De Novo Design, Synthesis, and Mechanistic Evaluation of Short Peptides That Mimic Heat Shock Protein 27 Activity

Abstract

We report the first small molecule peptides based on the N-terminal sequence of heat shock protein 27 (Hsp27, gene HSPB1) that demonstrates chaperone-like activity. The peptide, comprising the SWDPF sequence located at Hsp27's amino (N)-terminal domain, directly regulates protein aggregation events, maintaining the disaggregated state of the model protein, citrate synthase. While traditional inhibitors of protein aggregation act via regulation of a protein that facilitates aggregation or disaggregation, our molecules are the first small peptides between 5 and 8 amino acids in length that are based on the N-terminus of Hsp27 and directly control protein aggregation. The presented strategy showcases a new approach for developing small peptides that control protein aggregation in proteins with high aggregate levels, making them a useful approach in developing new drugs.

Figure 1

Overview of Hsp27 structure and activity. (a) There is evidence that the dissociated forms of Hsp27 (dimers) are the chaperone active species and that the large oligomeric forms are inactive. Thus, a potential mechanism to activate Hsp27 in the context of disease is to develop a molecule that dissociates the oligomers or a mini-chaperone that can mimic Hsp27 activity. (b) Protein structure of Hsp27 with key regions containing residues critical for Hsp27 function as discussed.

Figure 2

(a) Structure of the peptides A (GPSWDPFRDW), B (ALSRQLSSGV), C (YISRCFTRKY), D (SNEITIPVTF) and scrambled peptide (SP; SFNTTPEVLL). Effects of peptides A–D and SP on CS aggregation, as monitored by light scattering at 360 nm. CS (150 nM) was heated at 43 °C for 1 h in the presence of (b) 50 μM compound or (c) increasing concentrations of peptide A. Aggregation of CS with 1% DMSO was set to 100%, and Hsp27 (600 nM) reflects the impact of the chaperone on CS on the day that the experiments were run. All data and error bars represent mean ± SEM for at least three independent experiments.

Figure 3

(a) Structures of peptides 1–3. (b) Effects of 1–3 on the CS aggregation at 43 °C, as monitored by light scattering at 360 nm. All peptides were tested at 200 μM unless otherwise indicated. Aggregation of CS (150 nM) with 1% DMSO was set to 100% and Hsp27 (600 nM). All data and error bars represent mean ± SEM for at least three independent experiments.

Figure 4

(a) Structure of peptides 4–8. (b) Effects of peptides 4–8. (c) Structure of peptides 9 and 10. (d) Effects of 9 and 10 on CS aggregation. (e) Structure of peptides 11–13. (f) Effects of peptides 11–13 on CS aggregation. All peptides were tested at 200 μM unless otherwise indicated. CS aggregation was monitored by light scattering at 360 nm. Aggregation of CS (150 nM) with 1% DMSO was set to 100%, and Hsp27 (600 nM) reflects the impact of the chaperone on CS on the day that the experiments were run. All data and error bars represent mean ± SEM for at least two independent experiments.

Figure 5

Effect of Hsp27 and peptides 2, 12, and 13 on CS aggregation, as monitored by light scattering at 320 nm. Chemically denatured CS (150 nM) was monitored for 1 h in the presence of Hsp27 (600 nM) and peptides 2, 12, and 13 (200 μM). Aggregation of CS with 1% DMSO was set to 100%, and Hsp27 (600 nM) reflects the impact of the chaperone on CS on the day the experiments were run. All data and error bars represent mean ± SEM for at least two independent experiments.