Biology subjects

Cereghetti, G.

Publications and source records attributed to Cereghetti, G..

5 recordsLinked to original sources

bioRxiv2026

Systematic identification of pH-sensing amyloid core motifs reveals a widespread mechanism for reversible protein assembly upon stress

Unlike irreversible pathological amyloids, reversible fibrils can be regulated via pH-sensing core motifs, characterized by amyloid properties unleashed upon stress-induced protonation of critical residues. Here, we combined bioinformatic predictions and an in vitro validation pipeline to search for pH-responsive, reversible amyloid core peptides in yeast and human proteomes. This approach uncovered biophysical properties distinguishing pH-sensing and constitutive amyloid cores and established reliable criteria to identify novel reversible assemblies based on sequence data. Selected full-length candidate proteins with evolutionarily conserved pH-sensing motifs were analyzed in Saccharomyces cerevisiae using fluorescence microscopy and SDS-resistance assays, revealing multiple proteins forming reversible assemblies in stationary phase. We found that protonation of a specific histidine in the amyloid core motif of the asparagine synthase Asn1 is necessary and sufficient for assembling catalytically inactive, reversible structures called cytoophidia. Interestingly, mutant cells that fail to assemble Asn1-cytoophidia show defects to recover from stationary phase, demonstrating functional relevance of pH-sensing amyloid core motifs in vivo. Taken together, we uncovered a widespread and conserved pH-sensing mechanism that regulates the reversible assembly and function of structurally diverse fibrils upon stress.

biochemistry↗

bioRxiv2025

Mapping high resolution, multidimensional phase diagrams of physiological protein condensates

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the ALS-linked protein FUS remodels condensate phase landscapes. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

biophysics↗

bioRxiv2023

Oxidation-sensitive cysteines drive IL-38 amyloid formation

Cytokines of the interleukin (IL)-1 family are widely expressed in epithelial surfaces, including the epidermis, where they play a key role in the maintenance of barrier integrity and host defense. A recent report associated the IL-1 family member IL-33 with stress granules (SGs) in epithelial cells. Formation of SGs is promoted by the aggregation of proteins harboring low complexity regions (LCRs). In this study, using computational analyses, we predicted the presence of LCRs in six of the eleven IL-1 family members. Among these, IL-38 contained a long LCR and localized to Ras GTPase-activating protein binding protein 1 (G3BP1) positive SGs, as well as to G3BP1 negative intracellular protein condensates in keratinocytes exposed to oxidative stress (OS). In addition, we identified two highly aggregation-prone amyloid core (AC) motifs in the IL-38 LCR and detected the formation of amyloid IL-38 aggregates in response to OS in cells and in vitro. Disulfide bond mapping, in silico modelling and the analysis of specific cysteine mutants supported a model in which specific oxidation-sensitive cysteines act as redox switches to modify the conformation of IL-38 and thus the surface exposure of its ACs, shuttling it from a soluble state into biomolecular condensates. Finally, the presence of IL-38 granules in human epidermal layers highly exposed to environmental OS suggests that oxidation-induced formation of amyloid aggregates, as a previously unrecognized intrinsic biological property of IL-38, may be physiologically relevant at this epithelial barrier.

cell biology↗

bioRxiv2022

A conserved mechanism regulates reversible amyloids via pH-sensing regions

Amyloids were long viewed as irreversible, pathological aggregates, often associated with neurodegenerative diseases1. However, recent insights challenge this view, providing evidence that reversible amyloids can form upon stress conditions and fulfil crucial cellular functions2. Yet, the molecular mechanisms regulating functional amyloids and the differences to their pathological counterparts remain poorly understood. Here we investigate the conserved principles of amyloid reversibility by studying the essential metabolic enzyme pyruvate kinase (PK) in yeast and human cells. We demonstrate that PK forms stress-dependent reversible amyloids through a pH-sensitive amyloid core. Stress- induced cytosolic acidification promotes aggregate formation via protonation of specific glutamate (in yeast) or histidine (in human) residues within the amyloid core. Our work thus unravels a conserved and potentially widespread mechanism underlying amyloid functionality and reversibility, fine-tuned to the respective physiological cellular pH range.

cell biology↗

bioRxiv2019

Ultra High-Throughput Multiparametric Imaging Flow Cytometry: Towards Diffraction-Limited Sub-Cellular Detection

Flow cytometry is widely recognized as the gold-standard technique for the analysis and enumeration of heterogeneous cellular populations and has become an indispensable tool in diagnostics,1 rare-cell detection2 and single-cell proteomics.3 Although contemporary flow cytometers are able to analyse many thousands of cells per second, with classification based on scattering or fluorescence criteria, the vast majority require unacceptably large sample volumes, and do not allow the acquisition of spatial information. Herein, we report a sheathless, microfluidic imaging flow cytometer that incorporates stroboscopic illumination for blur-free fluorescence and brightfield detection at analytical throughputs in excess of 60,000 cells/s and 400,000 cells per second respectively. Our imaging platform is capable of multi-parametric fluorescence quantification and subcellular (co-)localization analysis of cellular structures down to 500 nm with microscopy image quality. We demonstrate the efficacy of our approach by performing challenging high-throughput localization analysis of cytoplasmic RNA granules in yeast and human cells. Results suggest significant utility of the imaging flow cytometer in the screening of rare events at the subcellular level for diagnostic applications.

bioengineering↗