Bio-effectiveness of Tat-catalase conjugate: a potential tool for the identification of H2O2-dependent cellular signal transduction pathways.
Reactive oxygen species such as hydrogen peroxide (H(2)O(2)) have taken center stage as bona fide second messengers in various signaling pathways. Here, we report the synthesis, metabolic fate, and effectiveness in modulating such pathways of a Tat-catalase conjugate. Incubation of L2 cells with Tat-catalase greatly increased cell-associated enzymatic activity, reaching close to a plateau by 30 min. The cell-associated catalase activity and antibody-detectable Tat-derivatives declined over time after changing medium, although still remaining at significantly higher levels than baseline even at 4h. While most cell-associated Tat-catalase was apparently tightly attached to the cell surface, a small fraction entered the cells as the proteasome inhibitor MG-132 slightly prevented the disappearance of the enzyme. Tat-catalase, either membrane-bound or intracellular, but not native catalase, inhibited serum-induced Elk phosphorylation and anisomycin- and/or MG-132-induced ERK phosphorylation, suggesting the involvement of H(2)O(2). Thus, Tat-catalase should be a useful tool to dissect H(2)O(2)-dependent events in signaling pathways.
Watanabe
Nobuo
N
Department of Environmental Health Sciences, School of Public Health and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Iwamoto
Takeo
T
Bowen
Kathy D
KD
Dickinson
Dale A
DA
Torres
Martine
M
Forman
Henry Jay
HJ
D000818:Animals; D000841:Anisomycin / Q000494:pharmacology; D002374:Catalase / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008168:Lung / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors
D015696:Gene Products, tat; D007976:Leupeptins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D000841:Anisomycin; D006861:Hydrogen Peroxide; D002374:Catalase; D020928:Mitogen-Activated Protein Kinases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde