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Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide

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Abstract

The structure and function of a 27-a.a. fragment of the N-terminal sequence of human endostatin (ES-Zn) were compared to those of the mutant peptide (ES-SSZn) obtained by adding Cys-Pro-Ala to the endostatin N-terminus and substituting Asn16 for Cys ensuring formation of a disulfide bond. Structural comparison of ES-Zn and ES-SSZn by far-UV circular dichroism (CD), intrinsic fluorescence, and molecular dynamics simulation methods revealed significant structural perturbations in ES-SSZn, such as elimination of the β-sheet conformer, modification of the N-terminal loop structure, and reorganization of dynamic properties of the entire peptide backbone. ES-SSZn was approximately 2 and 3 times less efficient than ES-Zn and the full-length human endostatin, respectively, in the induction of caspase-3-dependent apoptosis in human umbilical vein endothelial cells (HUVECs) in vitro (p < 0.05). In contrast, treatment of metastatic 4T1 breast tumors in mice with ES-Zn and ES-SSZn (5 mg/kg body weight daily) for 14 days resulted in similar regression of tumor size, comparable downregulation of angiogenesis (CD31 and CD34) and cell proliferation (Ki67), and therefore, the same extent of apoptosis induction (TUNEL, p53, and Bcl-2) for both peptides (as compared to the untreated controls). Western blot analysis of HUVEC and 4T1 tumor lysates revealed the same levels of suppression of key signaling mediators Akt and ERK1/2 by ES-Zn and ES-SSZn. Contrary to the earlier studies, our results showed that the function of the 1-27 endo-statin fragment is independent of its overall structure. Stabilization of the N-terminal loop structure by the disulfide bond incorporation causes relief from structural deviations.

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Abbreviations

Akt:

serine/threonine protein kinase B

DMEM:

Dulbecco’s modified Eagle’s medium

DSSP:

dictionary of secondary structure of proteins (hydrogen bond estimation algorithm)

ERK1/2:

extracellular signal-regulated kinase 1/2

ES-Zn:

a 27-a.a. fragment of the N-terminal sequence of endo-statin

ES-SSZn:

mutant peptide derived by addition of Cys-Pro-Ala to the N-terminus of ES-Zn peptide and substitution of Asn16 for Cys required for the disulfide bond formation

HUVECs:

human umbilical vein endothelial cells

MAPK:

mitogen-activated protein kinase

MD:

molecular dynamics

PBS:

phosphate buffered saline

PI3K:

phosphoinositide 3-kinase

RMSD:

root mean square deviation

RMSF:

root mean square fluctuation

TUNEL:

terminal deoxynucleotidyl trans-ferase-mediated dUTP nick end labeling

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Authors and Affiliations

  1. Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

    S. Ehtesham & A. Eidi

  2. Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

    R. Sariri

  3. Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

    S. Hosseinkhani

Authors
  1. S. Ehtesham
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  2. R. Sariri
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  3. A. Eidi
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  4. S. Hosseinkhani
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Corresponding authors

Correspondence to R. Sariri or S. Hosseinkhani.

Additional information

Published in Russian in Biokhimiya, 2018, Vol. 83, No. 11, pp. 1708–1721.

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Ehtesham, S., Sariri, R., Eidi, A. et al. Effect of Disulfide Bond Incorporation on the Structure and Activity of Endostatin Peptide. Biochemistry Moscow 83, 1388–1398 (2018). https://doi.org/10.1134/S0006297918110093

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  • DOI: https://doi.org/10.1134/S0006297918110093

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