Monomer-Dimer Equilibrium of Human Cystatin C During Internalization Into Cancer Cells

Graphical Abstract

The experimental pathway followed within the manuscript. Human cystatin C (hCC) was labeled with sulfo-Cyanine 5.5 NHS ester (Cy5.5) and incubated with HeLa cells. The internalization of hCC in the cells was monitored with confocal microscopy to understand the influence of the oligomeric state of hCC on the cell membrane-protein interaction.

Abstract

Human cystatin C (hCC) is a physiologically important protein that serves as intra- and extracellular cysteine proteinase inhibitor in homeostasis. However, in pathological states it dimerizes and further oligomerizes accumulating into a toxic amyloid. HCC forms an active monomer in the extracellular space and becomes an inactive dimer when internalized in cellular organelles. However, hCC cell penetration and its oligomeric state during this process are not well understood. To determine if and how the oligomeric state influences hCC transmembrane migration, we investigated the internalization of the hCC wild type protein as well as three different mutants, which exclusively exist in the monomeric or multimeric state into HeLa cells via confocal fluorescence microscopy. Our results showed that the preferred pathway was endocytosis and that the oligomeric state did not significantly influence the internalization because both monomeric and dimeric hCC migrated into HeLa cells. Considering the differences of the active monomeric and the passive dimeric states of hCC, our findings contribute to a better understanding of the intra and extra cellular functions of hCC and their interaction with cysteine proteases.