Biology subjects

Di Meo, F.

Publications and source records attributed to Di Meo, F..

9 recordsLinked to original sources

bioRxiv2026

The immunosuppressant tacrolimus (FK506) inhibits C. glabrata Cdr1 efflux pump function by stabilizing the inward-facing conformation.

The pathogenic yeast Candida glabrata is intrinsically resistant to azole antifungals through the overexpression of the multidrug transporter Cdr1. CgCdr1 detoxifies the yeast by expelling azoles out of the cell, thereby decreasing their intracellular concentration. Tacrolimus (FK506), one of the most widely used immunosuppressant medications used world-wide, has been identified as a broad-spectrum inhibitor of Cdr1 homologs in several Candida species. However, its mechanism of action remains unknown. We solved the cryoEM structure of CgCdr1 in complex with FK506, with or without ATP. The structure revealed that FK506 binds within the drug-binding site of CgCdr1, occupying the space occupied by Itraconazole. The hydrophobic face of FK506 stacks against the TMD1 and forms hydrogen bonds with TMD2, stabilizing a different conformation from the one adopted in FK-binding-proteins. FK506 binding triggered structural rearrangements bringing the nucleotide-binding-domains closer to the trans-membrane-domains, while stabilizing the inward-facing conformation. While ATP can still bind to the catalytic nucleotide-binding site, FK506 prevents the conformational transition required for ATP hydrolysis, thereby effectively blocking azole transport. Inter-particle variability analysis (3DVA) revealed significant conformational flexibility of FK506 within the binding pocket, with minimal transporter mobility. It allowed to visualize the conformational space occupied by the inhibitor within its binding-pocket, serving as a useful tool for inhibitor rational design. Overall, these findings demonstrate that FK506s inhibition extends beyond competitive binding, involving allosteric modulation of the ATPase cycle. Significance statementThe pathogenic yeast Candida glabrata exhibits intrinsic resistance to azole antifungals via the multidrug transporter Cdr1, which expels azoles from the cell. Tacrolimus (FK506), a widely used immunosuppressant, inhibits Cdr1 homologs across Candida species, yet its mechanism remained unknown. Here, we resolved the cryoEM structure of CgCdr1 in complex with FK506, revealing that FK506 binds to the drug-binding site, like itraconazole. Its hydrophobic face interacts with TMD1, while hydrogen bonds form with TMD2. FK506 stabilizes the inward-facing conformation, preventing ATP hydrolysis despite ATP binding, thereby blocking azole transport. Variability analysis highlighted FK506s conformational flexibility within the pocket, offering insights for rational inhibitor design. These findings demonstrate that FK506s inhibition involves both competitive binding and allosteric modulation of the ATPase cycle.

biochemistry↗

bioRxiv2024

Membrane-Dependent Dynamics and Dual Translocation Mechanisms of ABCB4: Insights from Molecular Dynamics Simulations

ABCB4 is an ATP-binding cassette transporter expressed at the canalicular membrane of hepatocytes and responsible for translocating phosphatidylcholine into bile. Despite the recent cryo-EM structures of ABCB4, knowledge about the molecular mechanism of phosphatidylcholine transport remains fragmented. In this study, we used all-atom molecular dynamics simulations to investigate ABCB4 dynamics during its transport cycle, leveraging both symmetric and asymmetric membrane models. Our results demonstrate that membrane composition influences the local conformational dynamics of ABCB4, revealing distinct lipid-binding patterns across different conformers, particularly for cholesterol. We explored the two potential mechanisms for phosphatidylcholine translocation: the canonical ATP-driven alternating access model and the "credit-card swipe" model. Critical residues were identified for phosphatidylcholine binding and transport pathway modulation, supporting the canonical mechanism while also indicating a possible additional pathway. The conformer-specific roles of kinking in transmembrane helices (TMH4 and TMH10) were highlighted as key events in substrate translocation. Overall, ABCB4 may utilize a cooperative transport mechanism, integrating elements of both models to facilitate efficient phosphatidylcholine motion across the membrane. This study provides new insights into the relationship between membrane environment and ABCB4 function, contributing to our understanding of its role in bile physiology and susceptibility to genetic and xenobiotic influences.

