Analysis of Nickel-Binding Peptides in a Human Hepidermoid Cancer Cell Line by Ni-NTA Affinity Chromatography and Mass Spectrometry

Abstract

To elucidate whether eukaryotic elongation factor 1A (eEF-1A) in a human hepidermoid cancer cell line (H1355) belonged to the family of the Ni-interacting protein, we analyzed the sequence of peptides obtained by on-Ni-NTA-agarose tryptic digestion of proteins from H1355 cell extract. LC/MS analysis showed the presence of several peptides mainly from abundant cellular proteins corresponding to eEF-1A, tubulin and actin. The results indicated that F-actin strongly binds to Ni-NTA-agarose whereas the other proteins are indirectly bound to the resin because of the formation of a protein-protein complex with actin.

Translational Control in Tumour Progression and Drug Resistance

“…A recent example of eEF1A1 involvement in apoptosis suggests that this protein mediates lipotoxic cell death through a mechanism independent from changes in the rates of protein synthesis. Since eEF1A1 plays an important role in remodelling microtubules and filamentous actin [56,69,70] and because the cytoskeleton undergoes dramatic changes during apoptosis and cell death, eEF1A1 may mediate cytoskeletal changes required to execute cell death programs in response to lipotoxic conditions [71].…”

Structural Biomedicine: Characterization of the Structural Basis in Protein-Drug Recognition in Different Human Diseases

“…Several studies have been carried out in which different molecules have been identified to interact with eEF1A, either involving the entire protein or just localized regions within specific domains, some reviewed in section 1.3.3.1. Thus, it has been shown that (i) F-actin interacts with domains I and III of Dictyostelium EF1α (Liu, et al, 1996) and domain II of human eEF1A from an epidermoid cancer cell line (Lamberti, et al, 2008); (ii) activation-induced deaminase (AID) binds to Domain III of eEF1A leading to cytoplasmic retention (Hälser, Rada and Neuberger, 2012); (iii) SH3 domain-containing adaptor protein SORBS2, involved in the assembly of signaling complexes, interacts with domain II of eEF1A1 near the membrane (Lamberti, et al, 2011); and (iv) SH2 and SH3 domains of diverse signaling molecules, such as Crk, Fgr, Fyn, Grb2, RasGAP, Shc and Shp2, possess different abilities to bind to phosphotyrosine-containing sites in domain I of both eEF1A1 and eEF1A2 (Panasyuk, et al, 2008). Further studies directed at characterizing eEF1A complexes and the PTMs involved are required to clarify, at the molecular level, the plethora of events in which this elongation factor is involved.…”

“…As actin cytoskeleton shown significant perturbations in cancer cells), eEF1A2 may contribute to the oncogenic transformation of cells (Stevenson, Veltman and Machesky, 2012. Furthermore, it has been shown that F-actin interacts with domains I and III of Dictyostelium EF1α (Liu, et al, 1996) and domain II of human eEF1A from an epidermoid cancer cell line (Lamberti, et al, 2008).…”