Biology subjects

Petris, G.

Publications and source records attributed to Petris, G..

5 recordsLinked to original sources

bioRxiv2026

ER-associated protein degradation initiates by retrotranslocation from the ER quality control compartment

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD), a process requiring their exit from the ER and delivery to cytosolic proteasomes via the key retrotranslocation step. Conventional assays detect fully extracted substrates, overlooking early initiation events. Here, we use an Escherichia coli-derived Biotin Acceptor Peptide (BAP)-biotin ligase BirA system to detect ERAD substrates upon cytosolic exposure. Using the model ERAD substrate H2a, we show that cytosolic BirA selectively biotinylates luminal BAP tags during early cytosolic exposure, enabling detection of substrates engaging the mammalian retrotranslocation machinery independently of subsequent extraction. Combined with high-resolution expansion fluorescence microscopy, this approach reveals active initiation of ERAD at the specialized ER-derived quality control compartment (ERQC). Substrates become transiently exposed to the cytosol while remaining membrane-associated, followed by tight coupling of polyubiquitination, transmembrane segment extraction, deglycosylation and degradation. Functional perturbations indicate that mannose trimming enables initiation, whereas p97 and HRD1 support later extraction and ubiquitination.

cell biology↗

bioRxiv2025

ecDNA replication is disorganised and vulnerable to replication stress

Extrachromosomal DNA (ecDNA) is a critical driver of cancer progression, contributing to tumour growth, evolution, and therapeutic resistance through oncogene amplification. Despite its significance, the replication of ecDNA remains poorly understood. In this study, we investigated the replication dynamics of ecDNA using high-resolution replication timing analysis (Repli-seq) and DNAscent, a method for measuring origin firing and replication fork movement based on ultra-long read Oxford Nanopore Sequencing, that we applied to both bulk DNA and to ecDNA isolated with FACS-based Isolation of Native ecDNA (FINE), a new method for isolating intact, chromatinised ecDNA without DNA or protein digestion. We demonstrate that ecDNA in the COLO 320DM colorectal cancer cell line exhibits largely asynchronous replication throughout the S phase, contrasting with the conserved replication timing of the corresponding normal linear chromosomal DNA in RPE-1 cells and the chromosomally reintegrated ecDNA in COLO 320HSR, which forms a homogeneously staining region. Replication origins on ecDNA are redistributed, and replication forks exhibit reduced velocity and increased stalling, particularly near the c-MYC oncogene. Under replication stress induced by hydroxyurea treatment, ecDNA replication is further compromised, leading to altered origin activation, reduced fork velocity and eventual ecDNA depletion from cells. Our findings reveal fundamental differences in the replication dynamics of ecDNA, providing insights that could inform the development of therapies targeting ecDNA-associated oncogene amplification in cancer.

cancer biology↗

bioRxiv2024

Functional rescue of F508del-CFTR through revertant mutations introduced by CRISPR base editing

Cystic Fibrosis (CF) is a life-shortening autosomal recessive disease caused by mutations in the CFTR gene, resulting in functional impairment of the encoded ion channel. F508del mutation, a trinucleotide deletion, is the most frequent cause of CF affecting approximately 80% of patients. Even though current pharmacological treatments alleviate the F508del-CF disease symptoms there is no definitive cure. Here we leveraged revertant mutations (RMs) in cis with F508del to rescue CFTR protein folding and restore its function. We developed CRISPR base editing strategies to efficiently and precisely introduce the desired mutations in the F508del locus. Both editing and CFTR function recovery were verified in CF cellular models including primary epithelial cells derived from CF patients. The efficacy of the CFTR recovery strategy was validated in cultures of pseudostratified epithelia from patients cells showing full recovery of ion transport. Additionally, we observed an additive effect by combining our strategy with small molecules that enhance F508del activity, thus paving the way to combinatorial therapies.

bioengineering↗

bioRxiv2020

PHARMACOLOGICAL PROTEIN INACTIVATION BY TARGETING FOLDING INTERMEDIATES

Recent computational advancements in the simulation of biochemical processes allow investigating the mechanisms involved in protein regulation with realistic physics-based models, at an atomistic level of resolution. Using these techniques to study the negative regulation of the androgen receptor (AR), we discovered a key functional role played by non-native metastable states appearing along the folding pathway of this protein. This unexpected observation inspired us to design a completely novel drug discovery approach, named Pharmacological Protein Inactivation by Folding Intermediate Targeting (PPI-FIT), based on the rationale of negatively regulating protein expression by targeting folding intermediates. Here, PPI-FIT was tested for the first time on the cellular prion protein (PrP), a cell surface glycoprotein playing a key role in fatal and transmissible neurodegenerative pathologies known as prion diseases. We predicted the all-atom structure of an intermediate appearing along the folding pathway of PrP, and identified four different small molecule ligands for this conformer, all capable of selectively lowering the expression of the protein by promoting its degradation. Our data support the notion that the level of target proteins could be modulated by acting on their folding pathways, implying a previously unappreciated role for folding intermediates in the biological regulation of protein expression.

biochemistry↗

bioRxiv2020

CRISPR-Csy4-mediated editing of rotavirus double-stranded RNA genome

CRISPR-nucleases have been widely applied for editing cellular and viral genomes, but nuclease-mediated genome editing of double-stranded RNA (dsRNA) viruses has not yet been reported. Here, by engineering CRISPR-Csy4 nuclease to localise to rotavirus viral factories, we achieved the first nuclease-mediated genome editing of rotavirus, an important human and livestock pathogen with a multi-segmented dsRNA genome. Rotavirus replication intermediates cleaved by Csy4 were repaired through the formation of defined deletions in the targeted genome segments in a single replication cycle. Using CRISPR-Csy4-mediated editing of rotavirus genome, we labelled for the first time the products of rotavirus secondary transcription made by newly assembled viral particles during rotavirus replication, demonstrating that this step largely contributes to the overall production of viral proteins. We anticipate that the nuclease-mediated cleavage of dsRNA virus genomes will promote a new level of understanding of viral replication and host-pathogen interactions, offering the opportunity to develop new therapeutics.

microbiology↗