Biology subjects

Quadroni, M.

Publications and source records attributed to Quadroni, M..

8 recordsLinked to original sources

bioRxiv2026

Endogenous lipid droplets exhibit profound proteomic and lipidomic differences across neural stem cell states

Lipid droplets (LDs) are protein-coated organelles that store neutral lipids and regulate diverse cellular processes beyond energy metabolism. In neural stem/progenitor cells (NSPCs), LD abundance and morphology vary across cellular states, yet whether LD molecular composition is similarly state-dependent remains unknown. Here, we define the first endogenous LD proteome and lipidome atlas of NSPCs and their progeny. State-resolved analyses reveal extensive differences in both LD-associated proteins and stored lipids, allowing for identification of LD signatures that distinguish quiescent and proliferative states, and uncovering selective enrichment of numerous proteins on quiescent NSPC LDs. Functional interrogation of one such protein, CIDEB, showed that its knockdown alters LD morphology and induces senescence-associated transcriptional programs, implicating CIDEB in the maintenance of NSPC quiescence. These findings establish LDs as dynamically specialized organelles in NSPCs and their progeny and provide a resource for investigating LD-mediated regulation of stem cell state and lineage progression.

cell biology↗

bioRxiv2025

Pus7 mutation links tRNA dysregulation to aggressive behavior through activation of the integrated stress response and glycolytic reprogramming

Pseudouridine ({Psi}) is a prevalent RNA modification found in multiple RNA species. It is deposited by {Psi} synthases (Pus) and it can stabilize RNA structures. Patients carrying alterations in the Pus7 gene suffer from developmental delay, intellectual disability, microcephaly, hyperactivity and increased aggression levels. Here we show that the Pus7 mutation in human patient cells and in a Drosophila model is associated with a specific decrease of tRNA:Aspartate (tRNA-Asp) levels, which leads to slow decoding at Aspartate codons. This in turn activates the integrated stress response and induces a metabolic shift towards increased glycolysis and reduced mitochondrial respiration. Elevating tRNA-Asp expression, inhibiting the integrated stress response or dampening the glycolytic pathway is sufficient to rescue the aggressiveness phenotype, demonstrating the involvement of the tRNA-Asp-ISR-glycolysis axis in this behavior. Together our data provide new insights into the molecular defects associated with the loss of Pus7 and suggest potential new avenues for therapeutic treatment.

genetics↗

bioRxiv2022

A database of accurate electrophoretic migration patterns for human proteins in cell lines.

Native molecular weight (MW) is one of the defining features of proteins. Denaturing gel electrophoresis (SDS-PAGE) is a very popular technique for separating proteins and determining their MW. Coupled with antibody-based detection, SDS-PAGE is widely applied for protein identification and quantitation. Yet, electrophoresis is poorly reproducible and the MWs obtained are often inaccurate. This hampers antibody validation and negatively impacts the reliability of western blot data, resulting worldwide in a considerable waste of reagents and labour. To alleviate these problems there is a need to establish a database of reference MWs measured by SDS-PAGE. Using mass spectrometry as an orthogonal detection method, we acquired electrophoretic migration patterns for approximately 10000 human proteins in five commonly used cell lines. We applied a robust internal calibration of migration to determine accurate and reproducible molecular weights. This in turn allows merging replicates to increase accuracy, but also enables comparing different cell lines. Mining of the data obtained highlights structural factors that affect migration of distinct classes of proteins. We also show that the information produced recapitulates known post-translational modifications and differential splicing and can be used to formulate hypotheses on new or poorly known processing events. The full information is freely accessible as a web resource through a user friendly graphical interface (https://pumba.dcsr.unil.ch/). We anticipate that this database will be useful to investigators worldwide for troubleshooting western blot experiments, but could also contribute to the characterization of human proteoforms.

biochemistry↗

bioRxiv2022

Translational reprogramming in response to accumulating stressors ensures critical threshold levels of Hsp90 for mammalian life

The cytosolic molecular chaperone Hsp90 is essential for eukaryotic life1, 2. It is involved in multiple branches of proteostasis2, 3, and as a molecular capacitor in morphological evolution4. Although reduced Hsp90 levels cause phenotypic variations5, 6 and correlate with aging7, whether eukaryotic cells and organisms can tune the basal Hsp90 protein levels to alleviate physiologically accumulated stress is unknown. To begin to explore this question, we investigated whether and how mice adapt to the deletion of three out of four alleles encoding cytosolic Hsp90, one Hsp90{beta} allele being the only remaining one. While the vast majority of such mouse embryos die during gestation, survivors apparently manage to increase their Hsp90{beta} protein to at least wild-type levels. Further mechanistic studies revealed an internal ribosome entry site in the 5UTR of the Hsp90{beta} mRNA allowing translational reprogramming to compensate for the genetic loss of Hsp90 alleles and in response to stress. We found that the minimum amount of total Hsp90 that is required to support viability of mammalian cells and organisms is 50-70% of what is normally there. Those that fail to maintain a threshold level are subject to accelerated senescence, proteostatic collapse, and ultimately death. Therefore, considering that Hsp90 levels can be reduced [≥]100-fold in the unicellular budding yeast, critical threshold levels of Hsp90 have been markedly increased during eukaryotic evolution. The incompressible part of the steady-state levels of Hsp90 may have increased to accommodate the ever-growing complexity of the proteome8 on the path towards mammals.

