Biology subjects

Binolfi, A.

Publications and source records attributed to Binolfi, A..

7 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↗

bioRxiv2024

An inducible and reversible system to regulate unsaturated fatty acid biosynthesis in C. elegans

Unsaturated fatty acids (UFAs) play crucial roles in various physiological and pathological processes. In animals, these lipids are synthesized from saturated fatty acids through the action of delta 9 ({Delta}9) desaturases. In C. elegans, three {Delta}9 desaturases are encoded by the genes fat-5, fat-6, and fat-7. The presence of multiple {Delta}9 desaturases has posed a significant challenge in developing a rapid and efficient approach to control UFA production in C. elegans and other model organisms. Utilizing the auxin-inducible degradation system, we specifically targeted the C. elegans fat-7 gene, responsible for the major stearoyl-CoA desaturase (SCD), while deleting fat-5 and fat-6. This design resulted in a strain that can be reversibly depleted of UFAs in the cells of interest. Conditional depletion in all somatic cells exhibited a pronounced auxin-dependent defect in UFA production. Using this system, we uncovered an essential requirement for de novo UFA production during L1 and L2 stage. Moreover, our results support a direct connection between UFA levels, fat storage and increased lipid turnover. This system will enable further studies exploring the cellular and physiological consequences of impairing UFA biosynthesis at different developmental stages or in specific tissues. SummaryUnsaturated fatty acids (UFAs) are essential for life. In animals, UFAs are synthesized by {Delta}9 desaturase enzymes. Caenorhabditis elegans possesses three {Delta}9 desaturase genes: fat-5, fat-6, and fat-7. We engineered a strain where fat-7 can be reversibly switched off while fat-5 and fat-6 are deleted, allowing precise control of UFA levels throughout the life cycle. Our findings demonstrate the critical role of UFA biosynthesis in early development and its direct link to fat storage and lipid turnover. This strain enables the study of UFA-related physiological and pathological processes in animals.

genetics↗

bioRxiv2023

G-quadruplexes regulate miRNA biogenesis in live zebrafish embryos

RNA guanine quadruplexes (G4s) regulate RNA functions, metabolism, and processing. G4s formed within precursors of microRNAs (pre-miRNAs) may impair pre-miRNAs maturation by Dicer, thus repressing mature miRNA biogenesis. As miRNAs are essential for proper embryonic development, we studied the role of G4s on miRNA biogenesis in vivo during zebrafish embryogenesis. We performed a computational analysis on zebrafish pre-miRNAs to find putative G4 forming sequences (PQSs). The precursor of the miRNA 150 (pre-miR-150) was found to contain an evolutionarily conserved PQS formed by three G-tetrads and able to fold in vitro as G4. MiR-150 controls the expression of myb, which shows a well-defined knock-down phenotype in zebrafish developing embryos. We microinjected zebrafish embryos with in vitro transcribed pre-miR-150 synthesized using either GTP (G-pre-miR-150) or 7-Deaza-GTP, a GTP analogue unable to form G4s (7DG-pre-miR-150). Compared to embryos injected with G-pre-miR-150, embryos injected with 7DG-pre-miR-150 showed higher levels of miRNA 150 (miR-150) and lower levels of myb mRNA and stronger phenotypes associated with myb knock-down. The incubation of pre-miR-150 prior to the injection with the G4 stabilizing ligand pyridostatin (PDS) reverted gene expression variations and rescued the phenotypes related to myb knock-down. Overall, results suggest that the G4 formed in pre-miR-150 functions in vivo as a conserved regulatory structure competing with the stem-loop structure necessary for miRNA biogenesis.

molecular biology↗

bioRxiv2022

Genetic variations in G-Quadruplex forming sequences affect the transcription of human disease-related genes

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4s). G4s folded in proximal promoter regions (PPR) are associated either with positive or negative transcriptional regulation. Given that single nucleotide variants (SNVs) affecting G4 folding (G4-Vars) may alter gene transcription, and that SNVs are associated with the human diseases onset, we undertook a comprehensive study of the G4-Vars genome-wide (G4-variome) to find disease-associated G4-Vars located into PPRs. We developed a bioinformatics strategy to find disease-related SNVs located into PPRs simultaneously overlapping with putative G4-forming sequences (PQSs). We studied five G4-Vars disturbing in vitro the folding and stability of the G4s located into PPRs, which had been formerly associated with sporadic Alzheimers disease (GRIN2B), a severe familiar coagulopathy (F7), atopic dermatitis (CSF2), myocardial infarction (SIRT1), and deafness (LHFPL5). Results obtained in cellulo for GRIN2B and F7 suggest that the G4 disruption due to the identified G4-Vars affect the transcription and are responsible for the mentioned diseases. Collectively, data suggest that G4-Vars may account for the different susceptibilities to human genetic diseases onset, and could be novel targets for diagnosis and drug design in precision medicine.

molecular biology↗

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

Megadalton-sized dityrosine aggregates of α-synuclein retain high degrees of structural disorder and internal dynamics.

Heterogeneous aggregates of the human protein -synuclein (Syn) are abundantly found in Lewy body inclusions of Parkinsons disease patients. While structural information on classical Syn amyloid fibrils is available, little is known about the conformational properties of disease-relevant, non-canonical aggregates. Here, we analyze the structural and dynamic properties of megadalton-sized dityrosine adducts of Syn that form in the presence of reactive oxygen species and cytochrome c, a proapoptotic peroxidase that is released from mitochondria during sustained oxidative stress. In contrast to canonical cross-{beta} amyloids, these aggregates retain high degrees of internal dynamics, which enables their characterization by solution-state NMR spectroscopy. We find that intermolecular dityrosine crosslinks restrict Syn motions only locally whereas large segments of concatenated molecules remain flexible and disordered. Indistinguishable aggregates form in crowded in vitro solutions and in complex environments of mammalian cell lysates, where relative amounts of free reactive oxygen species rather than cytochrome c are rate limiting. We further establish that dityrosine adducts inhibit classical amyloid formation by maintaining Syn in its monomeric form and that they are non-cytotoxic despite retaining basic membrane-binding properties. Our results suggest that oxidative Syn aggregation scavenges cytochrome cs activity into the formation of amorphous, high molecular-weight structures that may contribute to aggregate diversity in Lewy body deposits.

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↗