Biology subjects

Radi, M.

Publications and source records attributed to Radi, M..

3 recordsLinked to original sources

bioRxiv2025

Membrane proteins ClcB, PtsI and YcaM mediate the bactericidal effects of colistin in Escherichia coli

Bacteria can be killed very effectively by targeting their first line of protection. The cell membranes (outer and inner membranes) of Gram-negative bacteria are directly targeted by antibiotics, such as polymyxins, via electrostatic interactions with their lipopolysaccharide (LPS) fraction. The downstream effects - preceding the cell death - of the disruptions of the bacterial membranes upon the intercalation of these antibiotics are unknown. By screening a set of E. coli membrane protein knockouts, three membrane transporters were shown to mediate the growth-inhibitory effects of colistin. This was corroborated with growth assays on gain-of-function strains, cytotoxic assays and in vivo infections in an invertebrate animal model. This is first-time evidence that the disruption caused to membrane proteins, such as the chloride channel ClcB, the sugar transport system component PtsI and the hypothetical Glutamate:GABA antiporter YcaM, is part of the cytotoxic pathway that follows or is concomitant to the electrostatic intercalations of polymyxins with the Gram-negative bacteria membrane.

microbiology↗

bioRxiv2024

RNA Polymerase II subunits overexpressions induce genome instability and deregulate transcription

Independently of the pathways or circuits deregulated in cancer cells, these present altered transcription patterns, often also direct consequence of deregulation of transcription factors. In this sense, also the RNA Polymerase complexes responsible for transcription can be affected in cancers. We find that upregulations of RNA Polymerase II subunits, especially the largest ones, correlates with poor cancer patients outcome across a range of tumor types, presenting increased genome instability. Overexpressing the subunits RPB1, RPB3 and RPB4 in cells we find that these induce DNA damage. However, the mechanisms behind this increased genome instability are specific for each subunit, linked to the unique transcription alterations generated by the subunit overexpression. Importantly, we find significant overlap between the genes with more DNA damage in our cell line models and those more affected in cancers with subunit upregulation, indicating that upregulations could be responsible for some of the phenotypes present in these patients.

molecular biology↗

bioRxiv2024

Exploring the druggability of the UEV domain of human TSG101 in search for broad-spectrum antivirals

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low M range, and able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions. ImportanceMany viruses rely on the interaction between TSG101 and viral proteins containing PTAP motifs for their proliferation. Here we show that these interactions can be efficiently blocked by drug-like compounds that impair budding and replication of viruses from different families. We have also provided valuable insights into the determinants of high affinity for these small molecule inhibitors that open new avenues for developing the identified candidates into broad-spectrum antivirals with low susceptibility to resistance.

molecular biology↗