Biology subjects

Gaetani, M.

Publications and source records attributed to Gaetani, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Transcriptomic-guided compound prioritization and proteomics validation for HNRNPU deficiency identify signalling correction

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) deficiency is a rare genetic cause of neurodevelopmental disorders (NDDs) lacking targeted therapies. Here, we developed a transcriptomic-guided compound prioritization pipeline using Connectivity Map (CMap) analysis on multi-model transcriptomic signatures from HNRNPU-deficient human cells and mouse models. Ten compounds were selected through manual curation and functionally screened in patient-derived HNRNPU-deficient neuroepithelial stem (NES) cells with earlier observed cellular phenotypes. Two of the compounds, AS601245 and Lenalidomide, significantly reduced the elevated neural progenitor population during differentiation, and their combination further decreased primary cilia incidence, indicating partial rescue of the patient-specific cellular phenotypes. To understand the mechanisms underlying the partial rescue, we employed proteome integral solubility alteration (PISA) and expression proteomics. PISA assay identified TMEM150C and GSK3A as proximal targets of combined treatment. Additionally, we observed reversal of multiple biological pathways including downregulation of Wnt signalling and upregulation of mitochondrial pathways and transmembrane proteins. Altogether, we established a computational-experimental pipeline for transcriptomic-guided drug repurposing for a monogenic NDD, and demonstrated that the network-level modulation partially rescues the delayed neural differentiation in HNRNPU-deficient neural cells.

molecular biology↗

bioRxiv2026

Ritonavir-Induced Cellular Stress Alters Viral HBs Glycoprotein Biogenesis and Production of Infectious Hepatitis D Virions

Chronic co-infections by HBV and its satellite virus HDV are associated with a high risk of progression to cirrhosis and liver cancer, and therapeutic options for achieving a cure are still unsatisfactory. HBs is the main surface glycoprotein of both viruses, and is also massively secreted by infected hepatocytes in the form of empty subviral particles which suppress the host immune responses. This makes HBs an attractive target to develop therapeutic strategies. Here, we took advantage of the known interaction between the Large form HDV antigen (HDAg-L) and the small form of HBs (S-HBs) to develop a non-infectious, minimalistic reporter assay for the assembly and secretion of HDV particles. By screening the existing pharmacopeia for drugs that could interfere with S-HBs and HDAg-L co-secretion, we found that ritonavir and other Cytochrome P450 inhibitors affect the biogenesis of HBs and impair the production of infectious HDV virions. Mechanistically, we established that these drugs induce oxidative stress which dysregulates disulfide bond formation in the endoplasmic reticulum. As a consequence, the production of HBs, which depends on a dense network of disulfide bonds, is markedly affected as evidenced by an abnormal glycosylation profile, altered antigenic properties, and a poor expression of the largest form of HBs (L-HBs) which is essential to virus entry into target cells. This is associated with induction of the unfolded protein response, with the upregulation of CHOP/DDIT3 and key enzymes involved in the synthesis of the reducing metabolite glutathione (PHGDH, SHMT2, MTHFD2). Overall, our results indicate that alterations in redox homeostasis significantly impact HBs biogenesis, and reveal a druggable pathway that could be exploited to eliminate HDV in chronically infected patients. IMPACT AND IMPLICATIONSMore effective therapies are still needed to achieve a functional cure in patients chronically co-infected by HBV and HDV. In this study, we discovered that ritonavir, along with other cytochrome P450 inhibitors, can affect the production of infectious HDV particles in human hepatocyte cultures. Mechanistically, ritonavir induces oxidative stress and the unfolded protein response in the endoplasmic reticulum, thereby altering the biogenesis of HBs, the surface glycoprotein of both viruses. This work highlights the potential benefit and mechanism of action of ritonavir and related molecules in the treatment of co-infected patients.

microbiology↗

bioRxiv2025

NUDT5 regulates the global efficacy of nucleoside analog drugs by coordinating purine synthesis and PRPP allocation

