Biology subjects

Perez-Gil, J.

Publications and source records attributed to Perez-Gil, J..

3 recordsLinked to original sources

bioRxiv2025

Vehiculation and functional delivery of lipophilic therapeutics and antibiotics via pulmonary surfactant in a lung-on-chip model

Pulmonary surfactant forms a dynamic proteolipid thin-film at the alveolar air-liquid interface. It is both a barrier to but also a "last-mile" carrier for inhaled particulate matter to the distal lung. The role of vehiculation in pulmonary delivery of inhaled therapeutics remains poorly understood, as it cannot be easily studied in animal models, and is not recapitulated in liquid cell culture. Here we adapted a thin-film bridge (TFB) delivery method from vehiculation experiments in acellular models to a lung-on-chip (LoC) platform under breathing-like stretch. Using confocal live-imaging, we demonstrate vehiculation and cellular uptake of fluorescently-labelled cargoes such as Tacrolimus or Beclomethasone. We systematically compared TFB vehiculation to existing in vitro delivery approaches. TFB vehiculation promoted sustained retention of functionally effective Tacrolimus, strong co-localization with surfactant lipids, and accumulation in macrophages in human LoCs reconstituted with in vitro-differentiated alveolar macrophage-like cells. This contrasted with the unphysiological dominant uptake of formulations in liquid by alveolar epithelial cells. Finally, in proof-of-concept experiments, TFB-vehiculated bedaquiline effectively supressed subsequent growth of Mycobacterium tuberculosis in a prophylactic manner. Overall, our work demonstrates an approach for studying drug vehiculation in models of the alveolar interface in vitro, and the feasibility of surfactant-containing therapeutic formulations for direct and effective pulmonary delivery.

bioengineering↗

bioRxiv2023

Multipotent ubiquitin/ubiquitin-like deconjugation activity of the rhizobial effector NopD

Post-translational modification of proteins by ubiquitin-like modifiers (UbL), such as SUMO, ubiquitin or Nedd8, contribute to regulate most pathways in the cell. Protein modification can be reversed by dedicated UbL deconjugating proteases families. During bacterial infection, a repertoire of effector proteins, including deconjugating proteases, are released to perturb the host cell defense to favor bacterial survival. NopD, an effector protein from rhizobia involved in nodule symbiosis in legumes, possesses deSUMOylation, and unexpectedly, deubiquitination and deNeddylation activities. Here we show two crystal structures of Bradyrhizobium NopD in complex with either Arabidopsis SUMO2 and ubiquitin at 1.50 or 1.94 [A], respectively. Despite their low sequence identity, SUMO and ubiquitin interact with a similar NopD interface by means of a unique loop insertion in the NopD sequence. Biochemical and infiltrations in tobacco leaves reveal specific residues that discriminate between deubiquitination and deSUMOylation. These unusual multiple deconjugating activities against SUMO, ubiquitin and Nedd8, represent a paradigmatic example of an optimized protease to perturb distinct UbL post-translational modifications during host cell infection. Significance StatementDuring bacterial infection, including rhizobia symbiosis in legume plants to fix atmospheric nitrogen, a set of effector proteins, such as ubiquitin/ubiquitin-like deconjugating proteases, are released to perturb the host cell defense to favor bacterial survival. We have discovered that the rhizobial effector protein, NopD, encompasses triple deconjugation activities against SUMO, ubiquitin and Nedd8. Structural analysis of NopD in complex with SUMO and ubiquitin reveals the presence of a loop insertion in the protease-substrate interface to allow this multiple substrate binding capability. Such unusual deconjugating activities in NopD for ubiquitin, SUMO and Nedd8 modifiers, represent a paradigmatic example of an optimized protease domain to perturb distinct UbL post-translational modifications during host cell infection.

biochemistry↗

bioRxiv2022

Allosteric feedback inhibition of deoxy-D-xylulose-5-phosphate synthase involves monomerization of the active dimer.

Isoprenoids are a very large and diverse family of metabolites required by all living organisms. All isoprenoids derive from the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are produced by the methylerythritol 4-phosphate (MEP) pathway in bacteria and plant plastids. Understanding the regulation of the MEP pathway, probably the main metabolic pathway elucidated in this century, is a must for the rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems. It has been reported that IPP and DMAPP feedback regulate the activity of deoxyxylulose 5-phosphate (DXS), a dimeric enzyme catalyzing the main flux-controlling step of the MEP pathway. Here we provide experimental insights on the underlying mechanism. Our data show that direct allosteric binding of IPP and DMAPP to bacterial and plant DXS promotes monomerization of the enzyme. This allows a fast response to a sudden increase or decrease in IPP/DMAPP supply by rapidly shifting the dimer-monomer equilibrium accordingly. DXS monomers expose hydrophobic domains that are hidden in the dimer, resulting in aggregation and eventual degradation. Removal of monomers that would otherwise be available for dimerization and enzyme reactivation appears as a more drastic response in case of persistent IPP/DMAPP overabundance (e.g., by a blockage in their conversion to downstream isoprenoids). Our model provides a mechanistic explanation of how IPP and DMAPP supply can be adapted to changes in their demand and it also explains the changes in DXS protein levels observed after long-term interference of the MEP pathway flux. Significance StatementIsoprenoids are a vast family of organic compounds with essential roles in respiration, photosynthesis, photoprotection, membrane structure, and signaling. Many of them have great economic and nutritional relevance as pigments, aromas, drugs or phytonutrients. Despite their functional and structural diversity, they all derive from the same five-carbon precursors. We show that these precursors feedback-regulate their own synthesis in bacteria and plant plastids by allosterically shifting the dimer:monomer equilibrium of the enzyme that catalyzes the first step of their biosynthetic pathway towards the inactive monomeric form. This evolutionary conserved mechanism allows for both short-term (immediate) and long-term (sustained) control of the pathway flux, and its manipulation could be critical for the rational engineering of high-value isoprenoid products in bacterial and plant systems.

biochemistry↗