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Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation - PubMed

  • ️Tue Jan 01 2019

Roseltide rT7 is a disulfide-rich, anionic, and cell-penetrating peptide that inhibits proteasomal degradation

Antony Kam et al. J Biol Chem. 2019.

Abstract

Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S-alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies.

Keywords: Hibiscus sabdariffa; cell-penetrating peptide (CPP); cystine-knot peptide; drug action; drug discovery; enzyme inhibitor; natural product; peptide chemical synthesis; peptides; plant peptide; protease inhibitor; proteasome; proteasome inhibitor; roseltide rT7; structure-function.

© 2019 Kam et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.

A, the calyx of H. sabdariffa. B, sequence comparison between roseltide rT1 and rT7 as described previously (2). C, sequence comparison between the IIML motif of roseltide rT7, epoxomicin, and carmaphycin.

Figure 2.
Figure 2.

Roseltide rT7 structure based on NMR. A, left panel, 17 lowest energy structures of roseltide rT7. Right panel, cartoon view of roseltide rT7 with disulfide bonds in yellow. The side chains of the three proline residues located in loop 4 are shown. B, structure of roseltide rT1 (PDB entry 5GSF) and rT7 (PDB entry 6KLM). Loop 4 is colored in green. IIML motif is colored in red.

Figure 3.
Figure 3.

Synthesis and site-specific labeling of roseltide rT7. Synthetic scheme for roseltide rT7 by solid-phase peptide synthesis, as well as N-terminally fluorescence-labeled roseltide rT7.

Figure 4.
Figure 4.

Roseltide rT7 is stable against proteolytic degradation. A–D, comparison of the peptide stability of roseltide rT7 and S-alkylated rT7 (iodoacetamido-) under trypsin (A), Pronase (B), pepsin (C), and human neutrophil elastase (D) treatment as analyzed by RP-HPLC (n = 3). N.D., not detected.

Figure 5.
Figure 5.

Cellular uptake of TAMRA-rT7 is endocytosis-dependent. A, A549 cells after incubation with TAMRA-rT7 (1 μ

m

) using live-cell confocal microscopy at 37 °C. B, flow cytometry analysis of A549 cells after incubation with TAMRA-rT7 (1 μ

m

) at 37 °C. C, flow cytometry analysis of A549 cells incubated at 4 °C for 30 min prior to incubation with TAMRA-rT7 (1 μ

m

) for 1 h at 4 °C (n = 3). p < 0.05 compared with control.

Figure 6.
Figure 6.

Roseltide rT7 is a human 20S proteasome inhibitor. A, representative Western blotting analysis on the ubiquitinated protein expressions in A549 cells for 2 h with 10 μ

m

roseltide rT7; 1 μ

m

MG132 was used as positive control. B, dose-response effect of roseltide rT7 and MG132 on human 20S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 20S chymotrypsin-like proteasome activities are 3.3 ± 0.04 and 0.13 ± 0.03 μ

m

, respectively. C, in-gel fluorescence image of human 20S proteasome treated with roseltide rT7 or MG132 for 1 h followed by 30 min with Me4BodipyFL-Ahx3Leu3VS probe. D, MALDI-TOF MS profiles of roseltide rT7 (10 μ

m

) without (upper panel) or with human 20S proteasome (5 n

m

) (lower panel) for 24 h at 37 °C. E, dose-response effect of roseltide rT7 and MG132 on human 26S chymotrypsin-like proteasome activities using Proteasome-GloTM chymotrypsin-using assay kit. The IC50 values for roseltide rT7 and MG132 against human 26S chymotrypsin-like proteasome activities are 14.93 ± 0.04 and 0.05 ± 0.02 μ

m

, respectively. F, effects of linear pentapeptides, IIMLP, IIMLA, IIMLK, IIMPA, and IIMPK, on human 20S chymotrypsin-like proteasome activities using a Proteasome-GloTM chymotrypsin-using assay kit. The proteasome cleavage of P1′-P1 site was determined using RP-HPLC and LC-MS analysis. P(X) refers to N-terminal to the substrate cleavage site. P(X)′ refers to C-terminal to the substrate cleavage site.

Figure 7.
Figure 7.

Roseltide rT7 inhibits TNF-induced IκBα degradation and ICAM-1 expressions. A, representative Western blotting analysis on the IκBα expression in A549 cells following 30 min of TNF (50 ng/ml) incubation with or without 10 μ

m

roseltide rT7 for 2 h; MG132 was used as positive control. B, the gene expressions of ICAM-1 in A549 cells following 4 h TNF (50 ng/ml) incubation with or without 10 μ

m

roseltide rT7 for 2 h as determined by real-time qPCR; 1 μ

m

MG132 was used as positive control. C, the surface protein expressions of ICAM-1 in A549 cells following 4 h of TNF (50 ng/ml) incubation with or without 10 μ

m

roseltide rT7 for 2 h as determined by flow cytometry using anti–ICAM-1–PE antibody; 1 μ

m

MG132 was used as positive control. D, schematic diagram for the inhibitory effects of roseltide rT7 on TNF-mediated IκBα proteasomal degradation and ICAM-1 expressions. All results are expressed as means ± S.E. (n = 3). *, p < 0.05 compared with control group.

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References

    1. Riaz G., and Chopra R. (2018) A review on phytochemistry and therapeutic uses of Hibiscus sabdariffa L. Biomed. Pharmacother. 102, 575–586 10.1016/j.biopha.2018.03.023 - DOI - PubMed
    1. Loo S., Kam A., Xiao T., Nguyen G. K., Liu C. F., and Tam J. P. (2016) Identification and characterization of roseltide, a knottin-type neutrophil elastase inhibitor derived from Hibiscus sabdariffa. Sci. rep. 6, 39401 10.1038/srep39401 - DOI - PMC - PubMed
    1. Kam A., Loo S., Dutta B., Sze S. K., and Tam J. P. (2019) Plant-derived mitochondria-targeting cysteine-rich peptide modulates cellular bioenergetics. J. Biol. Chem. 294, 4000–4011 10.1074/jbc.RA118.006693 - DOI - PMC - PubMed
    1. Groll M., Kim K. B., Kairies N., Huber R., and Crews C. M. (2000) Crystal structure of epoxomicin: 20S proteasome reveals a molecular basis for selectivity of α′,β′-epoxyketone proteasome inhibitors. J. Am. Chem. Soc. 122, 1237–1238 10.1021/ja993588m - DOI
    1. Meng L., Mohan R., Kwok B. H., Elofsson M., Sin N., and Crews C. M. (1999) Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. Proc. Natl. Acad. Sci. U.S.A. 96, 10403–10408 10.1073/pnas.96.18.10403 - DOI - PMC - PubMed

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