Amyloid-like RIP1/RIP3 RHIM Fragments' Characterization and Application as a Drug Depot

Abstract

Amyloid aggregates play a major role in diseases as well as in normal physiological function. Receptor-interacting protein kinases 1 and 3 (RIP1/RIP3) aggregates complexes in cellular necroptosis is one example of protein aggregation in normal cellular function. Although recently there have been several studies on full kinase proteins aggregation, the aggregation potential of small peptide sequences of RIP1/RIP3, the physicochemical properties, and the potential in biomedical applications have not been explored. Hence, in this paper, we study the aggregation propensity of peptides consisting of four and twelve amino acid sequences in the RHIM region of RIP1/RIP3 proteins that are known to drive the beta-sheet formation and the subsequent aggregation. The aggregation kinetics, physicochemical characterization, mechanosensitive properties, cellular effects, and potential as a cancer drug depot have been investigated. The results show that the number and concentration of amino acids play a role in amyloid-like aggregates' properties. Further, the aggregates when formulated with cisplatin-induced significant lung cancer cell toxicity compared to an equal amount of cisplatin with and without ultrasound. The study would serve as a platform for further investigation on RIP1/RIP3 peptide and protein aggregates, their role in multiple cellular functions and diseases, and their potential as drug depots.

Keywords: Amyloid; RIP1; RIP3; aggregation.

Figure 1

(A) Thioflavin-T characterization of aggregation of RHIM fragments of RIP1, RIP3, and RIP1/RIP3 complex after 1 day. RHIM fragments composed of 4 amino acid residues (RIP1(4), RIP3(4), RIP1(4)/RIP3(4)) and 12 amino acid residues (RIP1(12), RIP3(12), RIP1(12)/RIP3(12)) were used for the study. (B) Turbidity measurement of the corresponding RHIM fragment aggregates after 1 day. n = 3 ± SEM, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Peptide aggregation detection with proteostat assay. RHIM fragments were aggregated for 1 day in 100 mM ammonium acetate buffer, pH 7. Lysozyme monomers and aggregates were used as controls. (A) Four amino acid fragments exhibited a monomers-like scattering profile similar to lysozyme monomers. (B) Twelve amino acid fragments exhibited a protein aggregates-like scattering profile similar to the lysozyme aggregates.

Figure 3

Effect of concentration on 12 amino acid RHIM fragments RIP1, RIP3, and RIP1/RIP3 aggregation. (A). ThT fluorescence of 100 µM peptides aggregation. (B) ThT fluorescence of 1 mM peptides aggregation. n = 3 ± SEM, * p < 0.05, ** p < 0.01.

Figure 4

(A) Transmission electron microscopy (TEM) images of 12 amino acid RHIM fragments RIP1, RIP3, and RIP1/RIP3 aggregation kinetics. The images reveal significant aggregation after 1 day compared to 30 min. Scale bar 100 nm. (B) Ultrasound (US) mediated mechanosensitive effects of aggregates assessed with TEM. The images reveal the aggregates mechano sensitivity to ultrasound 2.2 W/cm², 5 min. Scale bar 100 nm.

Figure 5

Ultrasound mediated uptake of the aggregates assessed with flowcytometry. The scattering measurements reveal cellular uptake of RIP1, RIP3, and RIP1/RIP3 aggregates were increased in the presence of ultrasound 2.2 W/cm², 5 min.

Figure 6

(A,B) Effect of aggregates on normal cell toxicity and ROS. Normal lung BEAS-2B cells were plated in 96 wells at a density of 10,000 cells/well. RIP1/RIP3 aggregates were added at a concentration of 10 µM and incubated for 48 h, and Alamar blue/ROS measurements were performed. (C) TNF alpha assay. Raw264.7 cells were plated at a density of 10,000 cells/well in 96 well plates. RIP1/RIP3 aggregates were added at a concentration of 100 µM and incubated for 48 h. TNF-alpha assay was performed according to the manufacturers protocol, and measurements were obtained using a M3 plate reader.

Figure 7

(A) LDH toxicity assay of H1299 cells. Cells were treated with 5 µM and 10µM cisplatin alone or encapsulated with RIP1/RIP3 peptide aggregates. Supernatant from the treated cells were treated with LDH reagents, and the absorbance measurements were taken at 490 nm. Control consisted of lysate from control cells in the absence of peptide aggregates or cisplatin. (B) Ultrasound-mediated delivery of RIP1/RIP3 peptide aggregates encapsulated with cisplatin significantly improve cellular toxicity. LDH toxicity assay was of H1299 cells treated with cisplatin conditions with and without ultrasound 1.6 W/cm², for 3 min. Controls consisted of lysate from control cells in the absence of peptide aggregates or cisplatin with and without ultrasound. n = 3 ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.