Biology subjects

Langelier, M.-F.

Publications and source records attributed to Langelier, M.-F..

5 recordsLinked to original sources

bioRxiv2024

A PARP2-specific active site alpha-helix melts to permit DNA damage-induced enzymatic activation

PARP1 and PARP2 recognize DNA breaks immediately upon their formation, generate a burst of local PARylation to signal their location, and are co-targeted by all current FDA-approved forms of PARP inhibitors (PARPi) used in the cancer clinic. Recent evidence indicates that the same PARPi molecules impact PARP2 differently from PARP1, raising the possibility that allosteric activation may also differ. We find that unlike for PARP1, destabilization of the autoinhibitory domain of PARP2 is insufficient for DNA damage-induced catalytic activation. Rather, PARP2 activation requires further unfolding of an active site -helix absent in PARP1. Only one clinical PARPi, Olaparib, stabilizes the PARP2 active site -helix, representing a structural feature with the potential to discriminate small molecule inhibitors. Collectively, our findings reveal unanticipated differences in local structure and changes in activation-coupled backbone dynamics between PARP1 and PARP2.

biochemistry↗

bioRxiv2022

Clinical PARP inhibitors allosterically induce PARP2 retention on DNA

PARP1 and PARP2 detect DNA breaks, which activates their catalytic production of poly(ADP-ribose) that recruits repair factors and releases PARP1/2 from DNA. PARP inhibitors (PARPi) are used in cancer treatment and target PARP1/2 catalytic activity, interfering with repair and increasing PARP1/2 persistence on DNA damage. Additionally, certain PARPi exert allosteric effects that increase PARP1 retention on DNA. However, no clinical PARPi exhibit this allosteric behavior toward PARP1. In contrast, we show that certain clinical PARPi exhibit an allosteric effect that retains PARP2 on DNA breaks in a manner that depends on communication between the catalytic and DNA-binding regions. Using a PARP2 mutant that mimics an allosteric inhibitor effect, we observed increased PARP2 retention at cellular damage sites. The new PARPi AZD5305 also exhibited a clear reverse allosteric effect on PARP2. Our results can help explain the toxicity of clinical PARPi and suggest ways to improve PARPi moving forward.

biochemistry↗

bioRxiv2022

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, simultaneous assessment of drug-associated phenotypes has proven challenging. CeTEAM (cellular target engagement by accumulation of mutant) is a platform that can concomitantly evaluate drug-target interactions and phenotypic responses for holistic assessment of drug pharmacology using conditionally-stabilized drug biosensors. We observe that drug-responsive proteotypes are prevalent among reported mutants of known drug targets. CeTEAM-compatible mutants follow structural and biophysical logic that permits intra-protein and paralogous expansion of the biosensor pool, as exemplified by alanine scanning of leucines within the PARP1 helical domain and transfer of PARP1 destabilization to the analogous PARP2 residue. We then apply CeTEAM to uncouple target engagement from divergent cellular activities of MTH1 inhibitors, dissect NUDT15-associated thiopurine metabolism with the R139C pharmacogenetic variant, and profile the live-cell dynamics of PARP1/2 binding and DNA trapping by PARP inhibitors. Further, PARP1-derived biosensors facilitated high-throughput screening of drug-like libraries for PARP1 binders, as well as multimodal ex vivo analysis and non-invasive tracking of PARPi binding in live animals. Our data suggests that CeTEAM can facilitate real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences.

biochemistry↗

bioRxiv2021

HPF1 dynamically controls the PARP1/2 balance between initiating and elongating ADPribose modifications

Upon detecting DNA strand breaks, PARP1 and PARP2 produce the posttranslational modification poly(ADP-ribose) to orchestrate the cellular response to DNA damage. Histone PARylation factor 1 (HPF1) binds to PARP1/2 to directly regulate their catalytic output. HPF1 is required for the modification of serine residues with ADP-ribose, whereas glutamate/aspartate residues are modified in the absence of HPF1. PARP1 is an abundant nuclear protein, whereas HPF1 is present in much lower amounts, raising the question of whether HPF1 can pervasively modulate PARP1 activity. Here we show biochemically that HPF1 efficiently regulates PARP1/2 catalytic output at the sub-stoichiometric ratios matching their relative cellular abundances. HPF1 rapidly associates and dissociates from multiple PARP1 molecules, initiating ADP-ribose modification of serine residues before modification can initiate on glutamate/aspartate residues. HPF1 accelerates the rate of attaching the first ADP-ribose, such that this initiation event is comparable to the rate of the elongation reaction to form poly(ADP-ribose). This "hit and run" mechanism ensures that HPF1 contributions to the PARP1/2 active site during initiation do not persist and interfere with PAR chain elongation at sites of DNA damage. HPF1 thereby balances initiation and elongation events to regulate PARP1/2 output. Structural analysis of HPF1 in complex with PARP1 provides first insights into the assembly on a DNA strand break, and the HPF1 impact on PARP1 retention on DNA. Our data support the prevalence of the serine-ADP-ribose modification in cells and establish that HPF1 imparts the efficiency of serine-ADP-ribose modification required for an acute response to DNA damage.

biochemistry↗

bioRxiv2020

Dynamics of the HD regulatory subdomain of PARP 1; substrate access and allostery in PARP activation and inhibition.

PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1s F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.

molecular biology↗