Evaluation of exogenous therapeutic protein activity under confinement and crowding effects

The dysfunction of intracellular proteins is frequently associated with various diseases, such as cancer. The exogenous proteins in cells are usually assembled with specific configurations due to physiological confinement/crowding to exhibit novel features in protein structure, folding or conformational stability, distinguished with their behaviors in buffer solutions. Here, we synthesized exogenous proteins in a confined/crowded condition, to explore protein activity within the cells. The findings suggested that the confinement and crowding effects on protein activity are heterogeneous; they showed an inhibitory consequence on HRP by decreasing Km (from ~9.5- and ~21.7-fold) and Vmax (from ~6.8- and ~20.2-fold) lower than that of dilute solutions. Interestingly, the consequences on Cyt C seem more complicated, and the crowding exerts a positive effect by increasing Km ~3.6-fold and Vmax ~1.5-fold higher than that of dilute solutions; however, the confinement takes a negative consequence by decreasing Km ~2.0 and Vmax ~8.3 times. Additionally, in contrast to traditional nanoparticles-based confinement models, we synthesized a biodegradable nanoparticle to mimic the confined space, and the biggest advantage of this novel model is that the particles can be degraded and thus it can provide more intuitive observations of the target protein properties under the confinement and after release. Furthermore, we also evaluated protein activity in different cellular environments, indicating the exogenous protein activity was closely related to the crowdedness of cellular environments, and the inhibition of protein activity in MDA-MB-231 cancer cells was more obvious than HEK293 normal cells. Finally, SAXS analysis revealed the correlation between the protein conformation and the different environments. Our work will provide a unique way for precisely assessing whether the target cellular environments are native matrix in which specific exogenous protein drugs delivered to function or whether they display a therapeutic role, which is of great significance for screening and development of new drugs.