Biology subjects

Gourdon, P.

Publications and source records attributed to Gourdon, P..

7 recordsLinked to original sources

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2023

Structure of the hepatitis C virus E1/E2 envelope proteins in a homodimeric complex

Worldwide, 58 million individuals suffer from chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer. The HCV envelope proteins, E1 and E2, form a heterodimer, which is the target for neutralizing antibodies. Despite high-resolution structural models of partial heterodimer elements, the structural landscape of higher-order E1/E2 oligomers remains unexplored. We determined a ~3.5 [A] cryo-electron microscopy structure of membrane-extracted HCV E1/E2 in a homodimeric arrangement. This structure includes detailed information on the homodimer interface, the E2-binding pocket for hypervariable region 1, antigenic site 412 conformation, and the organization of the E1/E2 transmembrane regions, including one internal to E1. This higher-order E1/E2 assembly could play a pivotal role in the design of novel vaccine antigens better mimicking E1/E2 complexes on the HCV particle.

molecular biology↗

bioRxiv2023

Ex vivo structures from spinach leaves

Ex vivo structure determination of macromolecules from native source is gaining increasing attention from the scientific community, as the method can be employed to dissect the function of important, multi-component molecular machines. However, the existing ex vivo procedures often require genome manipulation or availability high-affinity binders, limiting the general applicability. Here, we report simple yet robust principles for isolation of protein complexes from enriched native biological material, enabling cryoEM-facilitated high-resolution structure determination. We report the structures of ten separate membrane and soluble protein complexes determined from spinach leaves. Moreover, the developed pipeline is likely adaptable to essentially any biological system. As such, the approach may represent an attractive avenue for future structural proteomics efforts.

biochemistry↗

bioRxiv2022

FK506-binding protein 2 participates in proinsulin folding

Apart from chaperoning, disulphide bond formation and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94, and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high molecular weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic {beta}-cells increased apoptosis without detectable upregulation of ER-stress response genes. Interestingly, FKBP2 mRNA was overexpressed in {beta}-cells from pancreatic islets of T2D patients. Based on molecular modelling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B chain (P28) is the substrate of FKBP2s isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

molecular biology↗

bioRxiv2022

Copper binding leads to increased dynamics in the regulatory N-terminal domain of full-length human copper transporter ATP7B

ATP7B is a human copper-transporting P1B-type ATPase that is involved in copper homeostasis and resistance to platinum drugs in cancer cells. ATP7B consists of a copper-transporting core and a regulatory N-terminal tail that contains six metal-binding domains (MBD1-6) connected by linker regions. The MBDs can bind copper, which changes the dynamics of the regulatory domain and activates the protein, but the underlying mechanism remains unknown. To identify possible copper-specific structural dynamics involved in transport regulation, we constructed a model of ATP7B spanning the N-terminal tail and core catalytic domains and performed molecular dynamics (MD) simulations with (holo) and without (apo) copper ions bound to the MBDs. In the holo protein, MBD2, MBD3 and MBD5 showed enhanced mobilities, which resulted in a more extended N-terminal regulatory region. The observed separation of MBD2 and MBD3 from the core protein supports a mechanism where copper binding activates the ATP7B protein by reducing interactions among MBD1-3 and between MBD1-3 and the core protein. Instead, an increased interaction between MBD5 and the core protein was observed that brought the copper-binding site of MBD5 closer to the high-affinity internal copper-binding site in the core protein. The simulation results assign specific, mechanistic roles to the metal-binding domains involved in ATP7B regulation that are testable in experimental settings.

biophysics↗

bioRxiv2021

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.2 [A] structure, revealing a classical {beta}-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion conducting pathway as also supported by metal binding quantification via ICP-MS and MD simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrated that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

biochemistry↗

bioRxiv2021

Structure and ion-release mechanism of PIB-4-type ATPases

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB-4-ATPases, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release, and as an internal counter ion, of an invariant, central histidine. We also establish that the turn-over of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

biochemistry↗