Consequences of point mutations in melanoma-associated antigen 4 (MAGE-A4) protein: Insights from structural and biophysical studies.

Yoshio Hagiwara,Jensy Anton,Ernesto Cota,Farina Hanif,Eleanor R. Dickinson,Lina Sieverling,Tam T. T. Bui,Antonina Andreeva,Perdita E. Barran,Penka V. Nikolova

doi:10.1038/srep25182

Abstract

The Melanoma-Associated Antigen A4 (MAGE-A4) protein is a target for cancer therapy. The function of this protein is not well understood. We report the first comprehensive study on key cancer-associated MAGE-A4 mutations and provide analysis on the consequences of these mutations on the structure, folding and stability of the protein. Based on Nuclear Magnetic Resonance and Circular Dichroism, these mutations had no significant effects on the structure and the folding of the protein. Some mutations affected the thermal stability of the protein remarkably. Native mass spectrometry of wild-type MAGE-A4 showed a broad charge state distribution suggestive of a structurally dynamic protein. Significant intensity was found in relatively low charge states, indicative of a predominantly globular form and some population in more extended states. The latter is supported by Ion Mobility measurements. The MAGE-A4 mutants exhibited similar features. These novel molecular insights shed further light on better understanding of these proteins, which are implicated in a wide range of human cancers.

Highlights

Cancer/Testis (CT) antigens are a large family of proteins typically expressed in germ cells
In order to assess the folding state and secondary structure of Melanoma-Associated Antigen A4 (MAGE-A4) and examine the effects of mutations on them, the proteins were subjected to circular dichroism (CD) in the Far-UV region (260–195 nm)
MAGE-A2 has been reported to repress the activity of tumor suppressor protein p53 through recruitment of histone deacetylase (HDAC) or by inhibiting the interaction between p53 and DNA23,24

Summary

Introduction

Cancer/Testis (CT) antigens are a large family of proteins typically expressed in germ cells. Extensive chromosomal sequencing identified more than 50 genes of the MAGE family[9,10,11,12,13,14,15,16,17,18]. Many reports correlate over-expression of type-I MAGEs with cancer malignance, tumor growth and poor patient prognosis. MAGE-A2 was reported to promote tumor growth in normal oral keratinocytes and inhibits cell cycle arrest www.nature.com/scientificreports/. This report showed that in tamoxifen-treated breast cancer patients, there was a significant link between MAGE expression and reduced overall survival. A truncated form of MAGE-A4 (the C-terminal 107 amino acids) was reported to induce apoptosis by interacting with POZ domain/zinc finger transcription factor Miz-127

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Conclusion