p41 as a possible marker for cell death is generated by caspase cleavage of p42/SETbeta in irradiated MOLT-4 cells.
We have reported previously that X-irradiated MOLT-4 cells during their rapid cell death exhibited dose and time dependently a new protein named p41 detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). An antibody, AM-1, raised against partial peptide of p41 stained two spots, p41 and p42, unexpectedly. Amino acid sequence analysis of partial peptides showed homology between p41 and a putative oncogene, set. In the present study, N-terminal amino acid sequencing of p41 and p42, and polyclonal antibodies newly prepared against different partial peptide sequences revealed that p41 was a N-terminal truncation form of p42, and p42 was identified as SETbeta. The cleavage site was at carboxyl end of SNHD 18 of p42. Radiation-induced p42 cleavage as well as apoptotic cell death was suppressed by a caspase-specific inhibitor Ac-DEVD-CHO but not by Ac-YVAD-CHO. Further in vitro cleavage experiments with recombinant p42 and either irradiated cell extracts or recombinant caspases concluded that the cleavage of p42 into p41 was catalyzed by caspase(s) mainly by caspase-7. One of newly raised antibodies, AM-4, specific to p41 or specific to cleavage site of p42, was found useful enabling simple detection of p41 by 1-D PAGE instead of laborious 2-D PAGE. p41 may serve as a marker of apoptotic cell death.
Morita
A
A
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Suzuki
N
N
Matsumoto
Y
Y
Hirano
K
K
Enomoto
A
A
Zhu
J
J
Sakai
K
K
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D020169:Caspases / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology*; D002470:Cell Survival / Q000502:physiology* / Q000528:radiation effects; D002868:Chromosomal Proteins, Non-Histone; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011836:Radiation Tolerance; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D014965:X-Rays
D002868:Chromosomal Proteins, Non-Histone; D015853:Cysteine Proteinase Inhibitors; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins; D011994:Recombinant Proteins; C513473:SET protein, human; D014157:Transcription Factors; C108637:aspartyl-glutamyl-valyl-aspartal; D020169:Caspases