biophysics↗

bioRxiv2024

Proximal Tubule-on-Chip for Predicting Cation Transport: Dynamic Insights into Drug Transporter Expression and Function

Deciphering the sources of variability in drug responses requires to understand the processes modulating drug pharmacokinetics. However, pharmacological research suffers from poor reproducibility across clinical, animal, and experimental models. Predictivity can be improved by using Organs-on-Chips, which are more physiological, human-oriented, micro-engineered devices that include microfluidics. OoC are particularly relevant at the fundamental and preclinical stages of drug development by providing more accurate assessment of key pharmacokinetic events. We have developed a proximal tubule-on-a-chip model combining commercial microfluidic and chip technologies. Using the RPTEC/TERT1 cell line, we set up a dual-flow system with antiparallel flows to mimic the dynamics of blood and urine. We assessed transporters mRNA expression using RT-qPCR, cellular polarization and protein expression via immunofluorescence and confocal microscopy, and monitored the transcellular transport of a list of prototypic xenobiotics by determining their efflux ratios with LC-MS/MS. Our results show that flow exposure significantly modulate mRNA expression of drug membrane transporters compared to static conditions. Dynamic conditions also enhance cell polarization, as evidenced by preferential basal and apical expressions of Na+/K+-ATPase, P-gp, OCT2, and MATE1, as well as the cellular secretory profile. We demonstrated unidirectional transcellular transport of a cationic substrate (metformin) with a higher efflux than influx ratio, inhibited with a specific OCT2 inhibitor, thus confirming the relevance of our proximal tubule- on-a-chip set up for cation transport investigations. Our proximal tubule-on-a-chip can also be used to explore the interactions between transporters, xenobiotics, and endogenous metabolites, possibly involved in the variability of individual drug responses. This study provides additional evidence that OoC can bridge the gaps between systemic and local pharmacokinetics, i.e., drug concentration close to its target, at the fundamental and preclinical stages. HighlightsO_LICell exposure to flow shear stress modulate mRNA drug membrane transporters and cell polarization of proximal tubule C_LIO_LIProximal tubule-on-chip relying on RPTEC/TERT1 cell line is a suitable platform for assessing transcellular cationic transport C_LIO_LIOCT2 and MATE are involved in potential drug-endogenous metabolite interactions C_LIO_LICell exposure to xenobiotics and endogenous metabolites modulate the drug transporters expression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/617976v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@7562b7org.highwire.dtl.DTLVardef@a409c0org.highwire.dtl.DTLVardef@17c1d9aorg.highwire.dtl.DTLVardef@17b9a6f_HPS_FORMAT_FIGEXP M_FIG C_FIG Data StatementOriginal microscopy pictures, raw data for metabolomics and other data are available upon reasonable request.

pharmacology and toxicology↗

bioRxiv2022

Computational and structural insights into the pre- and post-hydrolysis states of multidrug resistance associated protein 1 in lipid bilayer membranes

ATP-binding cassette C-family drug membrane transporters play an important role in local pharmacokinetics, i.e., drug concentration in cellular compartments. From the structural point of view, only the bovine ortholog of the multidrug-resistance associated protein 1 (bMRP1) has been resolved. We here used s-scaled molecular dynamics simulations to investigate the structure and dynamics of bMRP1 in pre- and post-hydrolysis functional states. The present work aims to examine the slight but likely relevant structural differences between pre- and post-hydrolysis states of outward-facing conformations as well as the interactions between MRP1 and the surrounding lipid bilayer. Global conformational dynamics show unfavourable extracellular opening associated with nucleotide binding domain dimerization indicating that the post-hydrolysis state adopts a close-cleft conformation rather than an outward-open conformation. Our present simulations also highlight persistent interactions with annular cholesterol molecules and the expected active role of lipid bilayer in the allosteric communication between distant domains of MRP1 transporter.

pharmacology and toxicology↗

bioRxiv2022

Substrate binding and lipid-mediated allostery in the human organic anion transporter 1 at the atomic-scale

