Control of Enzyme Reactivity in Response to Osmotic Pressure Modulation Mimicking Dynamic Assembly of Intracellular Organelles

Abstract

In response to variations in osmotic stress, in particular to hypertonicity associated with biological dysregulations, cells have developed complex mechanisms to release their excess water, thus avoiding their bursting and death. When water is expelled, cells shrink and concentrate their internal bio(macro)molecular content, inducing the formation of membraneless organelles following a liquid–liquid phase separation (LLPS) mechanism. To mimic this intrinsic property of cells, functional thermo‐responsive elastin‐like polypeptide (ELP) biomacromolecular conjugates are herein encapsulated into self‐assembled lipid vesicles using a microfluidic system, together with polyethylene glycol (PEG) to mimic cells’ interior crowded microenvironment. By inducing a hypertonic shock onto the vesicles, expelled water induces a local increase in concentration and a concomitant decrease in the cloud point temperature (Tcp) of ELP bioconjugates that phase separate and form coacervates mimicking cellular stress‐induced membraneless organelle assemblies. Horseradish peroxidase (HRP), as a model enzyme, is bioconjugated to ELPs and is locally confined in coacervates as a response to osmotic stress. This consequently increases local HRP and substrate concentrations and accelerates the kinetics of the enzymatic reaction. These results illustrate a unique way to fine‐tune enzymatic reactions dynamically as a response to a physiological change in isothermal conditions.

Macromolecular Crowding Effect on Chitosan-Hyaluronic Acid Complexation and the Activity of Encapsulated Catalase

“…The macromolecular crowding of the intracellular environment could also dramatically modulate the kinetics of biochemical reactions via its influence on the diffusion rate, binding kinetics, and binding strength of biomacromolecules, enzymes, and substrates. − To investigate the effect of PEG crowding and PEC encapsulation on enzyme activity, we used CAT as a model enzyme and tested its activity under dilute, crowded, CS/HA encapsulated, and crowding/encapsulation combined conditions (Figures , S9, and S10). When fixing the CS (4.0 g/L) and CAT (0.40 g/L) concentrations, the encapsulation efficiency of catalase decreased from 87 to 29% as the HA concentration increased from 2.0 to 6.0 g/L (Figure a).…”

Protocells Featuring Membrane-Bound and Dynamic Membraneless Organelles

“…Therefore, a thick-shell microfluidic system was used to generate a W/O/W double emulsion (Figure ). By using this technique, partially dewetted liposomes were produced, as described in our prior research, , which we use as an artificial cell-like chassis.…”

Contribution of the ELRs to the development of advanced in vitro models

“…By creating cytomimetic protocellular models using water-in-oil-in-water (W/O/W) double emulsions and employing monoblock ELRs and ELR-bioconjugates with PEG and horse radish peroxidase as a model multicomponent enzymatic system, a study model for the kinetics of enzymatic reactions under different osmotic pressures was established. This approach demonstrated that synthetic condensates significantly improved the rate of enzymatic reactions after hypertonic shock ( Schvartzman et al, 2023 ).…”