Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference

Abstract

The human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.

Three-Dimensional Proteome-Wide Scale Screening for the 5-Alpha Reductase Inhibitor Finasteride: Identification of a Novel Off-Target

“… 91 The ff14SB ( 92 ) and gaff ( 93 ) force fields were adopted for protein and ligands, respectively. A protocol consisting in multiple equilibration steps up to a final temperature of 300 K at a constant volume and temperature ( NVT ) and constant pressure and temperature ( NPT ) was adopted as detailed in previous works, 94 , 95 followed by 200 ns of NPT production run. After analysis of the variation of the RMSD versus time during the whole trajectory ( Figure S1 ), H-bond (donor–acceptor distance cutoff = 3.5 Å; donor-H-acceptor angle cutoff = 135 deg) and clustering (five clusters based on mass-weighted RMSD were generated using the average linkage algorithm) analyses were performed using cpptraj ( 88 ) on the last 20 ns of each MD trajectory, where the RMSD was better converged.…”

Exploring the copper binding ability of Mets7 hCtr‐1 protein domain and His7 derivative: An insight in Michael addition catalysis

“…Both His7 and Mets7 peptides were synthetized by Fmoc solid phase peptide synthesis, using Rink‐amide resin (0.69 mmol/g) following standard procedures 41,44 . The complexes were prepared by mixing equimolar quantities of Mets7 or His7 with Cu (OAc) 2 in MeOH and adding two equivalent of ascorbic acid for inducing the Cu (II) reduction to Cu(I).…”

“…Furthermore, the HSAB principles establish border-line Cu (II) ion preference for the imidazole histidyl N-donor as the best match allowing thus a substantial change in the geometry of the related complex achieving a square-planar rather than tetrahedral complex. The obtainment of metal complexes with Cu (II) could give the possibility to extend the scope of reactions thanks to different coordination geometries and to improve stability towards oxidation.Both His7 and Mets7 peptides were synthetized by Fmoc solid phase peptide synthesis, using Rink-amide resin (0.69 mmol/g) following standard procedures 41,44. The complexes were prepared by mixing equimolar quantities of Mets7 or His7 with Cu (OAc) 2 in MeOH and adding two equivalent of ascorbic acid for inducing the Cu F I G U R E 1 CD spectra (50 μM in water) of (left) Mets7 and Mets7/Cu(I) complex and (right) His7, His7/Cu(I) and His7/Cu (II) complexes (II) reduction to Cu(I).…”

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight

“…The peptides identified by the Y2H strategy and the NoPv1 mutated derivatives were initially prepared by microwave assisted solid phase synthesis, based on Fmoc chemistry on pre-loaded Wang resin (0.4 meq/g substitution) using a fivefold molar excess of 0.2 M Fmoc-protected amino acids dissolved in NMP and using HOBT/HBTU/DIEA (5:5:10) as activators 72 , 73 ). Coupling reactions were performed for 5 min at 40 W with a maximum temperature of 75 °C.…”