W7FW14F apomyoglobin amyloid aggregates-mediated apoptosis is due to oxidative stress and AKT inactivation caused by Ras and Rac.
We have previously reported that addition of prefibrillar aggregates (PFAs) derived from W7FW14F apomyoglobin mutant to NIH-3T3 cells affects their viability. In this article, we have found that cytotoxicity induced by PFAs in NIH 3T3 and SH-SY5Y human neuroblastoma cells was due to early activation of apoptotic cell death dependent from a caspase-3- and -9-mediated mitochondrial pathway. A time-dependent increase of intracellular ROS and an about twofold decrease of mitochondrial localization of scavenger protein MnSOD was found. The use of the anti-oxidant agent N-acetyl-cysteine (NAC) antagonized both the increase of intracellular ROS and apoptosis induced by PFAs. PFAs caused an about 60% increase of the activity of both Ras and Erk-1/2 at 30 and 45 min while they were restored to basal levels at later time points. This effect was paralleled by a time-dependent decrease of the activity of the survival enzyme Akt. Effects similar to those on Ras activity were also recorded on the activity of the stress involved small GTP binding protein Rac that was about 75% increased after 30 min but resumed to basal levels at later time points. This effect was paralleled by a time-dependent activation of p38 kinase activity and HSP-70 expression. The use of both the ras farnesyltransferase inhibitor tipifarnib and the Rac geranyl-geranyltransferase GGTI-298, but not of the MEK-1 inhibitor U0126 partially antagonized the effects of PFAs on apoptosis occurrence. On the other hand, the PI3K/Akt inhibitor LY 294002 potentiated apoptosis induced by PFAs. Our results indicate a role for Ras and Rac in the induction of both intracellular ROS increased levels and apoptosis mediated by PFAs and disclose a new scenario of intervention in neurodegenerative diseases.
Sirangelo
Ivana
I
Dipartimento di Biochimica e Biofisica, Seconda Università degli Studi di Napoli, Napoli, Italy.
Iannuzzi
Clara
C
Vilasi
Silvia
S
Irace
Gaetano
G
Giuberti
Gaia
G
Misso
Gabriella
G
D'Alessandro
Annamaria
A
Abbruzzese
Alberto
A
Caraglia
Michele
M
D000682:Amyloid / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000737:chemistry / Q000494:pharmacology; D009211:Myoglobin / Q000737:chemistry*; D018384:Oxidative Stress / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015363:Quinolones / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000682:Amyloid; D001059:Apoproteins; D001549:Benzamides; C102521:GGTI 298; D050505:Mutant Proteins; D009211:Myoglobin; D015363:Quinolones; C010857:apomyoglobin; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D020744:rac GTP-Binding Proteins; D018631:ras Proteins; C402769:tipifarnib