Biology subjects

Chiesa, G.

Publications and source records attributed to Chiesa, G..

4 recordsLinked to original sources

bioRxiv2025

Organobodies: A robust and size-controllable system for generating scalable hiPSC-derived liver organoids for drug toxicity screening

Background and aimHepatic organoids generated from pluripotent and adult stem cells faithfully mimic the architecture and function of in vivo organs making them powerful tools for preclinical applications such as disease modelling and drug screening. However, developing robust and scalable models capable of preserving liver-specific functions long-term remains technically challenging. MethodsUsing a defined self-assembling peptide, we established a robust and reproducible method to generate hepatic organoids from human induced pluripotent stem cells (hiPSCs), termed hepatic organobodies (OBs), which remained stable in culture for several weeks. The hepatic identity and maturation of OBs were characterized by qPCR, histology, immunofluorescence, and bulk RNA sequencing. Their functional competence was evaluated by ELISA, CYP3A4 induction assays, and assessment of drug biotransformation capacity using HPLC-MS/MS, in parallel with three-dimensional human primary hepatocyte (3D PHH) cultures. Finally, the model was applied to predict the hepatotoxicity of a selected panel of known compounds and benchmarked against HepG2 cells and 3D PHH microtissues. ResultshiPSC-derived hepatocyte-like cells (HLCs) cultured in OBs acquired characteristic hepatic morphology and expressed several key hepatocyte-specific genes, some at levels comparable to those in freshly isolated primary human hepatocytes (PHHs). OBs secreted significantly higher amounts of albumin and 1-antitrypsin (A1AT) compared with parallel 2D-cultured HLCs. Bulk RNA sequencing revealed higher relative expression of major drug metabolism genes, including CYP3A4, CYP2C9, and CYP1A2, as well as enhanced maturation, evidenced by upregulation of the PPAR signalling and fatty acid {beta}-oxidation pathways in OBs relative to 2D HLCs. Moreover, OBs demonstrated CYP450 enzyme activity, with CYP3A4 and CYP2C9 activities comparable to those observed in 3D PHH microtissues. Finally, we show that our 3D model accurately predicted the hepatotoxicity of more than 10 tested chemical compounds. ConclusionHere, we demonstrate a novel, defined, robust and remarkably advanced 3D liver model as a valuable platform for scalable toxicity prediction and drug screening for personalised medicine.

bioengineering↗

bioRxiv2023

Nanoscale Structural Mapping of Protein Aggregates in Live Cells Modeling Huntington's Disease

Protein aggregation, in the form of amyloid fibrils, is intimately correlated with many neurodegenerative diseases. Despite recent advances in structural biology, it remains challenging to acquire structural information of proteins in live cells. Tagging with fluorescent proteins, like green fluorescent protein (GFP), is routinely used for protein visualization. Yet, this method alone cannot provide detailed structural information on the protein system of interest, and tagging proteins has the potential to perturb native structure and function. Here, by fluorescence-detected as well as label-free scattering-based mid-infrared photothermal (MIP) microscopy, we demonstrate nanoscale mapping of secondary structure of protein aggregates in a yeast model of Huntingtons disease. We first used GFP as a highly sensitive photothermal reporter to validate {beta}-sheet enrichment in huntingtin (htt) protein aggregates. We then obtained label-free structural maps of protein aggregates. Our data showed that the fluorescent protein tag indeed perturbed the secondary structure of the aggregate, evident by a spectral shift. Live cell MIP spectroscopy further revealed the fine spatial distribution of structurally distinct components in protein aggregates, featuring a 246-nm diameter core highly enriched in {beta}-sheet surrounded by a [a]-helix-rich shell. Interestingly, this structural partition exists only in presence of the [RNQ+] prion, a prion that acts to facilitate the formation of other amyloid prions. Indeed, when htt is induced to aggregate in the absence of this prion ([rnq-] state), it forms non-toxic amyloid aggregates exclusively. These results showcase the potential of MIP for unveiling detailed and subtle structural information on protein systems in live cells. SignificanceProtein aggregation is a hallmark of neurodegenerative diseases, such as Huntingtons Disease. Understanding the nature of neurotoxic aggregates could lead to better therapeutic approaches. The limited progress in this direction is partly due to the lack of tools for extracting structural information in the physiological context of the aggregates. Here, we report a photothermally detected mid-infrared micro-spectroscopy technique able to dissect the secondary structure of aggregates of the huntingtin protein in live cells. We describe for the first time a nanoscale partition of secondary structures between {beta}-rich core and [a]-rich shell of the aggregates. This work demonstrates the potential of mid-infrared photothermal microscopy for structural and functional mapping of proteins in live cells.

biophysics↗

bioRxiv2023

Human Herpesvirus 8 ORF57 protein is able to reduce TDP-43 pathology: Network analysis Identifies Interacting Pathways

Aggregation of TAR DNA-binding protein 43kDa (TDP-43) is thought to drive the pathophysiology of ALS and some Frontotemporal dementias. TDP-43 is normally a nuclear protein that in neurons translocates to the cytoplasm and forms insoluble aggregates upon activation of the integrated stress response (ISR). Viruses evolved to control the ISR. In the case of Herpesvirus 8, the protein ORF57 acts to bind protein kinase R, inhibit phosphorylation of eIF2 and reduce activation of the ISR. We hypothesized that ORF57 might also possess the ability to inhibit aggregation of TDP-43. ORF57 was expressed in the neuronal SH-SY5Y line and its effects on TDP-43 aggregation characterized. We report that ORF57 inhibits TDP-43 aggregation by 55% and elicits a 2.45-fold increase in the rate of dispersion of existing TDP-43 granules. These changes were associated with a 50% decrease in cell death. Proteomic studies were carried out to identify the protein interaction network of ORF57. We observed that ORF57 directly binds to TDP-43 as well as interacts with many components of the ISR, including elements of the proteostasis machinery known to reduce TDP-43 aggregation. We propose that viral proteins designed to inhibit a chronic ISR can be engineered to remove aggregated proteins and dampen a chronic ISR.

neuroscience↗

bioRxiv2018

Side chain to main chain hydrogen bonds stabilize polyglutamine helices in transcription factors

Polyglutamine (polyQ) tracts are regions of low sequence complexity of variable length found in more than one hundred human proteins. These tracts are frequent in activation domains of transcription factors and their length often correlates with transcriptional activity. In addition, in nine proteins, tract elongation beyond specific thresholds causes polyQ disorders. To study the structural basis of the association between tract length, transcriptional activity and disease, here we addressed how the conformation of the polyQ tract of the androgen receptor (AR), a transcription factor associated with the polyQ disease spinobulbar muscular atrophy (SBMA), depends on its length. We found that the tract folds into a helical structure stabilized by unconventional hydrogen bonds between glutamine side chains and main chain carbonyl groups. These bonds are bifurcate with the conventional main chain to main chain hydrogen bonds stabilizing -helices. In addition, since tract elongation provides additional interactions, the helicity of the polyQ tract directly correlates with its length. These findings suggest a plausible rationale for the association between polyQ tract length and AR transcriptional activity and have implications for establishing the mechanistic basis of SBMA.

biophysics↗