Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties

Abstract

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2′s activity remain unclear. To elucidate the structure–activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.

The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells

“…In accordance with recent reports, we have observed the dimerization of RL2 in breast cancer cells [11]. In addition, we have found out that RL2 co-localizes with mitochondria in breast cancer cells.…”

“…Efficient internalization of RL2 by cancer cells provides the essential prerequisite for its action [28]. The positively charged RL2 has a pI value of 9.85 and a hydrophobicity of −0.9, enabling RL2 to pass the plasma membrane of the cells [11,12]. Due to these properties, there seems to be no need for a specific RL2 receptor or an active transport through the cell membrane.…”

“…bridges, and therefore, should mostly diminish after SDS-PAGE under reducing conditions [11]. This is in contrast to the analysis of RL2 via SDS-PAGE under non-reducing conditions, in which the formation of the homodimers can be efficiently detected [6].…”

“…Importantly, RL2 was shown to spare normal tissue, such as non-malignant mesenchymal stem cells (MSCs) [6]. RL2 has been reported to exist as a mixture of both monomer and dimer forms, the latter are formed via formation of disulfide bonds [11,12]. However, the detailed molecular mechanisms of RL2 interference with cell death machinery are still not known.…”

Recombinant Lactaptin Induces Immunogenic Cell Death and Creates an Antitumor Vaccination Effect in Vivo with Enhancement by an IDO Inhibitor

“…The comparison of RL2 primary structure and the mechanism of its penetration into the cell suggests that it can be assigned to the cell penetrating peptide (supplementary materials) [7,8]. The N-terminal region, which is essential for proapoptotic activity, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure [9]. RL2 was shown to induce cell death with the hallmarks of various types of programmed cell death: apoptosis, ATP depletion-dependent necrosis, and autophagy [3,10,11].…”