The Organic Anion Transporter 1 is a membrane transporter known for its central role in drug elimination by the kidney. hOAT1 is an antiporter translocating substrate in exchange for -ketoglutarate. The understanding of hOAT1 structure and function remains limited due to the absence of resolved structure of hOAT1. Benefiting from conserved structural and functional patterns shared with other Major Facilitator Superfamily transporters, the present study intended to investigate fragments of hOAT1 transport function and modulation of its activity in order to make a step forward the understanding of its transport cycle. s-long molecular dynamics simulation of hOAT1 were carried out suggesting two plausible binding sites for a typical substrate, adefovir, in line with experimental observations. The well-known B-like motif binding site was observed in line with previous studies. However, we here propose a new inner binding cavity which is expected to be involved in substrate translocation event. Binding modes of hOAT1 co-substrate -ketoglutarate were also investigated suggesting that it may binds to highly conserved intracellular motifs. We here hypothesize that -ketoglutarate may disrupt the pseudo-symmetrical intracellular charge-relay system which in turn may participate to the destabilisation of OF conformation. Investigations regarding allosteric communications along hOAT1 also suggest that substrate binding event might modulate the dynamics of intracellular charge relay system, assisted by surrounding lipids as active partners. We here proposed a structural rationalisation of transport impairments observed for two single nucleotide polymorphisms, p.Arg50His and p.Arg454Gln suggesting that the present model may be used to transport dysfunctions arising from hOAT1 mutations. HighlightsO_LIAdefovir has at least two binding pockets on hOAT1 in the outward-facing conformation. C_LIO_LIThe highly conserved B-motif within MFS is strongly involved in substrate binding. C_LIO_LI-Ketoglutarate binds to the intracellular domain of hOAT1 and destabilizes its OF conformation. C_LIO_LIThe lipid membrane bilayer plays an active role in the allosteric communication between intracellular and extracellular domains of hOAT1. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/500056v2_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@18d092corg.highwire.dtl.DTLVardef@5538e9org.highwire.dtl.DTLVardef@10b62cforg.highwire.dtl.DTLVardef@84421e_HPS_FORMAT_FIGEXP M_FIG The present work (from left): (i) reveals binding modes of adefovir (top) and -ketoglutarate (bottom) to hOAT1; (ii) maps Single Nucleotide Polymorphisms on outward-facing (top) and inward-facing (bottom) conformation of hOAT1; (iii) asses the allosteric effect of lipidic environment and presence of substrates. C_FIG

pharmacology and toxicology↗

bioRxiv2022

On the interplay between lipids and asymmetric dynamics of an NBS degenerate ABC transporter

Multidrug resistance-associated proteins are ABC C-family exporters. They are crucial in pharmacology as they transport various substrates across membranes. However, the role of the degenerate nucleotide-binding site (NBS) remains unclear likewise the interplay with the surrounding lipid environment. Here, we propose a dynamic and structural overview of MRP1 from ca. 110 {micro}s molecular dynamics simulations. ATP binding to NBS1 is likely maintained along several transport cycles. Asymmetric NBD behaviour is ensured by lower signal transduction from NBD1 to the rest of the protein owing to the absence of ball-and-socket conformation between NBD1 and coupling helices. Even though surrounding lipids play an active role in the allosteric communication between the substrate-binding pocket and NBDs, our results suggest that lipid composition has a limited impact, mostly by affecting transport kinetics. We believe that our work can be extended to other degenerate NBS ABC proteins and provide hints for deciphering mechanistic differences among ABC transporters.

biophysics↗

bioRxiv2022

Insights into the structure and function of the human organic anion transporter 1 in lipid bilayer membranes

