Biology subjects

Kovacs, D.

Publications and source records attributed to Kovacs, D..

9 recordsLinked to original sources

bioRxiv2025

Chain length defines the spatial segregation and metabolic fate of fatty acids in adipocytes

Adipocytes primarily store fatty acids (FAs) as triacylglycerols (TGs) within lipid droplets, releasing them through lipolysis to meet systemic energy demands. While the metabolism of long-chain fatty acids (LCFAs) in adipocytes has been extensively characterized, it remains ill-defined how adipocytes utilize fatty acids depending on chain length and structure. Our work demonstrates that short- and medium-chain fatty acids (SMCFAs) are esterified into TGs within lipid droplets, rather than being incorporated into other FA-containing lipid species. During lipolytic activation, TGs enriched in SMCFAs are hydrolysed more rapidly than those containing LCFAs. This accelerated mobilization is facilitated by the preferential localization of SMCFA-containing TGs at the lipid droplet surface, which enhances accessibility to adipose triglyceride lipase. Unlike LCFAs, which are efficiently released for utilization by peripheral tissues, SMCFAs are predominantly oxidized within adipocytes. These findings reveal a unique metabolic routing of SMCFAs, indicating their preferential intracellular oxidation to support adipocyte energy requirements.

biochemistry↗

bioRxiv2025

Conserved transcriptional co-regulation of pyrophosphatehomeostasis genes governs systemic mineralization factorsin mice and humans

Inorganic pyrophosphate (PPi) is a critical inhibitor of ectopic calcification, yet transcriptional regulation of genes controlling its systemic production and degradation (ABCC6, ALPL, ANKH, ENPP1) remains elusive. We hypothesized that PPi homeostasis is governed by evolutionarily conserved transcription factor (TF) network. Promoter-motif analysis of PPi genes revealed conserved enrichment of nuclear receptor TFs (ESR1, NR4A1, RXRA, NR1H3/LXR) and metabolic regulators (SREBF1, CEBPB, HNF4A) across mouse and human orthologues. Supporting this, analysis of public RNA-seq datasets and RT-qPCR in wild-type and Abcc6-/- mice demonstrated tight co-expression of these genes in the liver and the kidney, as central transcriptional hub of systemic PPi regulation. Functionally, analyses in mice revealed age-dependent inverse coupling between plasma PPi concentration and serum alkaline phosphatase (AP) activity, with strongest impact during early life. Abcc6-/- mice exhibited persistently reduced although gradually increasing PPi and altered Pi/PPi ratios during aging. Translating these findings to humans, plasma PPi correlated inversely with AP activity and positively with Pi, though associations were weaker in ABCC6-deficient pseudoxanthoma elasticum patients. These results establish a conserved TF-driven program coordinating hepatic and renal expression of PPi homeostatic genes, highlight early-life sensitivity of PPi balance, and link gene-regulation to circulating mineralization factors, highlighting species-specific and pathology-driven differences.

physiology↗

bioRxiv2024

Surface tension-driven sorting of human perilipins on lipid droplets

Perilipins (PLINs), the most abundant proteins on lipid droplets (LDs), display similar domain organization including amphipathic helices (AH). However, the five human PLINs bind different LDs suggesting different modes of interaction. We established a minimal system whereby artificial LDs covered with defined polar lipids were transiently deformed to promote surface tension. Binding of purified PLIN3 and PLIN4 AH was dependent on tension, even with polar lipids favoring packing defects, and showed an inverse correlation between protein and phospholipid densities on LDs. In contrast, PLIN1 bound readily to LDs fully covered by phospholipids; PLIN2 showed an intermediate behavior. In human adipocytes, PLIN3/4 were found in a soluble pool and relocated to LDs upon stimulation of triglyceride synthesis, whereas PLIN1 and PLIN2 localized to pre-existing LDs, consistent with the huge difference in LD avidity observed in vitro. We conclude that the PLIN repertoire is adapted to handling LDs with different surface properties. Significance statementLipid droplets (LDs) are highly dynamic organelles, whose size and surface properties vary during their life-time and also differ between different tissues. Here, we analyze the mode of binding of human perilipins (PLINs), the most abundant LD proteins, to LDs. We have developed a new reconstitution method, which shows that the purified PLIN family members have very different affinities for LDs, which might explain how they handle LDs of different dynamics in the cell.

biochemistry↗

bioRxiv2021

OSBP-mediated cholesterol transfer determines epithelial polarity and associated cargo secretion

Golgi lipid environment regulates sorting and cargo secretion. However, the mechanisms that spatiotemporally control the lipid composition of the secretory membranes to drive cargo trafficking are poorly understood. Lipid transfer proteins regulate the concentration of specific lipids at membrane contact sites. We hypothesised that by catalysing cholesterol/PI(4)P exchange at ER-trans-Golgi membrane contact sites the lipid transfer protein oxysterol binding protein (OSBP) affects the secretion of a subset of cargoes. Here, we report that OSBP is a major epithelial protein as its inhibition leads to complete loss of apico-basal polarity. By mapping the OSBP proximity proteome with the biotin ligase TurboID, we found that OSBP controls the secretion of multiple membrane associated proteins, including key polarity determinants such as E-cadherin. Mechanistically, we established that OSBP contributes to E-cadherin secretion by supplying cholesterol to post-Golgi membranes. Importantly, when cells downregulate cell-cell junctions upon epithelial-to-mesenchymal transition, they re-wire their lipid homeostasis and downregulate OSBP as well, thus altering the trafficking of the OSBP-dependent secretory cargoes.

cell biology↗

bioRxiv2021

COVID-19: A need for new rather than repurposed antiviral drugs.

