ABSTRACT
Fatty acids have a great influence on the process of lymphocyte apoptosis which is considered as a modulating factor of immune response in both humans and animals. However the mechanism underlying the function of fatty acids in the process of lymphocyte apoptosis is not fully understood. In this study we show that the appearance of adipocyte-type fatty acid binding protein (A-FABP) is induced upon administration of dexamethasone (DEX) in both in vivo and cultured lymphocytes, and its distinct nuclear localization occurs in close relation to the DEX-induced apoptosis process. In immunohistochemistry of mouse spleen, A-FABP-immunoreactivity starts to occur 3 h after DEX stimulation, and it massively localizes in the nucleus 8 h after the treatment, while no A-FABP-immunoreactivity is discerned in the lymphocytes of normal as well as 24 h post-injection spleen. In the murine T-cell leukemia CTLL-2 cells, A-FABP-immunoreactivity is also induced in both of the cytoplasm and nucleus when the apoptosis is induced by IL-2 retrieval together with DEX treatment, while in the presence of IL-2 A-FABP-immunoreactivity is confined to the cytoplasm with DEX treatment. On the other hand, A-FABP-immunoreactivity is not detected by IL-2 retrieval alone. The present findings altogether suggest that A-FABP and its ligands, fatty acids, play an important role in the process of apoptosis and the immune modulation induced by DEX.
Subject(s)
Adipocytes/metabolism , Dexamethasone/pharmacology , Fatty Acid-Binding Proteins/metabolism , Lymphocytes/drug effects , Animals , Apoptosis/drug effects , Base Sequence , Cell Line, Tumor , DNA Primers , Lymphocytes/metabolism , Male , Mice , Mice, Inbred C57BLABSTRACT
We and others showed earlier that liver-type, epidermal-type and adipocyte-type (A-) fatty acid-binding proteins (FABPs) mediate peroxisome proliferator activated receptor (PPAR) dependent gene expression by channelling their ligand (fatty acid or drug) to the nuclear receptors via direct protein/protein interaction. To clarify mechanistic details of this signaling path, we address here A-FABP import into the nucleus and its interaction with PPARgamma. Making use of COS cells transfected with wild-type or mutant A-FABPs, we exclude posttranslational modification of A-FABP as import signal and provide evidence for both, ligand-dependent and ligand-independent nuclear translocation. With the aid of in vitro pull down assay we demonstrate that specific interaction of A-FABP with PPARgamma isoforms does not require ligand. Moreover, A-FABP binds not only to the ligand-binding domain including hinge domain (domains DEF), but also to the DNA-binding domain including AB domains (domains ABC) of PPARgamma.
Subject(s)
Fatty Acid-Binding Proteins/metabolism , PPAR gamma/agonists , Active Transport, Cell Nucleus , Animals , Base Sequence , Biological Transport, Active , COS Cells , Cell Compartmentation , Chlorocebus aethiops , DNA, Complementary/genetics , Fatty Acid-Binding Proteins/genetics , In Vitro Techniques , Ligands , Mice , Mutagenesis, Site-Directed , PPAR alpha/metabolism , PPAR gamma/chemistry , PPAR gamma/metabolism , PPAR-beta/metabolism , Protein Processing, Post-Translational , Protein Structure, Tertiary , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
The hepatitis C virus (HCV) nonstructural 3 (NS3) protein is composed of an amino terminal protease and a carboxyl terminal RNA helicase. NS3 contains major antigenic epitopes. The antibody response to NS3 appears early in the course of infection and is focused on the helicase region. However, this response cannot be defined by short synthetic peptides indicating the recognition of conformation-dependent epitopes. In this study, we have screened a dodecapeptide library displayed on phage with anti-NS3 mouse monoclonal antibodies (mAbs) that compete with each other and human anti-HCV NS3 positive sera. Two peptides (mimotopes) were selected that appeared to mimic an immunodominant epitope since they were recognized specifically by the different anti-NS3 mAbs of the study and by human sera from HCV infected patients. Homology search between the two mimotopes and the NS3 sequence showed that one of the two peptides shared amino acid similarities with NS3 at residues 1396-1398 on a very accessible loop as visualized on the three-dimensional structure of the helicase domain whereas the other one had two amino acids similar to nearby residues 1376 and 1378. Reproduced as synthetic dodecapeptides, the two mimotopes were recognized specifically by 19 and 22, respectively, out of 49 sera from HCV infected patients. These mimotopes allowed also the detection of anti-NS3 antibodies in sera of HCV patients at the seroconversion stage. These results suggest that the two NS3 mimotopes are potential tools for the diagnosis of HCV infection.
