ATP-induced loss of Alz-50 immunoreactivity with the A68 proteins from Alzheimer brain is mediated by ubiquitin.
The Alz-50 immunoreactive proteins, designated A68, are detected by electrophoretic blot analysis of 100,000 x g pellet fractions of brain tissue from individuals with Alzheimer disease (AD). In exploring the biochemical nature of these proteins, we have found that a preincubation of such fractions with 5 mM ATP results in loss of Alz-50 immunoreactivity on immunoblots. The loss of antigenicity is complete after a 1-hr incubation at 37 degrees C and is stringently dependent on ATP. Hydrolysis of ATP is required, since the inhibition is not supported by the nonhydrolyzable analog adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) and is prevented when the ATPase inhibitors o-vanadate and oligomycin are present. Upon further characterization, it was found that certain protease inhibitors, phenylmethylsulfonyl fluoride, antipain, tosylphenylalanine chloromethyl ketone, and aprotonin prevent the loss of the epitope. This suggests that hydrolysis of ATP is coupled with proteolysis of A68, leading to loss of Alz-50 immunoreactivity. Since a variety of proteins are believed to be degraded by an ATP/ubiquitin-dependent pathway, a possible role for ubiquitin (Ub) in this effect was investigated. Two polyclonal antibodies against Ub protected A68 from proteolysis and were also effective in immunoprecipitating A68 after incubation with ATP in the presence of Ub and phenylmethylsulfonyl fluoride. The proteolysis of A68 was also blocked by hemin, an inhibitor of the protease that cleaves Ub-protein conjugates. Taken together, these findings indicate that loss of Alz-50 immunoreactivity with A68 is due to ATP-dependent/Ub-mediated proteolysis. This mechanism may be relevant to the physiological role for A68 in AD or it may simply represent an attempt to abort an aberrant protein.
Vincent
I J
IJ
Department of Pathology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461.
Davies
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000544:Alzheimer Disease / Q000378:metabolism*; D000941:Antigens / Q000032:analysis*; D001921:Brain / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D012265:Ribonucleotides / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism*; D016875:tau Proteins
C056167:Alzheimer's disease antigen; D000941:Antigens; C054369:MAPT protein, human; D009419:Nerve Tissue Proteins; D011480:Protease Inhibitors; D012265:Ribonucleotides; D014452:Ubiquitins; D016875:tau Proteins; D000255:Adenosine Triphosphate