BackgroundSARS-CoV-2 infection, the causative agent of COVID-19, has resulted in over 2,500,000 deaths to date1. Although vaccines are becoming available, treatment options remain limited. Repurposing of compounds could reduce the time, cost, and risks associated with the development of new drugs and has been the focus of many clinical studies. Here, we summarise available evidence on 29 FDA-approved compounds, from in vitro results to clinical trials, focussing on remdesivir, galidesivir and favipiravir, and test 29 antiviral compounds activity in vitro. MethodsA comprehensive search strategy was used to retrieve trials and publications related to antiviral compounds with potential efficacy to treat coronaviruses. These data were used to prioritise testing of a panel of antiviral drugs in vitro against patient isolates of SARS-CoV-2. An in vitro screen was carried out to determine the activity of 29 FDA-approved compounds. Results625 clinical trials investigated 16 repurposed antiviral candidate compounds for the treatment of COVID-19. In vitro studies identified ten drug candidates with demonstrable anti-SARS-CoV-2 activity, including favipiravir, remdesivir, and galidesivir. To validate these findings, a drug screen was conducted using two cell lines and wildtype isolates of SARS-CoV-2 isolated from patients in the UK. While eight drugs with anti-SARS-CoV-2 activity were identified in vitro, activity in clinical trials has, as yet failed to demonstrate a strong effect on mortality. ConclusionsSo far, no repurposed antiviral has shown a strong effect on mortality in clinical studies. The urgent need for novel antivirals in this pandemic is clear, despite the costs and time associated with their development. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSRepurposing of existing compounds for the treatment of COVID-19 has been the focus of many in vitro studies and clinical trials, saving time, costs and risks associated with the research and development of new compounds. Added value of this studyWe reviewed the literature for 29 FDA-approved compounds with previously reported (or suspected) anti-SARS-CoV-2 activity and found 625 clinical trials that have been undertaken on 16 different drugs. We determined if repurposed antivirals are suitable for clinical trials based on previously published data, and conducted an additional in vitro screen using locally circulating strains in the UK (PHE2 and GLA1). We report the difference in IC50 from published data using Wuhan1/Wash1 strains with PHE2 and GLA1, including IC50 values below 100M for galidesivir in wild-type virus. Given the limited success of repurposed compounds in the treatment of COVID-19, we comment on the urgent need for new antivirals specifically targeting SARS-CoV-2. Implications of all the available evidenceOur data show that most prospective compounds for repurposing show no anti-SARS-CoV-2 activity, and antiviral activity in vitro does not always translate to clinical benefit. So far, no repurposed compound has shown a strong effect on mortality in clinical studies. Drugs, including monoclonal antibody therapies, that have been developed to target SARS-CoV-2 virus itself have shown most promise.

microbiology↗

bioRxiv2020

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterization in different cell types

Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g. Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. To facilitate further cellular investigations, notably by imaging techniques, we present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterization in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localization of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localization of Nsp13 to the centrosome, Orf3a to late endosomes, and Orf9b to mitochondria.

cell biology↗

bioRxiv2019

Molecular and cellular dissection of the OSBP cycle through a fluorescent inhibitor

ORPphilins, natural molecules that strongly and selectively inhibit the growth of some cancer cell lines, are proposed to target intracellular lipid-transfer proteins of the Oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phopsphatidylinositol-4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular level of OSBP. By taking advantage of the intrinsic fluorescence of SWG, we follow its fate in cell cultures and show that its incorporation at the TGN depends on OSBP cellular abundance. We report that SWG inhibits specifically the lipid exchange cycle of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels and PI(4)P turnover are affected. Finally, we demonstrate the direct binding of SWG into OSBP lipid-binding cavity by intermolecular FRET. Collectively these data describe for the first time a specific and intrinsically fluorescent pharmacological tool to dissect OSBP properties at the cellular and molecular levels.

cell biology↗

bioRxiv2019

Mast cells and γδ T cells are largely dispensable for adaptive immune responses after laser-mediated epicutaneous immunization

BackgroundThe skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like {gamma}{delta} T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, {gamma}{delta} T cell and MC function still remains to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI).\n\nMethods{gamma}{delta} T cell knock out, MC depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2-3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. The local cytokine/chemokine milieu after laser microporation was determined.\n\nResultsWhile the majority of inflammatory chemokines and cytokines induced by laser treatment was not affected by the presence of {gamma}{delta} T cells or MCs, RANTES, was elevated in {gamma}{delta} T cell knock out mice, and GRO and TSLP, were significantly decreased after MC depletion. However, absence of {gamma}{delta} T cells or depletion of MC had no substantial effect on adaptive humoral or cellular immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC depleted mice.\n\nConclusions{gamma}{delta} T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI, whereas MC depletion decreased numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI.\n\nHighlightsO_LILaser microporation induces an inflammatory chemokine milieu at the site of immunization\nC_LIO_LI{gamma}{delta} T cells and mast cells contribute to the steady-state or damage-induced cytokine milieu in the skin\nC_LIO_LI{gamma}{delta} T cells and mast cells are dispensable for adaptive immunity after laser-mediated immunization\nC_LI

immunology↗

bioRxiv2018

Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties

Collective phase transitions, including phase separation and gelation of multivalent protein and RNA molecules appears to underlie the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that are often organized into multi-layered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we investigate the role of spontaneous driving forces as determinants of protein-RNA condensates with complex morphologies and distinct material properties. Through the use of systematic in vitro experiments and simulations based on coarse-grained models we find that that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multi-layered condensates with distinct material properties. Our results demonstrate that key properties of protein-RNA condensates such as their overall morphologies, internal dynamics, and the selective partitioning of substrates are governed specific amino acid chemistries as well as RNA sequence and secondary structure. Our findings yield a clear set of heuristics regarding homo- and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

biophysics↗