Biology subjects

Schimpl, M.

Publications and source records attributed to Schimpl, M..

3 recordsLinked to original sources

bioRxiv2024

Direct β1/β2 AMPK activation reduces liver steatosis but not fibrosis in a mouse model of non-alcoholic steatohepatitis

5AMP-activated protein kinase (AMPK) activators show potential for treating Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) due to their inhibiting effects on fatty acid and cholesterol synthesis. The absence of treatments for NASH, and its propensity for progression to severe disease, lead us to identify and characterize BI9774, a small molecule AMPK activator, which we used to evaluate this potential, including its ability to reduce the NASH specific qualities of fibrosis and inflammation in a preclinical study. Male Lepob/Lepob mice on a control or NASH inducing (AMLN) diet, with liver fibrosis were given BI9774 or vehicle for 6 weeks while metabolic and NASH endpoints were evaluated. BI9774 treatment decreased plasma ALT, terminal liver weight, and liver lipids. RNA expression of collagen-related genes decreased, although collagen protein and inflammation remained unaltered. We also observed increased heart weight and glycogen levels, and increased expression of genes associated with cardiac hypertrophy. AMPK activation improved many metabolic endpoints, but lack of significant improvement in liver fibrosis and negative cardiac effects suggest systemic AMPK activation is not an ideal NASH therapy. Reductions in steatosis and fibrosis-related genes indicate that, with extended treatment, a liver specific AMPK activator has potential to resolve hepatic fibrosis. Summary StatementFatty liver disease affects up to 30 percent of adults worldwide with 30% of patients progressing to more sever liver disease. AMPK activation can help reduce liver fat.

pharmacology and toxicology↗

bioRxiv2024

Development of D-box peptides to inhibit the Anaphase Promoting Complex/Cyclosome

E3 ubiquitin ligases engage their substrates via degrons - short linear motifs typically located within intrinsically disordered regions of substrates. As these enzymes are large, multi-subunit complexes that generally lack natural small-molecule ligands and are hard to drug via conventional means, alternative strategies are needed to target them in diseases, and peptide-based inhibitors derived from degrons represent a promising approach. Here we explore peptide inhibitors of Cdc20, a substrate-recognition subunit and activator of the E3 ubiquitin ligase the anaphase promoting complex/cyclosome (APC/C) that is essential in mitosis and consequently of interest as an anti-cancer target. APC/C engages substrates via degrons that include the Destruction box (D-box) motif. We used a rational design approach to construct binders containing unnatural amino acids aimed at better filling a hydrophobic pocket on the surface of Cdc20. We confirmed binding by thermal-shift assays and surface plasmon resonance and determined the structures of a number of the Cdc20- peptide complexes. Using a cellular thermal shift assay, we confirmed that the D-box peptides also bind to and stabilise Cdc20 in the cell. We found that the D-box peptides inhibit ubiquitination activity of APC/CCdc20 and are more potent than the small molecule inhibitor Apcin. Lastly, these peptides function as portable degrons capable of driving the degradation of a fused fluorescent protein. Interestingly, we find that although inhibitory activity of the peptides correlates with Cdc20-binding affinity, degradation efficacy does not, which may be due to the complex nature of APC/C regulation and effects of degron binding of subunit recruitment and conformational changes. Our study lays the groundwork for the further development of these peptides as molecular therapeutics for blocking APC/C as well as potentially also for harnessing APC/C for targeted protein degradation.

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↗