Biology subjects

Lombardo, V. A.

Publications and source records attributed to Lombardo, V. A..

3 recordsLinked to original sources

bioRxiv2026

STARD3 mediates non-vesicular cholesterol transport in Caenorhabditis elegans

Cholesterol transport plays a pivotal role in regulating development and metabolism in Caenorhabditis elegans, a sterol-auxotrophic organism. Here, we identify the nematode cholesterol-binding protein STARD3 and provide structural and functional evidence for its role in non-vesicular sterol mobilization. Using biophysical and high-resolution structural methods, we show that the START domain (Ce-START) of C. elegans STARD3 binds cholesterol with high affinity and adopts a fold conserved with its human ortholog. Crystal structures of Ce-START in both apo and cholesterol-bound forms reveal key determinants of sterol recognition and conformational changes upon ligand binding. Functional analysis of a C. elegans stard3 knockout strain demonstrates that STARD3 is essential for cholesterol trafficking under sterol-limited conditions and that it genetically interacts with the NPC1/NPC2 pathway to sustain cholesterol mobilization. Collectively, these results establish STARD3 as a crucial cholesterol transporter in C. elegans and underscore the evolutionary conservation of START-domain proteins, reinforcing the utility of C. elegans as a model for studying intracellular cholesterol dynamics.

biophysics↗

bioRxiv2021

Unsaturated fatty acids profiling in live C. elegans using real-time NMR spectroscopy

Unsaturated fatty acids (UFAs) impact central cellular process in animals such as membrane function, development and disease. Perturbations of UFAs homeostasis contribute to the onset of metabolic, cardiovascular and neurodegenerative disorders. Nevertheless, links between lipid desaturation fluctuations and the dynamics of mono and polyunsaturated fatty acid synthesis in live animal physiology are poorly understood. To advance in the understanding of this process, we decided to study de novo UFAs synthesis with the highest resolution possible in live Caenorhabditis elegans. Conventional lipid analysis in this organism involves solvent extraction procedures coupled with analytical techniques such as chromatography and/or mass spectrometry. These methodologies are destructive and prevent the access of information, linking in vivo UFA dynamics and functionality. To overcome these limitations, we used uniform 13C isotope labeling and real-time 2D heteronuclear NMR spectroscopy in live C. elegans to identify their UFA compositions and the dynamic response of these fatty acids during cold adaptation. Our methodology allowed us to monitor in real time the upregulation of UFA synthesis when ambient temperature is decreased. The analysis of UFAs synthesis in worms lacking the adiponectin receptor AdipoR2 homolog PAQR-2 during a temperature drop supports the pivotal role of this protein in low temperature adaptation and survival. Our results provide new insights about the environmental regulation of UFAs and establish methodological benchmarks for future investigations of fatty acid regulation under experimental conditions that recapitulate human diseases.

biophysics↗

bioRxiv2020

An NMR-based biosensor to measure stereo-specific methionine sulfoxide reductase (MSR) activities in vitro and in vivo.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments that range from native cell lysates to zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways. Our approach further extends high-resolution in-cell NMR measurements of exogenously delivered biomolecules to an entire multicellular organism.

biophysics↗