Expression, Purification and Characterization of C-FADD

Abstract

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecular structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain an FADD variant that containing C-terminal region. In this study, mouse FADD (80-205) containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni 2+ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical α-helix spectrum in CD (circular dichroism) assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK I-binding activity which was important for its non-apoptotic function. Cellular & Molecular Immunology. 2009;6(4):295-301.

Protective effects of the aqueous extract ofScutellaria baicalensisagainst acrolein-induced oxidative stress in cultured human umbilical vein endothelial cells

“…HUVE cells have been extensively employed to study cardiovascular diseases (Breymann et al, 2006;Chen et al, 2009;Erdbrugger et al, 1989;Morikawa et al, 2002). The HUVEC model we used had duplicatable results when they were exposed to the cytotoxic acrolein, which is a major byproduct of oxidative stress and lipid peroxidation.…”

“…Protection against oxidative stress in cardiovascular disorders by using phytotherapy and western medicines has been extensively studied (Banerjee et al, 2003;Chen et al, 2009;Costa et al, 2007;Csiszar et al, 2008;He et al, 2008a;2008b;Kobayashi et al, 2002;Lakomkin et al, 2005;Liao et al, 2009;Nemoto et al, 2007;Saada et al, 2009;Vaage et al, 1997;Venardos et al, 2007;Yang et al, 1995). HUVE cells have been extensively employed to study cardiovascular diseases (Breymann et al, 2006;Chen et al, 2009;Erdbrugger et al, 1989;Morikawa et al, 2002).…”

β1,4-galactosyltransferase-I in synovial tissue of collagen-induced rat model of rheumatoid arthritis

“…It shows that a portion of b1,4-GalT-I resides on the cell surface as a cell adhesion molecule during a variety of cellular interactions by binding GlcNAc-containing oligosaccharide substrates or ligands, in the extracellular matrix [12]. In addition, it is thought that b1,4-GalT-I is associated with a variety of biological functions, including sperm-egg interactions, morula compaction, embryonic maturation, embryonal carcinoma cell spreading, neurite extension, and mesenchymal cell migration, in which it mediates specific cell-cell and cell-matrix adhesions [13][14][15][16][17][18].…”