Biology subjects

Giordano, F.

Publications and source records attributed to Giordano, F..

11 recordsLinked to original sources

bioRxiv2025

ORP5 controls the partitioning of phosphatidic acid between triacylglycerol and cardiolipin synthesis at mitochondria-ER-lipid droplet contact sites

Phosphatidic acid (PA) is a central metabolic intermediate that can fuel triacylglycerol (TAG) synthesis in lipid droplets (LDs) or cardiolipin production in mitochondria, but how cells partition PA between these competing fates has remained a fundamental unresolved question in lipid cell biology. We identify the lipid transfer protein ORP5 as a key regulator of PA partitioning at Mitochondria-Associated endoplasmic reticulum Membranes (MAM) that contact lipid droplets (LD), referred to as MAM-LD junctions. Cell imaging analysis shows that ORP5 stabilizes PA levels at MAM to promote TAG synthesis. On the other hand, loss of ORP5 causes PA accumulation on mitochondrial membranes, leading to excess cardiolipin synthesis and mitochondrial hyperfusion, while impairing triacylglycerol (TAG) synthesis and LD formation. Finally, reconstitution assays using liposomes or giant organelles further demonstrate that ORP5 can transfer PA from mitochondria to the ER via its ORD domain. Together, these findings reveal that ORP5 functions as a PA lipid transfer protein at tripartite MAM-LD contacts, where it balances LD formation with mitochondrial lipid metabolism, protecting mitochondria from cardiolipin overload.

cell biology↗

bioRxiv2025

Tripartite ER-Mitochondria-Lipid Droplets contact sites control adipocyte metabolic flexibility

Obesity is a major risk factor for cardiometabolic diseases, with adipocyte dysfunction playing a central role. Understanding how lipid storage and mobilization are regulated--and disrupted--in adipocytes is key to addressing obesity-associated complications. The ER-anchored protein Seipin controls lipid droplet (LD) biogenesis and maintenance, and its loss disrupts ER-LD contact sites. In humans, Seipin deficiency causes generalized lipodystrophy, a severe form of adipocyte dysfunction. We previously showed that Seipin also localizes at ER-mitochondria contact sites (MAM), where it regulates calcium exchange and mitochondrial function. Here, we examined whether Seipin targeting to MAM and ER-LD sites overlaps functionally. We analyzed subcutaneous adipose tissue (AT) from inducible Seipin-knockout mice using transmission electron microscopy (TEM) and proximity ligation assays (PLA) to quantify membrane contact sites (MCS) involving the ER, LDs, and mitochondria. In control mice, feeding reduced MAMs while increasing ER-LD and mitochondria-LD contacts, whereas Seipin deficiency abolished this remodeling. Specifically, under lipid loading, MAMs located in proximity to LDs--tripartite contact sites known as MAM-LD--were increased in control but not in Seipin-deficient adipocytes. Fluorescence recovery after photobleaching assays revealed that Seipin depletion impairs triglyceride transfer to LDs, an effect rescued by the MAM-LD-reinforcing synthetic peptide Linker-ER-Mi. Importantly, this rescue was abolished by silencing the mitochondrial calcium uniporter, demonstrating that calcium exchange is critical for triglyceride storage in LDs. We further investigated how MAM-LD remodeling influences adipocyte metabolic flexibility. Using TEM and PLA, we monitored two MAM subtypes: those forming MAM-LD and those engaging cytosolic mitochondria (MAM-CM). During adipogenesis, MAM-LD frequency increased while MAM-CM decreased. Similarly, in mouse AT and 3T3-L1 adipocytes, lipid loading selectively promoted MAM-LD. Notably, this adaptive remodeling of membrane contact sites was blunted in the adipose tissue of diet-induced obese mice. Genetic disruption of MCS in 3T3-L1 adipocytes altered lipid flux, impaired lipolysis, and reduced insulin signaling. Together, our findings identify MAM-LD contacts as key regulators of adipocyte lipid handling and metabolic flexibility, whose disruption may underlie the metabolic inflexibility of obesity.