The human SLC22A6/OAT1 plays an important role in the elimination of a broad range of endogenous substances and xenobiotics thus attracting attention from the pharmacological community. Furthermore, OAT1 is also involved in key physiological events such as the remote inter-organ communication. Despite its significance, the knowledge about hOAT1 structure and the transport mechanism at the atomic level remains fragmented owing to the lack of resolved structures. By means of protein-threading modeling refined by s-scaled Molecular Dynamics simulations, the present study provides the first robust model of hOAT1 in outward-facing conformation. Taking advantage of the AlphaFold 2 predicted structure of hOAT1 in inward-facing conformation, we here provide the essential structural and functional features comparing both states. The intracellular motifs conserved among Major Facilitator Superfamily members create a so-called "charge-relay system" that works as molecular switches modulating the conformation. The principal element of the event points at interactions of charged residues that appear crucial for the transporter dynamics and function. Moreover, hOAT1 model was embedded in different lipid bilayer membranes highlighting the crucial structural dependence on lipid-protein interactions. MD simulations supported the pivotal role of phosphatidylethanolamine components to the protein conformation stability. The present model is made available to decipher the impact of any observed polymorphism and mutation on drug transport as well as to understand substrate binding modes.

pharmacology and toxicology↗

bioRxiv2018

Cyclosporine A inhibits MRTF-SRF signalling through Na+/K+ ATPase inhibition & Actin remodelling

Calcineurin Inhibitors (CNI) are the pillars of immunosuppression in transplantation. However, they display a potent nephrotoxicity whose mechanisms remained widely unsolved. We used an untargeted quantitative proteomic approach (iTRAQ technology) to highlight new targets of CNI in renal proximal tubular cells (RPTCs). CNI-treated RPTCs proteome displayed an over-representation of Actin-binding proteins with a CNI-specific expression profile. Cyclosporine A (CsA) induced F-Actin remodelling and depolymerisation, decreased F-Actin-stabilizing, polymerization-promoting Cofilin (CFL) oligomers and inhibited the G-Actin-regulated serum responsive factor (SRF) pathway. Inhibition of CFL canonical phosphorylation pathway reproduced CsA effects; however, Ser3, an analogue of the phosphorylation site of CFL prevented the effects of CsA which suggests that CsA acted independently from the canonical CFL regulation. CFL is known to be regulated by the Na+/K+-ATPase. Molecular docking calculations evidenced 2 inhibiting sites of CsA on Na+/K+-ATPase and a 23% decrease in Na+/K+-ATPase activity of RPTCs was observed with CsA. Ouabain, a specific inhibitor of Na+/K+-ATPase also reproduced CsA effects on Actin organization and SRF activity. Altogether, these results described a new original pathway explaining CsA nephrotoxicity.

cell biology↗

bioRxiv2018

Nitrone-Trolox conjugate as an inhibitor of lipid oxidation: Towards synergistic antioxidant effects

1Free radical scavengers like -phenyl-N-tert-butylnitrone (PBN) and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) have been widely used as protective agents in various biomimetic and biological models. A series of three amphiphilic Trolox and PBN derivatives have been designed by adding to the parent molecules both a perfluorinated chain and a sugar group in order to render them amphiphilic. In this work, we have studied the interaction of these derivatives with lipid membranes and how it correlates to their antioxidant properties.\n\nThe three derivatives form monolayers at the air/water interface. We next investigated the ability of each derivative to interact with 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC) as well as their efficiency to inhibit the AAPH-induced oxidation of DLPC liposomes. The location of these derivatives in the membrane is a key parameter to rationalize their antioxidant efficiency. The derivative bearing both the PBN and the Trolox antioxidant moieties on the same fluorinated carrier exhibited a synergistic antioxidant effect by delaying the oxidation process. Molecular dynamics (MD) simulations supported the understanding of the mechanism of action, highlighting various key physical-chemical descriptors.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC=\"FIGDIR/small/323386_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@15e2ce5org.highwire.dtl.DTLVardef@1d58ee3org.highwire.dtl.DTLVardef@197f9d2org.highwire.dtl.DTLVardef@9279ff_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Amphiphilic fluorinated antioxidants efficiently inhibit lipid oxidation\n- The perfluorinated chain allows the insertion into membranes\n- The nature of the antioxidant is a key parameter in the antioxidant efficiency\n- The combination of Trolox and PBN results in a synergistic antioxidant effect\n\n\nCondensed running titleTrolox derivatives limit lipid oxidation

biophysics↗