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The HeLa cell line, a human adenocarcinoma, is used in many research fields, since it can be infected with a wide range of viruses and intracellular bacteria. Therefore, the mapping of HeLa cell proteins is useful for the investigation of parasite host cell interactions. Because of the recent improvements of two-dimensional gel electrophoresis with immobilized pH gradients (IPG) compared to isoelectric focusing with carrier ampholytes, a highly reproducible method for examining global changes in HeLa cell protein expression due to different stimuli is now available. Therefore, we have initiated the mapping of [35S]methionine/cysteine-labeled HeLa cell proteins with the 2-D PAGE (IPG)-system, using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and N-terminal sequencing for protein identification. To date 21 proteins have been identified and mapped. In order to make these and future data accessible for interlaboratory comparison, we constructed a 2-D PAGE database on the World Wide Web.

Interferon gamma (IFN-gamma) is a potent immunomodulatory lymphokine, secreted by activated T-lymphocytes and NK-cells during the cellular immune response. Actions of IFN-gamma are mediated through binding to the IFN-gamma-receptor, present on most cells, and the subsequent activation of a great magnitude of IFN-gamma responsive genes has been reported previously. Our goal is to identify and map IFN-gamma-regulated HeLa cell proteins to the two-dimensional polyacrylamide gel electrophoresis with the immobilized pH gradient (IPG) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) system. A semiconfluent layer of HeLa cells was grown on tissue culture plates, and changes in protein expression due to 100 U/mL IFN-gamma were investigated at different periods after treatment, using pulse labeling with [35S]methionine/cysteine in combination with 2-D PAGE (IPG). The identity of eight protein spots was elucidated by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), and several variants of the IFN-gamma-inducible tryptophanyl-tRNA synthetase (hWRS) were detected by immunoblotting.

Chlamydia trachomatis is an obligate intracellular bacterium causing human ocular and genital disease. The lymphokine interferon gamma (IFN-gamma) is an important immune effector exerting antimicrobial effects towards several intracellular parasites, the chlamydia included. IFN-gamma has been reported to inhibit the chlamydial replication in vitro in part by depleting intracellular levels of tryptophan in a dose-dependent manner. In addition, down-regulation of important immunogens has been described. These findings are extended in this paper, in which we are combining pulse labeling with [35S]methionine and two-dimensional gel electrophoresis with immobilized pH gradients in order to investigate changes in the protein expression of C. trachomatis serovar A and L2 caused by treatment with IFN-gamma. In contrast to what was observed in C. trachomatis L2, our results showed that, in C. trachomatis A, down-regulations of the chlamydia major outer membrane protein and of several other proteins were detectable upon IFN-gamma treatment. In addition, we report the up-regulations of C. trachomatis A and L2 proteins with molecular masses of approximately 30 kDa and 40 kDa which may be part of an, as yet, uncharacterized chlamydial response to IFN-gamma treatment.