molecular biology↗

bioRxiv2021

TRAIP regulates DNA double-strand break-induced ATM activation

DNA double-strand breaks (DSBs) affect cell survival and genomic integrity. They are repaired by a highly coordinated process called the DNA damage response. Here, we report that the ubiquitously expressed nucleolar E3 ubiquitin ligase TRAF-interacting protein (TRAIP), previously shown to regulate the spindle assembly checkpoint, has an essential role during the DNA damage response. A biotinylation proximity screening assay (BioID) identified Ku80, Ku70, SMARCA5 (SNF2H) and DNA-PKcs as novel TRAIP interactors. Co-immunoprecipitations demonstrated that the interaction of TRAIP with Ku80 was transiently increased while the one with SMARCA5 was strongly decreased after treatment of HeLa cells with neocarzinostatin (NCS). Treatment of fibroblasts from a microcephalic primordial dwarfism patient carrying a hypomorphic TRAIP mutation or shRNA-mediated knockdown of TRAIP in HeLa cells with NCS impaired the activation of ataxia-telangiectasia mutated (ATM), a protein kinase crucial for the DNA damage response. As consequence, the maintenance of {gamma}H2AX and Chk2-T68 phosphorylation, two downstream targets of ATM, was significantly abrogated after NCS-inflicted DSBs. DNA repair assays showed that TRAIP inhibits incorrect end utilization during non-homologous end joining. These observations highlight TRAIP as novel regulator of ATM activity in DNA damage signaling.

cell biology↗

bioRxiv2020

Human POT1 Prevents Severe Telomere Instability Induced by Homology Directed DNA Repair

The evolutionarily conserved POT1 protein binds single stranded G-rich telomeric DNA and has been implicated in contributing to telomeric DNA maintenance and the suppression of DNA damage checkpoint signaling. Here, we explore human POT1 function through genetics and proteomics discovering that the complete absence of POT1 leads to severe telomere maintenance defects that had not been anticipated from previous depletion studies. Conditional deletion of POT1 in HEK293E cells gives rise to rapid telomere elongation and length heterogeneity, branched telomeric DNA structures, telomeric R-loops and telomere fragility. We determine the telomeric proteome upon POT1-loss implementing an improved telomeric chromatin isolation protocol. We identify a large set of proteins involved in nucleic acid metabolism that engage with telomeres upon POT1-loss. Inactivation of the homology directed repair machinery suppresses POT1-loss mediated telomeric DNA defects. Our results unravel as major function of human POT1 the suppression of telomere instability induced by homology directed repair.

molecular biology↗

bioRxiv2019

The Hsp70/Hsp90 Co-Chaperone Hop/Stip1 Shifts the Proteostatic Balance from Folding Towards Degradation

Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70/Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures an alternate proteostatic equilibrium. Thus, cells may act on Hop to shift the proteostatic balance between folding and degradation.

cell biology↗

bioRxiv2018

Bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates preferentially by HslUV proteolysis

Whereas in eukaryotic cells, the Hsp90s are profusely-studied molecular chaperones controlling protein homeostasis together with Hsp70s, in bacteria, the function of Hsp90 (HtpG) and its collaboration with Hsp70 (DnaK) remains unknown. To uncover physiological processes depending on HtpG and DnaK, we performed comparative quantitative proteomic analyses of insoluble and total protein fractions from unstressed wild type E. coli, and from knockout mutants{Delta} dnaKdnaJ ({Delta}KJ),{Delta} htpG ({Delta}G) and{Delta} dnaKdnaJ{Delta}htpG ({Delta}KJG) and compared their growth rates under heat-stress also with{Delta} dnaKdnaJ{Delta}hslV. Whereas, expectedly, mutant {Delta}G showed no proteomic differences with wild-type, {Delta}KJ expressed more chaperones, proteases and ribosomes and dramatically less metabolic and respiratory enzymes. Unexpectedly, we found that {Delta}KJG showed higher levels of metabolic and respiratory enzymes and both {Delta}KJG and{Delta} dnaKdnaJ{Delta}hslV grew better at 37oC than {Delta}KJ. The results indicate that bacterial Hsp90 mediates the degradation of aggregation-prone Hsp70-Hsp40 substrates, preferably by the HslUV protease.\n\nSignificance statementThe molecular chaperones Hsp70 and Hsp90 are among the most abundant and well-conserved proteins in all realms of life, forming together the core of the cellular proteostasis network. In eukaryotes, Hsp90 functions in collaboration with Hsp70; we studied this collaboration in E. coli, combining genetic studies with label-free quantitative proteomics in which both protein abundance and protein solubility were quantified. Bacteria lacking Hsp70 (DnaK) and its co-chaperone DnaJ ({Delta}dnaKdnaJ) grew slower and contained significantly less key metabolic and respiratory enzymes. Unexpectedly, an additional deletion of the Hsp90 (htpG) gene partially restored the WT phenotype. Deletion of the HslV protease in the {Delta}dnaKdnaJ background also improved growth, suggesting that bacterial Hsp90 mediates the degradation of Hsp70 substrates, preferentially through HslV.\n\nAt 37oC{Delta} dnaKdnaJ E. coli mutants grow slower than wild type cells. Quantitative proteomics shows that compared to wild type cells,{Delta} dnaKdnaJ cells grown at 30oC contain significantly less key metabolic and respiratory enzymes. Unexpectedly, deletion of the HtpG gene in the {Delta} dnaKdnaJ background ameliorates growth at 37oC and partially restores the cellular levels of some metabolic and respiratory enzymes.

molecular biology↗