RESUMO
Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.
Assuntos
Fatores Inibidores da Migração de Macrófagos/química , Benzoquinonas/química , Domínio Catalítico , Cristalografia por Raios X , Dimerização , Iminas/química , Cinética , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Modelos Moleculares , Conformação Molecular , Fluoreto de Fenilmetilsulfonil/química , Ligação Proteica , Inibidores de Serina Proteinase/químicaRESUMO
BACKGROUND & AIMS: The cytokine macrophage migration inhibitory factor (MIF) is expressed throughout the human gastrointestinal tract. Recently, protumorigenic activity of MIF has been described in several cancer models. Therefore, we investigated the expression and function of MIF during the early stages of intestinal tumorigenesis. METHODS: MIF messenger RNA, protein, and tautomerase activity were measured in normal intestinal mucosa and adenomas from patients with sporadic colorectal adenomas and in the adenomatous polyposis coli (Apc)Min/+ mouse model of intestinal tumorigenesis. MIF function was investigated by using VACO-235 human colorectal adenoma cells in vitro and by testing the effect of genetic deletion of Mif on ApcMin/+ mouse intestinal tumorigenesis. RESULTS: MIF expression and tautomerase activity were increased in human and ApcMin/+ mouse intestinal adenomas compared with adjacent normal mucosa. Up-regulation of MIF occurred mainly in epithelial cells (associated with an increasing grade of dysplasia), but also in stromal plasma cells. Exogenous MIF inhibited apoptosis and promoted anchorage-independent growth of VACO-235 cells (maximal at 100 ng/mL). Homozygous deletion of Mif was associated with a reduction in the number and size of ApcMin/+ mouse adenomas (P = .025 for the difference in large [>7-mm] tumors) and decreased angiogenesis (43% decrease in mean tumor microvessel density), but there was no alteration in epithelial cell apoptosis or proliferation. CONCLUSIONS: MIF expression is increased in sporadic human colorectal adenomas, and exogenous MIF drives tumorigenic behavior of epithelial cells in vitro. Mif also promotes intestinal tumorigenesis (predominantly via angiogenesis) in the ApcMin/+ mouse. Therefore, MIF is a potential colorectal cancer chemoprevention target.
Assuntos
Adenoma/fisiopatologia , Polipose Adenomatosa do Colo/fisiopatologia , Neoplasias Colorretais/fisiopatologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/patologia , Animais , Apoptose , Divisão Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mucosa/metabolismo , Mucosa/patologia , Mucosa/fisiopatologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Células Tumorais CultivadasRESUMO
Macrophage migration inhibitory factor (MIF) is an immunoregulatory protein that is a potential therapeutic target for a number of inflammatory diseases. Evidence exists that an unexpected catalytic active site of MIF may have a biological function. To gain further insight into the role of the catalytic active site, a series of mutational, structural, and biological activity studies were performed. The insertion of an alanine between Pro-1 and Met-2 (PAM) abolishes a non-physiological catalytic activity, and this mutant is defective in the in vitro glucocorticoid counter-regulatory activity of MIF. The crystal structure of MIF complexed to (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF d-dopachrome tautomerase activity, reveals that ISO-1 binds to the same position of the active site as p-hydroxyphenylpyruvic acid, a substrate of MIF. ISO-1 inhibits several MIF biological activities, further establishing a role for the catalytic active site of MIF.
