RESUMO
Nucleophosmin (NPM1) is an abundant, nucleolar tumor antigen with important roles in cell proliferation and putative contributions to oncogenesis. Wild-type NPM1 forms pentameric oligomers through interactions at the amino-terminal core domain. A truncated form of NPM1 found in some hepatocellular carcinoma tissue formed an unusually stable oligomer and showed increased susceptibility to cleavage by granzyme B. Initiation of translation at the seventh methionine generated a protein (M7-NPM) that shared all these properties. We used deuterium exchange mass spectrometry (DXMS) to perform a detailed structural analysis of wild-type NPM1 and M7-NPM, and found dynamic conformational shifts or local "unfolding" at a specific monomer-monomer interface which included the ß-hairpin "latch." We tested the importance of interactions at the ß-hairpin "latch" by replacing a conserved tyrosine in the middle of the ß-hairpin loop with glutamic acid, generating Y67E-NPM. Y67E-NPM did not form stable oligomers and further, prevented wild-type NPM1 oligomerization in a dominant-negative fashion, supporting the critical role of the ß-hairpin "latch" in monomer-monomer interactions. Also, we show preferential cleavage by granzyme B at one of two available aspartates (either D161 or D122) in M7-NPM and Y67E-NPM, whereas wild-type NPM1 was cleaved at both sites. Thus, we observed a correlation between the propensity to form oligomers and granzyme B cleavage site selection in nucleophosmin proteins, suggesting that a small change at an important monomer-monomer interface can affect conformational shifts and impact protein-protein interactions.
Assuntos
Medição da Troca de Deutério , Granzimas/química , Granzimas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Proteínas Nucleares/genética , Nucleofosmina , Ligação Proteica , Multimerização Proteica , Homologia de Sequência de AminoácidosRESUMO
The increasing recognition that cancer is frequently associated with an autoantibody response, and observations that systemic autoimmunity is sometimes associated with the diagnosis of a variety of malignancies (many detected near the onset of autoimmune disease), strongly underscore a potential mechanistic connection between cancer immunity and systemic autoimmunity. Accumulating data suggest that autoantigens are critical partners in driving the autoimmune response. Furthermore, unique changes in antigen expression and conformation in the immunizing tumor and the target tissue may play a role in antigen selection and ongoing damage. This construct has important implications for diagnosis, monitoring, and treatment of autoimmunity and, potentially, cancer immunotherapy.
