ets1-KO mice display downregulated Hcrt expression, while pax5-ets1 knockdown in zebrafish causes decreased hcrt expression, decreased activity and sleep fragmentation, similar to narcolepsy patients. Our results suggest that HCRT neurons are alive, but epigenetically silenced, in the hypothalamus of narcolepsy patients, opening the possibility to reverse or cure narcolepsy.

neuroscience↗

bioRxiv2020

Potassium channels, megapolarization, and water pore formation are key determinants for cationic cell-penetrating peptide translocation into cells

Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. They provide efficient methodology to transfer bioactive molecules in cells, in particular in conditions when transcription or translation of cargo-encoding sequences is not desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (-150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying sizes, we assessed the diameter of the water pores in living cells and found that they readily accommodated the passage of 2 nm-wide molecules, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPPs cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membrane. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

cell biology↗

bioRxiv2019

Respiratory supercomplexes provide metabolic efficiency in zebrafish

The oxidative phosphorylation (OXPHOS) system is a dynamic system in which the respiratory complexes coexist with super-assembled quaternary structures called supercomplexes (SCs). The physiological role of SCs is still disputed. Here we used zebrafish to study the relevance of respiratory SCs. We combined immunodetection analysis and deep data-independent proteomics to characterize these structures and found similar SCs to those described in mice, as well as novel SCs including III2+IV2, I+IV and I+III2+IV2. To study the physiological role of SCs, we generated two null allele zebrafish lines for supercomplex assembly factor 1 (SCAF1). SCAF1-/- fish displayed altered OXPHOS activity due to the disrupted interaction of complex III and IV. SCAF1-/- fish were smaller in size, and showed abnormal fat deposition and decreased female fertility. These physiological phenotypes were rescued by doubling the food supply, which correlated with improved bioenergetics and alterations in the metabolic gene expression program. These results reveal that SC assembly by SCAF1 modulates OXPHOS efficiency and allows for the optimization of metabolic resources.

physiology↗