cell biology↗

bioRxiv2025

Renalase activates mitochondrial leak metabolism in response to cellular stress and to repair damage after injury

A variety of mechanisms enhance cell stress response and repair; however, the role of mitochondria in this activity is unclear. Here we show that exogenous renalase (RNLS), an intracellular flavin-dependent NADH oxidase, activates intramitochondrial RNLS activity to promote cell survival. RNLS interacts with the ATP synthase alpha and beta subunits (ATP5 and ATP5{beta}) and opens the ATP synthase c-subunit leak channel to increase complex I and II activities and protein synthesis rate. RNLS causes a selective, sustained, time-dependent increase in cellular protein synthesis without affecting cell proliferation, whereas RNLS deletion or direct inhibition of the mitochondrial leak blocks RNLS-mediated protein synthesis. Functional analysis of newly and differentially synthesized proteins over 24 hours reveals rapid changes in one-carbon metabolism and ribosomal biogenesis pathways as early as one hour after RNLS exposure. Mitochondrial injury is more severe in the RNLS KO kidney after acute stress, related to decreased protein synthesis rate and increased mitophagy. RNLS KO mice exposed to the stress of chronic cardiac pressure overload fail to develop cardiac hypertrophy, the physiological response, and die of heart failure and cardiac rupture. These data highlight the critical role RNLS has in activating mitochondrial leak metabolism to induce selective protein synthesis and protect against acute and chronic stress. HIGHLIGHTSO_LIRenalase interacts with the ATP synthase alpha and beta subunits C_LIO_LIRenalase activates mitochondrial leak metabolism C_LIO_LIRenalase and leak metabolism increase complex I and II activities C_LIO_LILeak metabolism increases protein synthesis rate C_LIO_LIRenalase protects against cell stress and organ injury C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/671117v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@96ea7eorg.highwire.dtl.DTLVardef@1fcffbaorg.highwire.dtl.DTLVardef@f946cdorg.highwire.dtl.DTLVardef@197498a_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

cell biology↗

bioRxiv2024

Seipin Regulates Caveolin-1 Trafficking and Organelle Crosstalk

Caveolin-1 (CAV1), the main structural component of caveolae, is essential in various biological processes, including mechanotransduction, lipid metabolism, and endocytosis1-4. Deregulation of CAV1 dynamics is linked to various pathologies, including cellular senescence, cancer, insulin resistance, and lipodystrophy5-9. However, mechanisms regulating CAV1 trafficking and function remain poorly understood. Here, we show that seipin, a crucial lipid droplet (LD) biogenesis factor10, modulates CAV1 trafficking. Deletion of seipin resulted in the accumulation of saturated lipids, leading to ceramide and sphingomyelin overproduction, which disrupted the membrane order of the trans-Golgi network (TGN). In seipin deficiency, CAV1 location to the plasma membrane (PM) was impaired, reducing caveolae. Instead, CAV1 accumulated in TGN and late endosome compartments, which fused with LDs and delivered the protein. In wild-type (WT) cells, this process was minimal but significantly enhanced by treatment with palmitate, ceramide, or Stearoyl-CoA desaturase-1 (SCD1) inhibition. Conversely, in seipin-deficient cells, inhibiting Fatty Acid Synthase (FASN) or overexpressing SCD1 restored CAV1 localization to the PM and reduced its accumulation in LDs. Our findings reveal that seipin controls the funneling of palmitate toward glycerolipids synthesis and storage in LDs versus conversion to ceramides in the ER. This balance is crucial to cellular protein trafficking by controlling the TGN membrane order. Therefore, our study identifies seipin as a critical regulator of cellular lipid metabolism, protein trafficking, and organelle homeostasis. These findings shed light on the processes regulating CAV1 trafficking and show that convergent pathophysiological mechanisms associated with defects in CAV1 and seipin contribute to metabolic disorders, including insulin resistance and lipodystrophies11-14.

