RESUMO
BACKGROUND/AIM: Colorectal cancer is the third most common cancer globally, and the poor prognosis of patients with metastatic colorectal cancer (mCRC) warrants urgent attention. We previously obtained 10 candidate serum biomarkers for mCRC. Our aim with this study was to determine the prognostic performance of the pre-treatment serum C-C motif chemokine ligand 7 (CCL7) concentration in patients with mCRC. PATIENTS AND METHODS: Protein concentrations of CCL7 were examined using ELISA and immunohistochemistry for serum (n=110) and surgical specimens (n=85), respectively, of patients with mCRC. The relationship between protein concentration and prognosis was examined using Cox regression analysis, receiver operator characteristic curve analysis and the Kaplan-Meier method. RESULTS: The overall survival (OS) of patients with high concentrations of serum CCL7 was significantly poorer than that of patients with low concentrations. Patients with a high CCL7 concentration in the stroma had significantly poorer outcomes than those with a low concentration. The concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 were significantly higher in the high-CCL7 group, compared to those in the low-CCL7 group. Univariate and multivariate analysis revealed that serum CCL7 concentration was a significant prognostic factor for mCRC. The combination of serum CCL and CEA concentrations was also useful in this regard (area under the curve=0.71). CONCLUSION: The combined pre-treatment serum levels of CCL7 and CEA are useful prognostic biomarkers for mCRC.
Assuntos
Quimiocina CCL7 , Neoplasias do Colo , Neoplasias Colorretais , Neoplasias Retais , Humanos , Biomarcadores Tumorais , Antígeno Carcinoembrionário , Quimiocina CCL7/sangue , Quimiocina CCL7/química , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/metabolismo , Ligantes , Prognóstico , Neoplasias Retais/diagnóstico , Neoplasias Retais/metabolismo , Estudos RetrospectivosRESUMO
FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.
Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Cálcio/metabolismo , Quimiocina CCL7/química , Proteínas de Choque Térmico/química , Processamento de Proteína Pós-Traducional , Monofosfato de Adenosina/química , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biocatálise , Cálcio/química , Cátions Bivalentes , Quimiocina CCL7/genética , Quimiocina CCL7/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Chaperona BiP do Retículo Endoplasmático , Enterococcus faecalis/genética , Enterococcus faecalis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Magnésio/química , Magnésio/metabolismo , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Chemokines and their receptors control cell migration associated with routine immune surveillance, inflammation and development. They are also implicated in a large number of inflammatory diseases, cancer and HIV. Here we describe a rapid and efficient way to express and purify milligram quantities of multiple chemokine ligands (CCL7/MCP-3, CCL14/HCC-1, CCL3/MIP-1α and CXCL8/IL-8) containing C-terminal modifications to enable coupling to fluorescent dyes or small molecules such as biotin, in vitro. These labeled chemokines display wild-type behavior in both receptor binding and calcium mobilization assays. The ability to rapidly and inexpensively produce labeled chemokines opens the way for their use in many applications, including non-traditional chemokine-receptor interaction studies, both on intact cells and with purified receptor reconstituted in artificial membranes in vitro. Furthermore, the ability to immobilize chemokines to obtain ligand affinity columns aids in efforts to purify chemokine receptors for structural and biophysical studies, by facilitating the separation of functional proteins from their non-functional counterparts.