Biology subjects

Vazquez-Ibar, J. L.

Publications and source records attributed to Vazquez-Ibar, J. L..

3 recordsLinked to original sources

bioRxiv2026

Probing the interaction between the WDR41 7CD loop of the C9ORF72 complex and purified PQLC2

The lysosomal cationic amino acid transceptor PQLC2 integrates transport with receptor-like signaling roles by recruiting the C9ORF72/SMCR8/WDR41 (CSW) complex in response to cellular cationic amino acid depletion. CSW recruitment depends on the interaction of PQLC2 with a disordered loop (7CD loop) extending from the WDR41 {beta}-propeller. Defining the molecular basis of this interaction is therefore essential for understanding how PQLC2 connects cellular amino acid availability to downstream signaling. Here, we used site-directed electron paramagnetic resonance (EPR) spectroscopy to characterize the interaction between purified PQLC2 and a synthetic WDR41 7CD loop peptide containing a site-specific nitroxide spin label. Using a consensus mutagenesis strategy, we stabilized detergent-purified PQLC2 and found that it assembles as a homotrimer, similar to other PQ-loop transporters. EPR experiments demonstrated the direct and specific association of the 7CD loop with PQLC2, and highlighted the key role of the the central region of the 7CD loop to establish this interaction via conserved aromatic residues. Spin labels introduced at different positions further provided local structural and dynamic information of the 7CD loop upon PQLC2 binding. The data supports a model in which the central region of the loop is anchored within the cytosolic cavity of PQLC2, whereas the flanking regions, which are dispensable for binding, retain substantial conformational flexibility. Together, we provide a direct biochemical evidence for PQLC2/WDR41 7CD loop recognition, a step forward towards understanding the transceptor mechanism of PQLC2.

biochemistry↗

bioRxiv2023

Detection of tumor-derived extracellular vesicles interactions with immune cells is dependent on EV-labelling methods

Cell-cell communication within the complex tumor microenvironment is critical to cancer progression. Tumor-derived extracellular vesicles (TD-EVs) are key players in this process. They can interact with immune cells and modulate their activity, either suppressing or activating the immune system. Understanding the interactions between TD-EVs and immune cells is essential for understanding immune modulation by cancer cells. Fluorescent labelling of TD-EVs is a method of choice to study such interaction. This work aims to determine the impact of EV labelling methods on the detection of EV interaction and capture by the different immune cell types within human Peripheral Blood Mononuclear Cells (PBMCs), analyzed by imaging flow cytometry and multicolor spectral flow cytometry. EVs released by the triple-negative breast carcinoma cell line MDA-MB-231 were labeled either with the lipophilic dye MemGlow-488 (MG-488), with Carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE), or through expression of a MyrPalm-superFolder GFP (sfGFP) that incorporates into EVs during their biogenesis using a genetically engineered cell line. Our results showed that these different labeling strategies, although analyzed with the same techniques, led to diverging results. While MG-488-labelled EVs incorporate in all cell types, CFSE-labelled EVs are restricted to a minor subset of cells and sfGFP-labelled EVs are mainly detected in CD14+ monocytes which are the main uptakers of EVs and other particles, regardless of the labeling method. Moreover, MG-488-labeled liposomes behaved similarly to MG-488 EVs, highlighting the predominant role of the labelling strategy on the visualization and analysis of TD-EVs uptake by immune cell types. Consequently, the use of different EV labeling methods has to be considered as they can provide complementary information on various types of EV-cell interaction and EV fate.

cell biology↗

bioRxiv2020

ATP2, the essential P4-ATPase of malaria parasites, catalyzes lipid-dependent ATP hydrolysis in complex with a Cdc50 β-subunit

Efficient mechanisms of lipid transport are indispensable for the Plasmodium malaria parasite along the different stages of its intracellular life-cycle. Gene targeting approaches have recently revealed the irreplaceable role of the Plasmodium-encoded type 4 P-type ATPases (P4-ATPases or lipid flippases), ATP2, together with its potential involvement as antimalarial drug target. In eukaryotic membranes, P4-ATPases assure their asymmetric phospholipid distribution by translocating phospholipids from the outer to the inner leaflet. As ATP2 is a yet putative transporter, in this work we have used a recombinantly-produced P. chabaudi ATP2, PcATP2, to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 heterodimerizes with two of the three Plasmodium-encoded Cdc50 proteins: PcCdc50B and PcCdc50A, indispensable partners for most P4-ATPases. Moreover, the purified PcATP2/PcCdc50B complex catalyses ATP hydrolysis in the presence of phospholipids containing either phosphatidylserine, phosphatidylethanolamine or phosphatidylcholine head groups, and that this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work provides the first study of the function and quaternary organization of ATP2, a promising antimalarial drug target candidate.

biochemistry↗