Kartoosh Heydari | PubMed Health

PubMed2005

The extracellular microenvironment plays a key role in regulating the redox status of cell surface proteins in HIV-infected subjects.

There is an overwhelming interest in the study of the redox status of the cell surface affecting redox signaling in the cells and also predicting the total redox status of the cells. Measuring the total surface thiols (cell surface molecule thiols, csm-SH) we have shown that the overall level of surface thiols is tightly controlled. In vitro, the total concentration of intracellular glutathione (iGSH) seems to play a regulatory role in determination of the amounts of reduced proteins on cells. In addition, short term exposure of the cell surface to glutathione disulfide (GSSG, oxidized GSH) seems to reduce the overall levels of csm-SH suggesting that the function of some cysteine containing proteins on the cell surface may be regulated by the amount of GSSG secreted from the cells or the GSSG available in the extracellular environment. Examination of peripheral blood mononuclear cells (PBMCs) from healthy or HIV-infected subjects failed to reveal a similar correlation between the intra- and extracellular thiol status of cells. Although there is a relatively wide variation between individuals in both csm-SH and iGSH there is no correlation between the iGSH and csm-SH levels measured for healthy and HIV-infected individuals. There are many reports suggesting different redox active proteins on the cell surface to be the key players in the total cell surface redox regulation. However, we suggest that the redox status of the cells is regulated through a complex and tightly regulated mechanism that needs further investigation. In the mean time, overall surface thiol measurements together with case specific protein determinations may offer the most informative approach. In this review, we discuss our own results as well as results from other laboratories to argue that the overall levels of surface thiols on the exofacial membrane are regulated primarily by redox status of the cell surface microenvironment.

Cell Membrane↗

PubMed2003

Lymphocyte surface thiol levels.

Recent studies have implicated reduced thiols (cysteine -SH) in the function of individual cell surface proteins. Studies presented here demonstrate that the overall level of reduced thiols on cell surface molecules differs on individual subsets of peripheral blood mononuclear cells and that these levels can be manipulated in vitro by altering the level of intracellular glutathione (iGSH). To quantitate cell surface thiols, we have developed a Hi-D (11-color) fluorescence-activated cell sorter method in which we covalently couple a fluorescent molecule, Alexa-maleimide, to free (reduced) -SH groups on proteins or other molecules exposed on the cell surface (exofacial membrane). In addition, to reveal changes in cell surface thiol levels in response to various in vitro treatments, we used a pair of fluorescent Alexa dyes with distinct excitation and emission spectra to stain the cells before and after treatments. These in vitro studies demonstrate that decreasing iGSH, by specifically inhibiting its synthesis, decreases cell surface molecule thiols (csm-SH) and that preventing loss of iGSH also prevents loss of csm-SH. However, examination of peripheral blood mononuclear cell subsets tested immediately after isolation from healthy or HIV-infected subjects failed to reveal a similar relationship between internal iGSH and csm-SH. Although there is a relatively wide variation between individuals in both csm-SH and iGSH, there is no correlation between median iGSH and csm-SH compared for 22 healthy and 36 HIV-infected subjects. Collectively, our findings indicate that local environment plays a greater role in determining the redox status of cell surface molecules than the internal redox status of the cells.

Antigens, CD↗