Cancer cells require nucleotides to meet proliferation demands, which has been effectively exploited by nucleoside analog (NA) drugs. Despite their success, we still do not completely understand factors dictating their anticancer effect. Here, we report that a NUDT5 scaffolding function indirectly regulates the global efficacy of anti-cancer NA drugs. Mechanistically, PROTAC- or RNAi-mediated NUDT5 depletion, not inhibition, desensitizes cells to 6-thioguanine (6TG) and other NA drugs proportionally with NUDT5 abundance. NUDT5-depleted cells appear locked into de novo purine biosynthesis (DNPB), thus impairing salvage of nucleotide precursors and NA drug activation. Specifically, NUDT5 interacts with phosphoribosyl amidotransferase (PPAT), the rate-limiting DNPB enzyme, to putatively regulate its activity, DNPB flux, and resultant phosphoribosyl pyrophosphate (PRPP) allocation. Collectively, these results suggest that NUDT5 controls NA drug efficacy by coordinating nucleotide synthesis and PRPP utilization, making it a potential biomarker of clinical efficacy and target for finetuning antimetabolite therapies. Statement of significanceNUDT5 non-enzymatically regulates the global efficacy of anti-cancer nucleoside analog drugs by interacting with PPAT to control DNPB and allocation of PRPP, thereby establishing it as a biomarker of clinical significance.

biochemistry↗

bioRxiv2025

Proteome integral solubility alteration assay for monitoring antibody-antigen cell interactions

The Proteome Integral Solubility Alteration (PISA) assay is commonly used to identify and characterize interactions between small molecules and the cellular proteome. In this study, a modified PISA approach identified tumor necrosis factor alpha (TNF) as the antigen of the monoclonal antibody infliximab in activated monocytes - uniquely among [~]8,000 cellular proteins. These results highlight the potential of PISA-based analysis not only for monitoring antibody-antigen interactions in therapeutic development, but also for identifying new antigens and characterizing the initial effector functions of antibodies within complex proteome environments. TeaserModified PISA pinpointed TNF- as Infliximabs antigen among 8000 proteins, highlighting broad potential for therapeutic antigen studies.

molecular biology↗

bioRxiv2025

Thioredoxin Reductase 1 inhibition triggers ferroptosis in KRAS-independent lung cancers

Lung cancers that harbor wild type KRAS (KRAS-WT) represent a molecularly diverse subset of tumors that often lack targeted therapeutic options. Using synthesized gold(I)-based inhibitors, a multi-omics approach, and functional validation, we identified Thioredoxin reductase 1 (TXNRD1), encoding as a selective vulnerability in KRAS-WT and oncogenic KRAS mutant (KM)-independent lung cancer (LC). Mechanistically, TRXR1 blockade induces ferroptosis through glutathione depletion, lipid reactive oxygen species (ROS) accumulation, and HMOX1-dependent iron overload in KRAS-WT LC both in vitro and in vivo. Furthermore, while KM LC cells are intrinsically resistant to TRXR1 inhibition, KMLC cells that acquire resistance to KRAS inhibitors (KRASi) undergo a redox shift that renders them sensitive to TRXR1 inhibition, uncovering a potential novel therapeutic vulnerability in KRASi-refractory tumors. These findings establish TRXR1 as a targetable redox checkpoint in KRAS-WT and KRASi-resistant lung cancers and support further development of TRXR1 inhibitors.

cancer biology↗

bioRxiv2025

High-ratiO partiaL proteolysiS with carriER proteome (HOLSER) Enables Global Structure Profiling and Site-resolved Elucidation of Ligand-Protein Interactions

Understanding how cellular proteins interact with their environment, including endogenous and exogeneous molecules, is critical for elucidating mechanisms of cellular regulation and drug action. Partial proteolysis-based techniques offer peptide-level resolution of ligand-induced conformational changes but are limited by modest proteome coverage and depth, as well as sensitivity to the experimental conditions. To overcome these limitations, we developed higH ratiO partiaL proteolysiS with carriER proteome (HOLSER), an efficient workflow that features extended digestion time for reduced peptide yield variability as well as tandem mass tag multiplexing that includes full digests for enhanced proteome depth and sequence coverage as well as higher precision of peptide abundance measurements. We demonstrate HOLSER capabilities of probing structural changes on the scale of specific binding sites for kinase target mapping, individual protein domains for structural mapping of the FKBP-mTOR complex in response to rapamycin as well as global proteome structure profiling.

