Redox regulation of surface protein thiols: identification of integrin alpha-4 as a molecular target by using redox proteomics.
Thiols affect a variety of cell functions, an effect known as redox regulation. We show here that treatment (1-2 h) of cells with 0.1-5 mM N-acetyl-L-cysteine (NAC) increases surface protein thiol expression in human peripheral blood mononuclear cells. This effect is not associated with changes in cellular glutathione (GSH) and is also observed with a non-GSH precursor thiol N-acetyl-D-cysteine or with GSH itself, which is not cell-permeable, suggesting a direct reducing action. NAC did not augment protein SH in the cytosol, indicating that they are already maximally reduced under normal, nonstressed, conditions. By using labeling with a non permeable, biotinylated SH reagent followed by two-dimensional gel electrophoresis and analysis by MS, we identified some of the proteins associated with the membrane that are reduced by NAC. These proteins include the following: integrin alpha-4, myosin heavy chain (nonmuscle type A), myosin light-chain alkali (nonmuscle isoform), and beta-actin. NAC pretreatment augmented integrin alpha-4-dependent fibronectin adhesion and aggregation of Jurkat cells without changing its expression by fluorescence-activated cell sorter, suggesting that reduction of surface disulfides can affect proteins function. We postulate that some of the activities of NAC or other thiol antioxidants may not only be due to free radical scavenging or increase of intracellular GSH and subsequent effects on transcription factors, but could modify the redox state of functional membrane proteins with exofacial SH critical for their activity.
Laragione
Teresa
T
Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy.
Bonetto
Valentina
V
Casoni
Filippo
F
Massignan
Tania
T
Bianchi
Giancarlo
G
Gianazza
Elisabetta
E
Ghezzi
Pietro
P
D000111:Acetylcysteine / Q000737:chemistry; D000199:Actins / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D039441:Integrin alpha4 / Q000378:metabolism / Q000502:physiology*; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D013058:Mass Spectrometry; D018995:Myosin Heavy Chains / Q000737:chemistry; D018994:Myosin Light Chains / Q000737:chemistry; D010084:Oxidation-Reduction; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds; D013997:Time Factors
D000199:Actins; D005609:Free Radicals; D018994:Myosin Light Chains; D020543:Proteome; D013438:Sulfhydryl Compounds; D039441:Integrin alpha4; D018995:Myosin Heavy Chains; D005978:Glutathione; D000111:Acetylcysteine