Biology subjects

Pierangelini, A.

Publications and source records attributed to Pierangelini, A..

3 recordsLinked to original sources

bioRxiv2025

Evolution tunes functional sub-state interconversion to boost enzyme function

Enzymes do not operate as static structures, but continuously fluctuate between different conformations. Enzymes therefore dynamically sample conformations with varying catalytic activity. However, it remains largely unexplored whether evolution can exploit the conformational dynamics between sub-states to improve activity. Here, we dissect the evolutionary trajectory of the {beta}-lactamase OXA-48 toward improved ceftazidime hydrolysis. Evolution relieved conformational bottlenecks by promoting alternate functional sub-states, gradually shifting the rate-limiting step from substrate binding to sub-state interconversion, and finally to the chemical step. Reorganization of the conformational landscape enhanced OXA-48s ability to hydrolyze ceftazidime and introduced a trade-off in its native activity against meropenem. This trade-off stemmed from catalytic incompatibility between the native and the evolved sub-state populations. Our findings highlight the transitions between functional sub-states as a mechanism of natural selection, shaping functional divergence and offering new strategies for enzyme and antibiotic engineering.

molecular biology↗

bioRxiv2025

Nucleophagy removes cytotoxic trapped PARP1

Poly (ADP-Ribose) Polymerase inhibitors (PARPi) induce cytotoxicity in homologous recombination repair (HRR)-deficient cancers by causing PARP1 to become trapped on chromatin, resulting in irreparable replication-associated DNA damage. Although increased clearance of trapped PARP1 from chromatin reduces the sensitivity of cancer cells to PARPi, details surrounding this process remain unclear. PARPi exposure is known to cause increased autophagy flux, whilst autophagy inhibition can hypersensitise cells to PARPi. Using various biochemical, cell biological and live imaging-based assays, we found that trapped PARP1 is cleared by nucleophagy, the selective autophagy of nuclear substrates. Specifically, the nucleophagy of trapped PARP1 was orchestrated by the selective autophagy receptor TEX264 and its partner segregase p97/VCP. TEX264 mediates this process by directly interacting with trapped PARP1, thus bridging PARP1 to the autophagosomal resident protein LC3 for processing via autophagy. Impeding this process, either chemically or genetically, heightened PARP1 trapping, leading to accumulation of protein aggregates, replication-associated DNA damage and cell lethality, re-sensitising PARPi-resistant cells to various PARPi. In conclusion, we show that nucleophagy acts in a cytoprotective manner to directly target PARPi-induced trapped PARP1 for degradation.

cell biology↗

bioRxiv2025

obABPP-HT*: A Precision-Engineered Activity Proteomics Pipeline for the Streamlined Discovery of Deubiquitinase Inhibitors

Deubiquitinases (DUBs), and the dysregulation thereof, are implicated in human disease. The recent inclusion of selective DUB inhibitors in clinical trials has heightened interest in DUB-focused drug discovery. Current DUB screening methods remain constrained, however, as they often rely on recombinant proteins that are truncated or derived from non-human sources, typically necessitating extensive optimisation of initial hits. We introduce a high-throughput, endogenous human DUB-focused activity proteomics workflow designed for the simultaneous screening and profiling of small, targeted libraries of catalytic group-reactive compounds. In a proof-of-concept screen, this innovative platform expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5. Remarkably, these inhibitors required minimal or no optimisation to confirm the previously reported biological roles of the three DUBs, underscoring the advantages of this methodology for drug discovery applications.

biochemistry↗