Biology subjects

Pascal, J. M.

Publications and source records attributed to Pascal, J. M..

3 recordsLinked to original sources

bioRxiv2021

Tankyrase interacts with the allosteric site of glucokinase and inhibits its glucose-sensing function in the beta cell

Insulin secretion in the pancreatic beta cell is rate-limited by glucokinase (GCK), the glucose sensor that catalyzes the first step of glucose metabolism. GCK consists of two lobes connected by a flexible hinge that allows the kinase to exhibit a spectrum of conformations ranging from the active, closed form to several inactive, less-compact forms. Activating GCK mutations can cause hyperinsulinemia and hypoglycemia in infants. A similar phenotype exhibited by tankyrase (TNKS)-deficient mice prompted us to investigate whether TNKS might modulate the glucose-sensing function of GCK. We found that TNKS colocalizes and directly interacts with GCK. Their interaction is mediated by two ankyrin-repeat clusters (ARC-2 and -5) in TNKS and a tankyrase-binding motif (TBM, aa 63-68) in the GCK hinge. This interaction is conformation sensitive, human GCK variants that cause hyperglycemia (V62M) or hypoglycemia (S64Y) enhance or diminish the interaction respectively, even though they have no impact on TNKS interaction in the context of a GCK peptide (V62M) or a peptide library (S64Y). Moreover, the TNKS-GCK interaction is inhibited by high glucose concentrations, which are known to stabilize GCK in the active (closed, glucose-avid) conformation. Conversely, glucose phosphorylation by GCK in vitro is inhibited by TNKS. To study this in vitro inhibitory effect in the MIN6 beta cells, we showed that glucose-stimulated insulin secretion is suppressed upon stabilization of the TNKS protein and is conversely enhanced upon TNKS knockdown. Based on these findings as well as by contrasting with hexokinase-2, we propose that TNKS is a physiological GCK inhibitor in pancreatic beta cells that acts by trapping the kinase in the open (inactive) conformation.

cell biology↗

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↗