Biology subjects

Ross-Kaschitza, D.

Publications and source records attributed to Ross-Kaschitza, D..

5 recordsLinked to original sources

bioRxiv2024

Large transient assemblies of Apaf1 constitute the apoptosome in cells

Upon cell death signals, the apoptotic protease-activating factor Apaf1 and cytochrome c interact to form the apoptosome complex. The apoptosome is crucial for mitochondrial apoptosis, as it activates caspases that dismantle the cell. However, the assembly mechanism and appearance of the apoptosome in vivo remain unclear. We show that upon onset of apoptosis, Apaf1 molecules accumulate into multiple foci per cell. Disassembly of the foci is linked to survival of the cell. Structurally, Apaf1 foci resemble organelle-sized, cloud-like assemblies. Foci form upon specific molecular interactions with cytochrome c and depending on procaspase-9. We propose that Apaf1 foci correspond to the apoptosome in cells. Transientness and ultrastructure of Apaf1 foci suggest that the dynamic spatiotemporal organisation of apoptosome components regulates progression of apoptosis.

cell biology↗

bioRxiv2024

Identification of a Binding Site for Small Molecule Inhibitors Targeting Human TRPM4

Transient receptor potential (TRP) melastatin 4 (TRPM4) protein is a calcium-activated monovalent cation channel associated with various genetic and cardiovascular disorders. The anthranilic acid derivative NBA is a potent and specific TRPM4 inhibitor, but its binding site in TRPM4 has been unknown, although this information is crucial for drug development targeting TRPM4. We determined three cryo-EM structures of full-length human TRPM4 embedded in native lipid nanodiscs without inhibitor, bound to NBA, and a new anthranilic acid derivative, IBA. We found that the small molecules NBA and IBA were bound in a pocket formed between the S3, S4, and TRP helices and the S4-S5 linker of TRPM4. Our structural data and results from patch clamp experiments enable validation of a binding site for small molecule inhibitors, paving the way for further drug development targeting TRPM4.

biochemistry↗

bioRxiv2022

Role of 14-3-3 proteins in human cardiac sodium channel Nav1.5 regulation

Background14-3-3 proteins are ubiquitous proteins that play a role in cardiac physiology (e.g., metabolism, development, and cell cycle). Furthermore, 14-3-3 proteins were proposed to regulate the electrical function of the heart by interacting with several cardiac ion channels, including the voltage-gated sodium channel Nav1.5. Given the many cardiac arrhythmias associated with Nav1.5 dysfunction, understanding its regulation by the protein partners is crucial. AimsIn this study, we aimed to investigate the role of 14-3-3 proteins in the regulation of the human cardiac sodium channel Nav1.5. Methods and ResultsAmongst the seven 14-3-3 isoforms, only 14-3-3{eta} (encoded by YWHAH gene) weakly co-immunoprecipitated with Nav1.5 when heterologously co-expressed in tsA201 cells. Total and cell surface expression of Nav1.5 was however not modified by 14-3-3{eta} overexpression or inhibition with difopein, and 14-3-3{eta} did not affect physical interaction between Nav1.5 - subunits. The current-voltage relationship and the amplitude of Nav1.5-mediated sodium peak current density were also not changed. ConclusionsOur findings illustrate that the direct implication of 14-3-3 proteins in regulating Nav1.5 is not evident in a transformed human kidney cell line tsA201. SummaryThis work shows that only 14-3-3{eta}, exhibits weak/transient interaction with Nav1.5, and does not modify its total protein expression, plasmalemmal trafficking, and basal biophysical properties of the whole-cell current. Furthermore, inhibition of endogenous 14-3-3/ligand interactions with difopein does not affect the dimerization of Nav1.5. Therefore, 14-3-3 proteins are suggested to be dispensable for the Nav1.5 regulation in a heterologous expression system.

biophysics↗

bioRxiv2021

Species-specific effects of cation channel TRPM4 small-molecule inhibitors

BackgroundThe Transient Receptor Potential Melastatin member 4 (TRPM4) gene encodes a calcium-activated non-selective cation channel expressed in several tissues. Mutations in TRPM4 have been reported in patients with different types of cardiac conduction defects. It is also linked to immune response and cancers, but the associated molecular mechanisms are still unclear. Thus far, 9-phenanthrol is the most common pharmacological compound used to investigate TRPM4 function. We recently identified two promising aryloxyacyl-anthranilic acid compounds (abbreviated CBA and NBA) inhibiting TRPM4. However, all afore-mentioned compounds were screened using assays expressing human TRPM4, whereas the efficacy on mouse TRPM4 has not been assessed. Mouse models are essential to investigate ion channel physiology and chemical compound efficacy. AimIn this study, we performed comparative electro-physiology experiments to assess the effect of these TRPM4 inhibitors on human and mouse TRPM4 channels heterologously expressed in TsA-201 cells. Methods and ResultsWe identified striking species-dependent differences in TRPM4 responses. NBA inhibited both human and mouse TRPM4 currents when applied intracellularly and extracellularly using excised membrane patches. CBA inhibited human TRPM4, both intracellularly and extracellularly. Unexpectedly, the application of CBA had no inhibiting effect on mouse TRPM4 current when perfused on the extracellular side. Instead, it increased mouse TRPM4 current at negative holding potentials. In addition, CBA on the intracellular side altered the outward rectification component of the mouse TRPM4 current. Application of 9-phenanthrol, both intracellularly and extracellularly, inhibited human TRPM4. For mouse TRPM4, 9-phenanthrol perfusion led to opposite effects depending on the site of application. With intracellular 9-phenanthrol, we observed a tendency towards potentiation of mouse TRPM4 outward current at positive holding potentials. ConclusionAltogether, these results suggest that pharmacological compounds screened using "humanised assays" should be extensively characterised before application in in vivo mouse models.

pharmacology and toxicology↗

bioRxiv2020

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 N-terminal domain (NTD): Y87C-here newly identified-, R104W and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.

physiology↗