SUMMARY Poly(ADP-ribose) polymerase-1 (PARP-1) creates the posttranslational modification PAR from substrate NAD+ to regulate multiple cellular processes. DNA breaks sharply elevate PARP-1 catalytic activity to mount a cell survival repair response, whereas persistent PARP-1 hyperactivation during severe genotoxic stress is associated with cell death. The mechanism for tight control of the robust catalytic potential of PARP-1 remains unclear. By monitoring PARP-1 dynamics using hydrogen/deuterium exchange-mass spectrometry (HXMS), we unexpectedly find that a specific portion of the helical subdomain (HD) of the catalytic domain rapidly unfolds when PARP-1 encounters a DNA break. Together with biochemical and crystallographic analysis of HD deletion mutants, we show that the HD is an autoinhibitory domain that blocks productive NAD+ binding. Our molecular model explains how PARP-1 DNA damage detection leads to local unfolding of the HD that relieves autoinhibition, and has important implications for the design of PARP inhibitors.
SUMMARY Tankyrase-1 (TNKS1/PARP-5a) is a poly(ADP-ribose) polymerase (PARP) enzyme that regulates multiple cellular processes creating a poly(ADP-ribose) posttranslational modification that can lead to target protein turnover. TNKS1 thereby controls protein levels of key components of signaling pathways, including Axin1, the limiting component of the destruction complex in canonical Wnt signaling that degrades β-catenin to prevent its coactivator function in gene expression. There are limited molecular level insights into TNKS1 regulation in cell signaling pathways. TNKS1 has a sterile alpha motif (SAM) domain that is known to mediate polymerization, but the functional requirement for SAM polymerization has not been assessed. We have determined the crystal structure of wild-type human TNKS1 SAM domain and used structure-based mutagenesis to disrupt polymer formation and assess the consequences on TNKS1 regulation of β-catenin dependent transcription. Our data indicate the SAM polymer is critical for TNKS1 catalytic activity and allows TNKS1 to efficiently access cytoplasmic signaling complexes.
The poly(ADP-ribose) polymerase enzyme Tankyrase-1 (TNKS) regulates multiple cellular processes and interacts with diverse proteins using five ankyrin repeat clusters (ARCs). There are limited structural insights into functional roles of the multiple ARCs of TNKS. Here we present the ARC1-3 crystal structure and employ small-angle X-ray scattering (SAXS) to investigate solution conformations of the complete ankyrin repeat domain. Mutagenesis and binding studies using the bivalent TNKS binding domain of Axin1 demonstrate that only certain ARC combinations function together. The physical basis for these restrictions is explained by both rigid and flexible ankyrin repeat elements determined in our structural analysis. SAXS analysis is consistent with a dynamic ensemble of TNKS ankyrin repeat conformations modulated by Axin1 interaction. TNKS ankyrin repeat domain is thus an adaptable binding platform with structural features that can explain selectivity toward diverse proteins, and has implications for TNKS positioning of bound targets for poly(ADP-ribose) modification.
Poly(ADP-ribose) (PAR) is a posttranslational modification predominantly synthesized by PAR polymerase-1 (PARP-1) in genome maintenance. PARP-1 detects DNA damage, and damage detection is coupled to a massive increase PAR production, primarily attached to PARP-1 (automodification). Automodified PARP-1 then recruits repair factors to DNA damage sites. PARP-1 automodification eventually leads to release from DNA damage thus turning off catalytic activity, although the effects of PAR on PARP-1 structure are poorly understood. The multiple domains of PARP-1 are organized upon detecting DNA damage, creating a network of domain contacts that imposes a major conformational transition in the catalytic domain that increases PAR production. Presented here are two novel fluorescent sensors that monitor the global and local structural transitions of PARP-1 that are associated with DNA damage detection and catalytic activation. These sensors display real-time monitoring of PARP-1 structural transitions upon DNA damage detection, and their reversal upon PARP-1 automodification. The fluorescent sensors are further used to investigate intramolecular and intermolecular PARP-1 activation, followed by the observation that intramolecular activation of PARP-1 is the predominant response to DNA strand breaks in cells. These results provide a unique perspective on the interplay between PARP-1 DNA damage recognition, allosteric regulation, and catalytic activity.
Heart disease is the leading cause of mortality in the United States. One cause of heart arrhythmia is calcium (Ca 2+) mishandling in cardiac muscle cells. We adapt Izu's et al. mathematical reaction-diffusion model of calcium in cardiac muscle cells, or cardiomyocytes, [14], implemented by Gobbert [12], and analyzed in Coulibaly et al. [8] to include calcium being released from the sarcoplasmic reticulum (SR), the effects of buffers in the SR, particularly calsequestrin, and the effects of Ca 2+ influx due to voltage across the cell membrane. Based on simulations of the model implemented in parallel using MPI, our findings aligned with known biological models and principles, giving us a thorough understanding of several factors that influence Ca 2+ dynamics in cardiac myocytes. Specifically, dynamic calcium store will cap previous calcium blow-up seen in the model. Calcium channels located in spatial opposition of calcium release units produce more predictable intracellular calcium propagation. And we used multi-parametric calcium dynamics tables, which act as a multidimensional bifurcation diagram, to visualize parameter boundaries between different biophysical dynamics.
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-1’s 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.
Human PARP-1, PARP-2, and PARP-3 are key players in the cellular response to DNA damage, during which their catalytic activities are acutely stimulated through interaction with DNA strand breaks. There are also roles for these PARPs outside of the DNA damage response, most notably for PARP-1 and PARP-2 in the regulation of gene expression. Here, we describe a general method to express and purify these DNA damage-dependent PARPs from E. coli cells for use in biochemical assays and for structural and functional analysis. The procedure allows for robust production of PARP enzymes that are free of contaminant DNA that can interfere with downstream analysis. The described protocols have been updated from our earlier reported methods, most importantly to introduce PARP inhibitors in the production scheme to cope with enzyme toxicity that can compromise the yield of purified protein.
AimsTo assess whether natriuretic peptides (NPs) can be used to reliably predict long-term therapeutic effect on clinical outcomes for patients with heart failure and reduced ejection fraction (HFrEF).HFrEF intervention trials with mortality data were identified. Subsequently, we identified trials assessing therapy-induced changes in NPs. We assessed the correlation between the average short-term placebo-corrected drug or device effect on NPs and the longer-term therapeutic effect on clinical outcomes. Of 35 distinct therapies with an identifiable mortality result (n = 105 062 patients), 20 therapies had corresponding data on therapeutic effect on NPs. No correlation was observed between therapy-induced placebo-corrected change in brain natriuretic peptide or N-terminal pro-brain natriuretic peptide and therapeutic effect on all-cause mortality (ACM) (Spearman r = −0.32, P = 0.18 and r = −0.20, P = 0.47, respectively). There was no correlation between therapy-induced placebo-corrected per cent change in NP and intervention effect on ACM or ACM-heart failure hospitalizations (r = −0.30, P = 0.11 and r = 0.10, P = 0.75, respectively).
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