The antiangiogenic and antitumor activities of the N-terminal fragment of endostatin augmented by Ile/Arg substitution: The overall structure implicated the biological activity Reyhane Chamani a , S. Mohsen Asghari a, ⁎, Ali Mohammad Alizadeh b , Kamran Mansouri c , Taher Doroudi d , Peir Hossein Kolivand d , Hossein Ghafouri a , Somayeh Ehtesham e , Hanieh Rabouti a , Faramarz Mehrnejad f a Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran b Cancer Biology Research Center, Tehran University of Medical Sciences, Tehran, Iran c Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran d Shefa Neuroscience Research Center, Khatam Alanbia Hospital, Tehran, Iran e Department of Biology, Faculty of Science, Islamic Azad University, Parand, Iran f Department of Life Sciences Engineering, Faculty of New Sciences & Technologies, University of Tehran, Iran abstract article info Article history: Received 2 June 2016 Received in revised form 25 August 2016 Accepted 25 September 2016 Available online 28 September 2016 The antiangiogenic and antitumor activities of the 27-amino acid fragment corresponding to the N-terminal do- main of endostatin were shown to be dependent on a Zn-binding loop in the N-terminus. To investigate whether the regions outside of the N-terminal loop play a role in the peptide function, the structure and function of a var- iant containing Ile26Arg mutation (ES-R) were compared with those of the native peptide (ES-Zn). Structural analysis using far-UV CD, intrinsic fluorescence and molecular dynamics simulation provided information re- garding the overall changes upon the mutation. In addition, the docking simulations predicted a higher affinity of ES-R to integrins α v β 3 and α 5 β 1 than ES-Zn and a profound reorganization of the binding residues throughout the sequence. In Human Umbilical Vein Endothelial Cells (HUVECs), ES-R inhibited the tube formation and acti- vated caspase-3 more strongly than do ES-Zn. Based on in vivo studies, the growth of breast tumor and expression of CD31, Bcl-2 and nonfunctional p53 were inhibited more effectively by ES-R than by ES-Zn. We conclude that the C-terminal region is involved in the peptide function through some global structural effects. © 2016 Elsevier B.V. All rights reserved. Keywords: Endostatin peptide Arginine Structure Angiogenesis Tumor Apoptosis 1. Introduction Angiogenesis, the formation of new blood vessels, plays a critical role in the growth and spread of tumors. Inhibition of angiogenesis is an ef- fective strategy for treatment of cancer. The growth of solid relies on a shift in the balance of proangiogenic and antiangiogenic factors. The most important proangiogenic factors are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Several antiangiogenic has been found including antiangiogenic factors, hor- mone metabolites, and apoptosis modulators. Extensive research has been conducted to inhibit the proangiogenic factors, e.g. using monoclo- nal antibodies [1] and endogenous inhibitors of angiogenesis [2]. Endostatin, a 20-kDa fragment derived from the C-terminus of colla- gen XVIII, is a potent endogenous inhibitor of angiogenesis and tumor growth [3]. The structure of human endostatin contains a basic patch of 11 Arg residues on the protein surface that clustered in two regions: a major site, including Arg 24, Arg 27, Arg 53, and Arg 139, and a minor site consisting of Arg 62 and 63 [4,5]. The Arg residues are critical for the antiangiogenic activity of endostatin [6–10]. For instance, it was found that a double mutation R27A/R139A strongly decreases the binding ca- pability of endostatin to integrins α 5 β 1 and α v β 3 [8], and R158A/R270A mutation in murine endostatin (corresponding to R27A/R139A in human endostatin) predominantly reduced its internalization to endo- thelial cells and the antiangiogenic and antitumor activities [11]. A 27-amino acid fragment from the N-terminus of endostatin was shown to preserve the antiangiogenic and antitumor activities of the molecule [12]. The biological activity of this fragment, termed ES-Zn in this study (Table 1), is crucially dependent on the Zn-binding loop formed by residues 1 to 11. We have recently demonstrated that loop formation rather than Zn-binding is relevant to the antiangiogenic and antitumor activities of ES-Zn [13]. In addition, the endostatin fragment was found to be less structured when the loop structure was destroyed. Importantly, ES-Zn comprises three out of eleven Arg residues of full- length endostatin, including Arg4, Arg24 and Arg27 that are expected to be involved in its antiangiogenic activity. Torres et al. predicted that Arg residues are determinant for the conformation of this fragment as well as for its binding to integrin α v β 3 [14]. Experimental investigations Biochimica et Biophysica Acta 1864 (2016) 1765–1774 ⁎ Corresponding author. E-mail addresses: sm_asghari@guilan.ac.ir, asghari.smohsen@gmail.com (S.M. Asghari). http://dx.doi.org/10.1016/j.bbapap.2016.09.014 1570-9639/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap