title: Anamorsin, a novel caspase-3 substrate in neurodegeneration.
abstract: Activated caspases play a central role in the execution of apoptosis by cleaving endogenous substrates. Here, we developed a high throughput screening method to identify novel substrates for caspase-3 in a neuronal cell line. Critical steps in our strategy consist of two-dimensional electrophoresis-based protein separation and in vitro caspase-3 incubation of immobilized proteins to sort out direct substrates. Among 46 putative substrates identified in MN9D neuronal cells, we further evaluated whether caspase-3-mediated cleavage of anamorsin, a recently recognized cell death-defying factor in hematopoiesis, is a general feature of apoptosis. In vitro and cell-based cleavage assays indicated that anamorsin was specifically cleaved by caspase-3 but not by other caspases, generating 25- and 10-kDa fragments. Thus, in apoptosis of neuronal and non-neuronal cells induced by various stimuli including staurosporine, etoposide, or 6-hydroxydopamine, the cleavage of anamorsin was found to be blocked in the presence of caspase inhibitor. Among four tetrapeptide consensus DXXD motifs existing in anamorsin, we mapped a specific cleavage site for caspase-3 at DSVD(209)↓L. Intriguingly, the 25-kDa cleaved fragment of anamorsin was also detected in post-mortem brains of Alzheimer and Parkinson disease patients. Although the RNA interference-mediated knockdown of anamorsin rendered neuronal cells more vulnerable to staurosporine treatment, reintroduction of full-length anamorsin into an anamorsin knock-out stromal cell line made cells resistant to staurosporine-induced caspase activation, indicating the antiapoptotic function of anamorsin. Taken together, our approach seems to be effective to identify novel substrates for caspases and has the potential to provide meaningful insights into newly identified substrates involved in neurodegenerative processes.
authors: From the Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea.
authors: Nuri
authors: N
authors: Yun
authors: From the Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea.
authors: Young Mook
authors: YM
authors: Lee
authors: From the Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea.
authors: Chiho
authors: C
authors: Kim
authors: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Hirohiko
authors: H
authors: Shibayama
authors: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Akira
authors: A
authors: Tanimura
authors: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Yuri
authors: Y
authors: Hamanaka
authors: Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan.
authors: Yuzuru
authors: Y
authors: Kanakura
authors: Department of Cellular and Molecular Medicine, College of Medicine, Chosun University, Gwangju 501-759, Korea.
authors: Il-Seon
authors: IS
authors: Park
authors: Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Gyeonggi-do, Korea, and.
authors: Areum
authors: A
authors: Jo
authors: Division of Pharmacology, Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Gyeonggi-do, Korea, and.
authors: Joo-Ho
authors: JH
authors: Shin
authors: Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, Korea.
authors: Chung
authors: C
authors: Ju
authors: Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, Korea wonki@korea.ac.kr.
authors: Won-Ki
authors: WK
authors: Kim
authors: From the Department of Systems Biology, Yonsei University College of Life Science and Biotechnology, Seoul 120-749, Korea, yjoh@yonsei.ac.kr.
authors: Young J
authors: YJ
authors: Oh
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D000544:Alzheimer Disease / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D001665:Binding Sites; D016022:Case-Control Studies; D053148:Caspase 3 / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D005260:Female; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D008875:Middle Aged; D008969:Molecular Sequence Data; D009410:Nerve Degeneration / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010300:Parkinson Disease / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C489114:CIAPIN1 protein, human; C557202:Ciapin1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; C505409:Casp3 protein, mouse; D053148:Caspase 3
keywords: Apoptosis; Caspase; Neurodegeneration; Neurotoxin; Proteomics