cell biology↗

bioRxiv2022

Extending and improving metagenomic taxonomic profiling with uncharacterized species with MetaPhlAn 4

Metagenomic assembly enables novel organism discovery from microbial communities, but from most metagenomes it can only capture few abundant organisms. Here, we present a method - MetaPhlAn 4 - to integrate information from both metagenome assemblies and microbial isolate genomes for improved and more comprehensive metagenomic taxonomic profiling. From a curated collection of 1.01M prokaryotic reference and metagenome-assembled genomes, we defined unique marker genes for 26,970 species-level genome bins, 4,992 of them taxonomically unidentified at the species level. MetaPhlAn 4 explains [~]20% more reads in most international human gut microbiomes and >40% in less-characterized environments such as the rumen microbiome, and proved more accurate than available alternatives on synthetic evaluations while also reliably quantifying organisms with no cultured isolates. Application of the method to >24,500 metagenomes highlighted previously undetected species to be strong biomarkers for host conditions and lifestyles in human and mice microbiomes, and showed that even previously uncharacterized species can be genetically profiled at the resolution of single microbial strains. MetaPhlAn 4 thus integrates the novelty of metagenomic assemblies with the sensitivity and fidelity of reference-based analyses, providing efficient metagenomic profiling of uncharacterized species and enabling deeper and more comprehensive microbiome biomarker detection.

bioinformatics↗

bioRxiv2022

The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites.

The precise control of phagosome maturation is critical for innate and adaptive immunity, determining whether phagocytosed material is destroyed or used to present antigens. We observed previously that non-fusogenic contacts between the endoplasmic reticulum (ER) and phagosomes, called membrane contact sites (MCS), are tethered by the calcium regulator STIM1 and fine-tune phagosomal maturation. The secretory pathway SNARE protein Sec22b has been implicated in controlling phagocytosis, phagosome maturation and antigen presentation, though its effects are controversial, and its mechanism of action poorly understood. Recently, Sec22b was shown to tether MCS at the plasma membrane without mediating membrane fusion. Here, we show that Sec22b localizes to and regulates the frequency of ER-phagosome contacts independently of STIM proteins. Sec22b knockdown and overexpression of a an MCS-disrupting mutant Sec22b-P33 induced only mild or no effect on global and local calcium signalling. However, Sec22b knockdown altered phagosomal phospholipids including PI(3)P, PI(4)P and PS, but not PI(4,5)P2. Increased PI(4)P in shSec22b cells was rescued by re-expression of Sec22b or the artificial MCS tether MAPPER but not the P33 mutant. Moreover, Sec22b co-precipitated and was co-recruited to phagosomes with the PS/PI(4)P lipid exchange protein ORP8. Expression of wild-type, but not mutant ORP8, also rescued phagosomal PI(4)P. Concordantly, Sec22b, MAPPER and ORP8 but not P33 or the ORP8 mutant decreased phagolysosome fusion in shSec22b cells. These results clarify a novel mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

cell biology↗

bioRxiv2021

ORP5 AND ORP8 ORCHESTRATE LIPID DROPLET BIOGENESIS AND MAINTENANCE AT ER-MITOCHONDRIA CONTACT SITES

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the Endoplasmic Reticulum (ER). The ER-protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at Mitochondria-Associated ER Membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulate seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for the ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at membrane contact sites. HIGHLIGHTSORP5 and ORP8 localize at MAM subdomains where LDs originate. Phosphatidic acid is enriched in MAM subdomains that are the birthplace of LDs. ORP5 and ORP8 knockdown impairs LD biogenesis. ORP5 and ORP8 regulate seipin recruitment to MAM-LD contact sites.

cell biology↗

bioRxiv2021

Neuroprotective, antioxidant and antiproliferative activity of grapefruit IntegroPectin on SH-SY5Y cells

