Abstract
Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.
Highlights
Human Angiogenin (Ang) is a 14 kDa protein belonging to the ribonucleases family, with a RNase catalytic activity about 106 lower than pancreatic RNase [1].Ang is a physiological constituent of human plasma but is over-expressed in patients affected by different types of cancers
Ang regulates the expression of other angiogenic factors, such as the vascular endothelial growth factor (VEGF), the epidermal growth factor (EGF), the acidic fibroblast growth factor and the basic fibroblast growth factor [8,9,10,11,12]
The protein angiogenin (Ang) is a potent angiogenic factor whose activity is influenced by copper ions
Summary
Human Angiogenin (Ang) is a 14 kDa protein belonging to the ribonucleases family, with a RNase catalytic activity about 106 lower than pancreatic RNase [1]. A strong correlation between the protein and the metal ion has been demonstrated: Cu2+ increases the expression of Ang and modulates its intracellular localization in HUVEC, affecting angiogenic activity of protein and ERK activation signaling [41] This contrast is likely related to the use of different forms of the Ang protein: the recombinant (r-Ang), mostly used in many literature reports, containing an extra methionine as first residue, and the native form, present in vivo (“wild-type”-angiogenin, wt-Ang), which lacks the free amino terminal group, owing to the spontaneous cyclization of the first glutamine group residue to pyroglutamate [1]. The effect of Ang(1–17) and AcAng(1–17) peptides on the actin aggregation has been tested on human neuroblastoma cells, in the presence or not of copper ions, to exploit their potential use as mimicking system of whole r-Ang and wt-Ang proteins, respectively
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