Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway.

In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.