Tested in vitro on SH-SY5Y neuroblastoma cells, grapefruit IntegroPectin is a powerful neuroprotective, antioxidant and antiproliferative agent. The strong antioxidant properties of grapefruit IntegroPectin, and its ability to preserve mitochondrial membrane potential and morphology, severely impaired in neurodegenerative disorders, make this new biopolymer highly soluble in water an attractive therapeutic agent for oxidative stress-associated brain disorders. Similarly, the ability of this new citrus pectin rich in naringin, linalool, linalool oxide and limonene adsorbed at the outer surface to inhibit cell proliferation or even kill, at high doses, neoplastic cells, coupled to its excellent health and safety profile, opens up new therapeutic strategies in cancer research. In order to take full advantage of its vast therapeutic and preventive potential, detailed studies of molecular mechanism involved in the antiproliferative and neuroprotective of IntegroPectin are urgently needed.

neuroscience↗

bioRxiv2021

Recruitment of Scc2/4 to double strand breaks depends on γH2A and DNA end resection

Homologous recombination (HR) enables cells to overcome the threat of DNA double strand breaks (DSB), allowing for repair without the loss of genetic information. Central to the HR repair process is the de novo loading of Cohesin around a DSB by its loader complex Scc2/4. Although Cohesins accumulation at these sites has been well studied, the prerequisites leading to Scc2/4 recruitment during the repair process are still elusive. Here we investigate which factors are required for recruitment of Scc2 around DSBs in Saccharomyces cerevisiae. To address this question, we combined ChIP-qPCR with a GAL-inducible HO-endonuclease system to generate a site specific DSB in vivo. We find that Scc2 recruitment relies on yH2A and Tel1, but as opposed to Cohesin, not on Mec1. We further demonstrate that binding of Scc2 depends on and coincides with DNA end resection. Although affected by the impact on resection, this recruitment of Scc2 is not directly facilitated by the RSC, SWR1 or INO80 complexes. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and the subsequent loading of Cohesin.

cell biology↗

bioRxiv2019

The interaction between non-fusogenic Sec22b-Stx complexes and Extended-Synaptotagmins promotes neurite growth and ramification

Axons and dendrites are long and often ramified neurites that need particularly intense plasma membrane (PM) expansion during the development of the nervous system. Neurite growth depends on non-fusogenic Sec22b-Stx1 SNARE complexes at endoplasmic reticulum (ER)-PM contacts. Here we show that Sec22b interacts with the endoplasmic reticulum lipid transfer proteins Extended-Synaptotagmins (E-Syts) and this interaction depends on the Longin domain of Sec22b. Overexpression of E-Syts stabilizes Sec22b-Stx1 association, whereas silencing of E-Syts has the opposite effect. Overexpression of wild-type E-Syt2, but not mutants unable to transfer lipids or attach to the ER, increase the formation of axonal filopodia and ramification of neurites in developing neurons. This effect is inhibited by a clostridial neurotoxin cleaving Stx1, expression of Sec22b Longin domain and a Sec22b mutant with extended linker between SNARE and transmembrane domains. We conclude that Sec22b-Stx1 ER-PM contact sites contribute to PM expansion by interacting with lipid transfer proteins such as E-Syts.

cell biology↗

bioRxiv2019

ORP5 REGULATES TRANSPORT OF LIPIDS AND CALCIUM TO MITOCHONDRIA AT ENDOPLASMIC RETICULUM-MITOCHONDRIA MEMBRANE CONTACT SITES

Mitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the mitochondrial intermembrane space. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at sites of close organelle apposition called membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that the main physiological localization of the lipid transfer proteins ORP5 and ORP8 is at mitochondria-associated ER membranes (MAMs) subdomains, physically linked to the MIB/MICOS complexes that bridge the two mitochondrial membranes. We also show that ORP5/8 mediate non-vesicular transport of phosphatidylserine (PS) lipids from the ER to mitochondria by cooperating with the MIB/MICOS complexes. Overall our study reveals a novel physical and functional link between ER-mitochondria contacts involved in lipid transfer and intra-mitochondrial membranes contacts maintained by the MIB/MICOS complexes.

cell biology↗