Biology subjects

Vidilaseris, K.

Publications and source records attributed to Vidilaseris, K..

5 recordsLinked to original sources

bioRxiv2025

Novel Binding Partners of the Vacuolar Transporter Chaperone (VTC) complex in Acidocalcisomes of Leishmania tarentolae

Acidocalcisomes are evolutionarily conserved acidic organelles that are rich in cations and inorganic phosphate, primarily polyphosphates. In kinetoplastid parasites, acidocalcisomes and their polyphosphate content are essential for osmoregulation and environmental adaptation during host switching. In this organelle, polyphosphate is synthesised and transported to the lumen by the vacuolar transporter chaperone (VTC) complex. Interestingly, unlike yeast VTC, which has five components, only two have been observed in kinetoplastids: Vtc1, which contains only a transmembrane domain and Vtc4, which, in addition to a transmembrane domain, also consists of SPX and catalytic domains. In this study, we used proximity-dependent biotinylation (BioID) in Leishmania tarentolae to identify proteins located close to the VTC complex. The complex was found near several known acidocalcisomal proteins, including membrane-bound pyrophosphatase (mPPase), vacuolar-type H-ATPase (V-H+-ATPase), Ca{superscript 2}-transporting P-type ATPase (Ca2+-ATPase), zinc transporter (ZnT), and palmitoyl acyltransferase 2 (PAT2). Importantly, this approach revealed three novel VTC binding partners (VBPs) that colocalise and interact with the complex in acidocalcisomes, as confirmed by confocal microscopy, pulldown assays, and AlphaFold3 structural predictions. Together, our results expand the acidocalcisome interactome and suggest that the newly identified VBPs may contribute to the structural organisation and regulatory function of the VTC complex in phosphate homeostasis of kinetoplastid parasites. Author summaryProtozoan parasites such as Leishmania and Trypanosoma cause serious diseases affecting millions of people worldwide. To better understand how these parasites survive environmental changes during transmission between hosts, we studied a specialised organelle called the acidocalcisome, which stores polyphosphates and helps regulate stress responses. In this work, we used the non-pathogenic Leishmania tarentolae as a safe and cost-effective model that shares key cellular features with disease-causing species. Using a combination of CRISPR-Cas9 genome editing, proximity-based labelling (BioID), confocal microscopy, pulldown assays and AlphaFold3 structure prediction, we investigated the vacuolar transporter chaperone (VTC) complex, which synthesises and transports polyphosphate into the acidocalcisome lumen. Proximity proteomics identified several known proteins located near the VTC complex, and importantly, led us to discover three novel proteins that interact with it. These findings open new directions for exploring the organisation and regulation of the VTC complex in protozoan parasites. By revealing novel protein interactions, our study contributes to a deeper understanding of parasite biology and may help identify therapeutic targets for treating neglected tropical diseases.

biochemistry↗

bioRxiv2025

Expression, purification and preliminary pharmacological characterization of the Plasmodium falciparum membrane-bound pyrophosphatase type 1

Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n-dodecyl-{beta}-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5- a]pyrimidine-based TmPPase inhibitors, retained micromolar IC50 activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

biochemistry↗

bioRxiv2024

Conformational dynamics and asymmetry in multimodal inhibition of membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H+/Na+ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na+ can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

biochemistry↗

bioRxiv2022

Functional and structural asymmetry suggest a unifying principle for catalysis in integral membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase, and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this we propose, for the first time, a unified and testable model for ion pumping, hydrolysis, and energy-coupling in all M-PPases, including those that pump both Na+ and H+.

biochemistry↗

bioRxiv2018

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and protist parasites, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first non-phosphorous allosteric inhibitor (Ki of 1.8 {+/-} 0.3 M) of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure at 3.7 [A] resolution together with the substrate analogue imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of {beta}-strand 1-2 during pumping, and thus preventing the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provide the first clear demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

molecular biology↗