ABSTRACT
We constructed a plasmid containing a protein transduction domain (PTD) and a human A20 (hA20) gene fragment; the fusion protein was obtained by highly expressing this plasmid in the yeast Pichia pastoris GS115. The plasmid was obtained by adding 9xArg and EcoRÐ recognition sites to the end of the primer, and 6xHis-Tag and NotÐ recognition sites to its end. After sequencing, the hA20 gene fragment was inserted into plasmid pPIC9k to construct expression vector pPIC9k-PTD-hA20; then, we transfected GS115 with the vector and induced PTD-hA20 protein expression. We purified protein from the yeast fermentation supernatant using a nickel column. Human umbilical vein endothelial cells (HUVECs) were cultured in high glucose medium (30 mM glucose) and in high glucose medium containing different concentrations of protein. Apoptosis of HUVECs was assayed by TUNEL 72 h later. The biological activity tests indicated that the fusion protein not only passed through the cell membrane freely, but also inhibited apoptosis of HUVECs induced by high glucose levels. We conclude that the fusion protein PTD-hA20 has potential for clinical use.
Subject(s)
Cytoprotection/drug effects , DNA-Binding Proteins/pharmacology , Endothelial Cells/cytology , Endothelial Cells/drug effects , Glucose/toxicity , Intracellular Signaling Peptides and Proteins/pharmacology , Nuclear Proteins/pharmacology , Recombinant Fusion Proteins/pharmacology , Apoptosis/drug effects , Blotting, Western , Electrophoresis, Agar Gel , Endothelial Cells/metabolism , Genome, Fungal/genetics , Human Umbilical Vein Endothelial Cells , Humans , Microscopy, Fluorescence , Mutagenesis, Insertional/genetics , Pichia/drug effects , Pichia/genetics , Plasmids/metabolism , Polymerase Chain Reaction , Protein Structure, Tertiary , Recombinant Fusion Proteins/isolation & purification , Recombination, Genetic/genetics , Tumor Necrosis Factor alpha-Induced Protein 3ABSTRACT
Diabetes mellitus causes vascular lesions and may ultimately lead to atherosclerosis. One of the earliest steps in the development of atherosclerotic lesions is the adhesion of monocytes to endothelial cells of the vessel wall. It is currently unknown whether zinc finger protein A20 is able to protect endothelial cells from injury caused by high levels of glucose and monocyte homing. In our study, adhesion of monocytes to the vessel wall endothelium was detected by measuring the rolling velocity of monocytes along human umbilical vein endothelial cells (HUVECs). Activation of NF-κB was analyzed through Western blot. HUVEC apoptosis was monitored by TUNEL in situ end-labeling and flow cytometry. High glucose concentrations (25 mM) stimulated monocytes, reducing the velocity at which they roll along HUVECs. Stimulation of monocytes with high levels of glucose also induced HUVEC apoptosis. Overexpression of the zinc finger protein A20 inhibited monocyte recruitment, NF-κB activation, P-selectin expression, and HUVEC apoptosis induced by high glucose levels. We conclude that zinc finger protein A20 can protect HUVECs from injury induced by high levels of glucose and potentially could be used to develop treatments against diabetic vascular lesions.
