Biology subjects

Reggiori, F.

Publications and source records attributed to Reggiori, F..

5 recordsLinked to original sources

bioRxiv2023

The endosomal sorting complex Retromer has a central role insystemic cholesterol homeostasis by controlling endo-lysosomal cholesterol transport in hepatocytes

BackgroundDisturbed hepatic cholesterol homeostasis is associated with multiple diseases, including atherosclerotic cardiovascular disease and metabolic dysfunction- associated steatotic liver disease. The endo-lysosomal system is essential for cholesterol uptake and intracellular distribution, yet the mechanisms governing these processes remain incompletely understood. Here, we investigated the impact of hepatic VPS35, a subunit of the endosomal sorting complex retromer, on hepatocellular and whole-body cholesterol homeostasis. MethodsWe generated a liver-specific Vps35 knockout mouse model (Vps35HepKO) and applied biochemical analyses, proteomics, and stable-isotope-labeled tracers to quantify critical processes of cholesterol metabolism. Human iPSC-derived liver organoids and CRISPR technology were used to translate our findings to humans. Mechanistic studies were performed in precision-cut liver slices from WT and Vps35HepKO mice. ResultsHepatic VPS35 deficiency led to an increase in endo-lysosomal degradative compartments and a marked reduction in specific lysosomal proteins, including lysosomal acid lipase (LAL), Scavenger Receptor Class B Member 2 (SCARB2), and Niemann-Pick type C1 (NPC1). Using pathway-specific inhibitors, we showed that VPS35 loss impairs the translation of SCABR2 and NPC1. Consistently, human iPSC-derived liver organoids lacking VPS35 also exhibited reduced expression of NPC1 and SCARB2 proteins. The decrease in these lysosomal proteins correlated with increased cholesterol levels in the plasma and liver of Vps35HepKO mice. This was likely explained by the disrupted cholesterol trafficking through the endo-lysosomal system, delayed plasma cholesterol turnover, and upregulated cholesterol biosynthesis. ConclusionThese findings uncover a previously unknown role for the hepatic retromer complex in maintaining systemic cholesterol homeostasis. Beyond its established function in endosomal cargo transport, we now demonstrate that retromer is also essential for lysosomal cholesterol handling. This role is mediated by regulating key lysosomal proteins involved in cholesterol metabolism, including LAL, NPC1, and SCARB2.

molecular biology↗

bioRxiv2022

BNIP3 regulates Chikungunya virus infection independently of autophagy and cell death

Chikungunya virus (CHIKV) is a human pathogen causing outbreaks of febrile illness for which vaccines and specific treatments remain unavailable. Autophagy-related (ATG) proteins and autophagy receptors are a set of host factors that participate in autophagy, but have also shown to function in other unrelated cellular pathways. Although autophagy is reported to both inhibit and enhance CHIKV replication, the specific role of individual ATG proteins remains largely unknown. Here, a siRNA screen was performed to evaluate the importance of the ATG proteome and autophagy receptors in controlling CHIKV infection. We observed that 7 out of 50 ATG proteins impact the replication of CHIKV. Among those, depletion of the mitochondrial protein and autophagy receptor BCL2 Interacting Protein 3 (BNIP3) increased CHIKV infection. Interestingly, BNIP3 controls CHIKV independently of autophagy and cell death. Detailed analysis of the CHIKV viral cycle revealed that BNIP3 interferes with the early stages of infection. Moreover, the antiviral role of BNIP3 was found conserved across two distinct CHIKV genotypes and the closely related Semliki Forest virus. Altogether, this study describes a novel and previously unknown function of the mitochondrial protein BNIP3 in the control of the early stages of the alphavirus viral cycle.

microbiology↗

bioRxiv2020

Targeting of integral membrane proteins to the surface of lipid droplets

Lipid droplets (LDs) are globular subcellular structures that mainly serve to store energy in form of neutral lipids, particularly triacylglycerols and steryl esters. LDs are closely associated with the membrane of the endoplasmic reticulum (ER), and are limited by a monolayer membrane of phospholipids harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here we address the question whether integral membrane spanning proteins could localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER resident proteins, such as Wbp1 (a N-glycosyl transferase complex subunit), Sec61 (a translocon subunit), and Pmt1 (a protein O-mannosyltransferase). The resulting fusion proteins localize to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, is due to redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners in cells coexpressing the membrane-anchored perilipin. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of OM14, an outer mitochondrial membrane protein. Expression of OM14-PLIN3 resulted in close apposition of mitochondria and LDs. Taken together, these data indicate that the LD surface constitutes a barrier for ER-localized integral membrane spanning proteins.

cell biology↗

bioRxiv2019

Lecithin:Retinol Acyl Transferase (LRAT) inducesthe formation of lipid droplets

Lipid droplets are unique and nearly ubiquitous organelles that store neutral lipids in a hydrophobic core, surrounded by a monolayer of phospholipids. The primary neutral lipids are triacylglycerols and steryl esters. It is not known whether other classes of neutral lipids can form lipid droplets by themselves. Here we show that production of retinyl esters by lecithin:retinol acyl transferase (LRAT) in yeast cells, incapable of producing triacylglycerols and steryl esters, causes the formation of lipid droplets. By electron microscopy, these lipid droplets are morphologically indistinguishable from those in wild-type cells. In silico and in vitro experiments confirmed the propensity of retinyl esters to segregate from membranes and to form lipid droplets. The hydrophobic N-terminus of LRAT displays preferential interactions with retinyl esters in membranes and promotes the formation of large retinyl ester-containing lipid droplets in mammalian cells. Our combined data indicate that the molecular design of LRAT is optimally suited to allow the formation of characteristic large lipid droplets in retinyl ester-storing cells.

biochemistry↗

bioRxiv2019

Pseudomonas aeruginosa lectin LecB impairs keratinocyte fitness by abrogating growth factor signalling

Lectins are glycan-binding proteins with no catalytic activity and ubiquitously expressed in nature. Numerous bacteria employ lectins to efficiently bind to epithelia, thus facilitating tissue colonisation. Wounded skin is one of the preferred niches for Pseudomonas aeruginosa, which has developed diverse strategies to impair tissue repair processes and promote infection.\n\nHere, we analyse the effect of the P. aeruginosa fucose-binding lectin LecB on human keratinocytes and demonstrate that it triggers events in the host, upon binding to fucosylated residues on cell membrane receptors, that extend beyond its role as an adhesion molecule. We found that LecB associates with several growth factor receptors and dampens their signalling pathways, leading to the arrest of cell cycle. Additionally, we describe a novel LecB-triggered mechanism to downregulate host cell receptors by showing that LecB leads to insulin-like growth factor receptor 1 internalisation, without receptor activation, and subsequent missorting towards intracellular endosomal compartments.\n\nOverall, these data highlight that LecB is a multitask virulence factor that, through subversion of several host pathways, has a profound impact on keratinocyte proliferation and survival.

cell biology↗