biochemistry↗

bioRxiv2025

A cell-based degrader assessment platform facilitates discovery of functional NUDT5 PROTACs

Targeted protein degradation (TPD) via PROTACs and molecular glues holds significant therapeutic promise but demands detailed mechanistic evaluation in live cells to fully understand compound behavior and optimize efficacy. Here, we present an integrated, cell-first platform that combines a modular degradation assay with E3 ligase target engagement readouts for comprehensive assessment of TPD molecules in cells and use it to evaluate PROTACs towards NUDT5. To mimic endogenous degradation conditions and TPD amenability, we established a fusion protein expression system consisting of a lysine-free FKBP12 F36V PROTAC handle (FKBPVK0) and used a HiBiT/akaLuc dual luciferase reporter to accurately measure degradation dynamics. This set-up identified a VHL-dependent NUDT5 PROTAC, DDD2, that induced robust NUDT5 degradation, despite impaired NUDT5 binding in vitro and in cellulo, but no CRBN-dependent degraders. NUDT5 lysine availability mapping with DDD2 and FKBP12 F36V-directed PROTACs suggested that the CRL4CRBN complex is more sensitive to target lysine accessibility than CRL2VHL, which may have implications for E3 ligase choice and therapeutic resistance. CeTEAM drug biosensors were also established towards CRBN and VHL to quantitatively monitor degrader engagement in living cells and confirmed that the tested CRBN-directed NUDT5 PROTACs poorly engaged the E3. All together, this platform provides a versatile and scalable framework for TPD molecule discovery in a cellular context.

biochemistry↗

bioRxiv2025

Towards a species search engine: KISSE offers a rigorous statistical framework for bone collagen tandem mass spectrometry data

DNA and bone collagen are two key sources of resilient molecular markers used to identify species from their remains. Collagen is more stable than DNA, and thus it is preferred for ancient and degraded samples. Current mass spectrometry-based collagen sequencing approaches are empirical and lack a rigorous statistical framework. Based on the well-developed approaches to protein identification in shotgun proteomics, we introduce a first approximation of the species search engine (SSE). Our SSE named KISSE is based on a species-specific library of collagenous peptides that uses both peptide sequences and their relative abundances. The developed statistical model can identify the species and the probability of correct identification, as well as determine the likelihood of the analyzed species not being in the library. We discuss the advantages and limitations of the proposed approach and the possibility of extending it to other tissues.

zoology↗

bioRxiv2025

Above-Filter Digestion Proteomics reveals drug targets and localizes ligand binding site

While a number of efficient chemical proteomics methods are available for determining protein targets of pharmaceutical drugs, each approach exhibits its own "blind spot" and development of complementary techniques is needed. Here, we introduce the Above-Filter Digestion Proteomics (AFDIP) approach based on the monitoring of the rate of trypsin digestion. Digestion rate is decreased at the site of ligand binding to its target protein, while other sites may simultaneously experience an increase the digestion rate. Molecular dynamics (MD) simulations revealed that this increase may be due to allosteric structural changes related to backbone flexibility. We showcase the utility of AFDIP for deconvoluting the targets of versatile drugs and metabolites. Like other techniques, AFDIP allows for a two-dimensional analysis, with the second dimension based on drug concentration. AFDIP exhibits structural resolution, identifying drug target binding sites within [≤]10 [A], and for larger proteins often [≤]5 [A] of the crystallography-determined binding sites. Compared to earlier introduced limited proteolysis approaches, AFDIP provides easier sample preparation, deeper proteome analysis and broader sequence coverage. AFDIP is expected to find its place among the most efficient chemical proteomics methods currently available.

biochemistry↗

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2024

One-Pot Time-Induced Proteome Integral Solubility Alteration (OPTI-PISA) assay for automated and sensitive drug target identification

Proteome Integral Solubility Alteration (PISA) assay is widely used for identifying drug targets, but it is labour-intensive and time-consuming, and requires a substantial amount of biological sample. Aiming at enabling automation and greatly reducing the sample amount, we developed One-Pot Time-Induced (OPTI)-PISA. Here we demonstrate OPTI-PISA performance on identifying targets of multiple drugs in cell lysate and scaling down the sample amount to sub-microgram levels, making PISA method suitable for NanoProteomics.

biochemistry↗

bioRxiv2024

ThermoTargetMiner as a proteome integral solubility alteration target database for prospective drugs against lung cancer

Knowledge of the targets of therapeutic compounds is vital for understanding their action mechanisms and side effects, but such valuable data is seldom available. The multiple complementary techniques needed for comprehensive target characterization must combine data reliability with sufficient analysis throughput. Here, we leveraged the Proteome Integral Solubility Alteration (PISA) assay to characterize the targets of 67 approved and experimental compounds against two main lung cancer subtypes, which is now provided through the website https://thermotargetminer.serve.scilifelab.se/app/thermotargetminer. Novel target candidates (pro-targets) were found for 77% of the tested molecules. Comparison of the protein solubility shifts in lysate vs. living cells highlighted the targets directly interacting with the compounds. We verified that the drug PEITC exerts cytotoxicity through the inhibition of a pro-target PAFAH1B. As PISA is now joining the arsenal of fast and reliable target characterization techniques, the presented database, ThermoTargetMiner, will become a useful resource in lung cancer research.

biochemistry↗

bioRxiv2024

Gel-Assisted Proteome Position Integral Shift (GAPPIS) Assay Returns Molecular Weight to Shotgun Proteomics and Identifies Novel Caspase 3 Substrates

Here we present a high-throughput virtual top-down proteomics approach that restores the molecular weight (MW) information in shotgun proteomics, and demonstrate its utility in studying proteolytic events in programmed cell death. With Gel-Assisted Proteome Position Integral Shift (GAPPIS), we quantified over 7000 proteins in staurosporine-induced apoptotic HeLa cells and identified 84 proteins exhibiting in a statistically significant manner at least two of the following features: 1) a negative MW shift; 2) an elevated ratio in a pair of a semi-tryptic and tryptic peptide, 3) a negative shift in the standard deviation of MW estimated for different peptides, and 4) a negative shift in skewness of the same data. Of these proteins, 58 molecules were novel caspase 3 substrates. Further analysis identified the preferred cleavage sites consistent with the known caspase cleavages after the DXXD motif. As a powerful tool for high-throughput MW analysis simultaneously with the conventional expression analysis, GAPPIS assay can prove useful in studying a broad range of biological processes involving proteolytic events.

biochemistry↗

bioRxiv2024

Do light eaters live shorter lives? The case of ultralight Caenorhabditis elegans

The idea that ingesting heavy stable isotopes can increase longevity emerged shortly after the discovery of deuterium in the early 1930s and has been extensively tested since then on animals. Here we present the first experimental evidence for the opposite. Growing C. elegans on bacteria E. coli that are in turn fed on a diet depleted of heavy isotopes of C, H, N and O produced ultralight worms that grow and mature faster but have a shorter lifespan. Based on the differences in expression and solubility of proteins, we established an aging pseudo-time scale. Notably, the newly born ultralight worms appear to be significantly "younger" than their normal counterparts, while at day 10 they are significantly "older". Pathway analysis revealed involvement of mitochondria; analysis of reactive oxygen species (ROS) confirmed significant ROS overproduction in ultralight worms that increases further with age. These findings provide a new modality of affecting the lifespan in this important animal model of human diseases and aging.

biochemistry↗

bioRxiv2023

Merging Multi-OMICs with Proteome Integral Solubility Alteration Unveils Antibiotic Mode of Action

Antimicrobial resistance is responsible for an alarming number of deaths, estimated at 5 million per year. To combat priority pathogens, like Helicobacter pylori, the development of novel therapies is of utmost importance. Understanding the molecular alterations induced by medications is critical for the design of multi-targeting treatments capable of eradicating the infection and mitigating its pathogenicity. However, the application of bulk omics approaches for unraveling drug molecular mechanisms of action is limited by their inability to discriminate between target-specific modifications and off-target effects. This study introduces a multi-omics method to overcome the existing limitation. For the first time, the PISA assay is utilized in bacteria in the PISA-express format to link proteome solubility with different and potentially immediate responses to drug treatment, enabling us the resolution to understand target-specific modifications and off-target effects. This study introduces a comprehensive method for understanding drug mechanisms and optimizing the development of multi-targeting antimicrobial therapies.

microbiology↗

bioRxiv2023

Multifaceted proteome analysis at solubility, redox, and expression dimensions for target identification

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, we devise the first high- throughput redox proteomics approach integrated with expression analysis (REX) and combine it with Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, we analyzed HCT116 cells treated with auranofin, showing great improvement compared with previous such studies. Then we applied PISA-REX to study proteome remodeling upon stimulation of human monocytes by interferon . We also studied the proteome changes in plasmacytoid dendritic cells isolated from wild type vs. Ncf1- mutant mice treated with interferon , showing that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

biochemistry↗

bioRxiv2023

Chemogenetic inhibition of IST1-CHMP1B interaction impairs endosomal recycling and promotes unconventional LC3 lipidation at stalled endosomes

The Endosomal Sorting Complex Required for Transport (ESCRT) machinery constitutes a multisubunit protein complex that plays an essential role in membrane remodeling and trafficking. ESCRTs regulate a wide array of cellular processes, encompassing cytokinetic abscission, cargo sorting into multivesicular bodies (MVBs), membrane repair and autophagy. Given the versatile functionality of ESCRTs and the intricate organizational structure of the ESCRT complex, the targeted modulation of distinct ESCRT-mediated membrane deformations for functional dissection poses a considerable challenge. This study presents a pseudo-natural product targeting IST1-CHMP1B within the ESCRT-III complex. This compound specifically disrupts the interaction between IST1 and CHMP1B, thereby inhibiting the formation of IST1-CHMP1B copolymers essential for normal-topology membrane scission events. While the compound has no impact on cytokinesis, MVB sorting and exosome biogenesis, it rapidly hinders transferrin receptor (TfR) recycling in cells, resulting in the accumulation of transferrin in perinuclear endosomal recycling tubules. Stalled recycling endosomes acquire unconventional LC3 lipidation, establishing a link between non-canonical LC3 lipidation and endosomal recycling.

cell biology↗

bioRxiv2022

Lineage-specific oncogenes drive growth of major forms of human cancer using common downstream mechanisms

Mutations in hundreds of genes have been associated with formation of human cancer, with different oncogenic lesions prevalent in different cancer types. Yet, the malignant phenotype is simple, characterized by unrestricted growth of cells that invade neighboring healthy tissue and in many cases metastasize to distant organs. One possible hypothesis explaining this dichotomy is that the cancer genes regulate a common set of target genes, which then function as master regulators of essential cancer phenotypes, such as growth, invasion and metastasis. To identify mechanisms that drive the most fundamental feature shared by all tumors - unrestricted cell proliferation - we used a multiomic approach to identify common transcriptional and posttranslational targets of major oncogenic pathways active in different cancer types, and combined this analysis with known regulators of the cell cycle. We identified translation and ribosome biogenesis as common targets of both transcriptional and posttranslational oncogenic pathways. By combining proteomic analysis of clinical samples with functional studies of cell cultures, we also establish NOLC1 as a key node whose convergent regulation both at transcriptional and posttranslational level is critical for tumor cell proliferation. Our results indicate that lineage-specific oncogenic pathways commonly regulate the same set of targets important for growth control, revealing novel key downstream nodes that could be targeted for cancer therapy or chemoprevention.

cancer biology↗