Linked Life Data

12097160

Source:http://linkedlifedata.com/resource/pubmed/id/12097160

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2002-7-4
pubmed:abstractText
We generated and characterized novel antibodies specific for a cleavage site of human caspase-8/FLICE and its substrate, FLICE-like inhibitory protein (FLIP). The synthetic peptides used as immunogens were CQGDNYQKGIPVETD (#791) and VSEGQLEDSSLLEVD (#1342), which corresponded to cleaved regions of N-terminal fragments of caspase-8 and FLIP generated by active caspase-8, respectively. Each antibody purified from rabbit antiserum reacted specifically with the immunogen but not with the peptide corresponding to the unproteolyzed form, as assessed by ELISA. In vitro cleavage of GST-FLIP by active caspase-8 generated an N-terminal fragment (GST-p43) and a C-terminal one (p12). Consistent with other in vivo data, the FLIP cleavage site follows the Asp residue, LEVD(376)GPAMKNVEF, identified on N-terminal sequencing of the p12 fragment. #1342-antibody (#1342-Ab) recognized the GST-p43 fragment but not the uncleaved protein, thus confirming its specificity. When the antibodies were used for immunoblotting, flow cytometry, and confocal laser microscopy, the proteolysis of caspase-8 and FLIP, and the subcellular localization of their digests could be monitored in apoptotic U937 cells. Interestingly, a significant increase in the percentage of cells exhibiting caspase-8 and FLIP cleavage was observed upon Fas stimulation in interferon-gamma-treated U937 cells, in which the susceptibility to Fas is extremely enhanced. In contrast, U937 cells treated with vitamin D(3) or all-trans retinoic acid showed Fas-resistance, and caspase-8 processing and FLIP cleavage were strongly inhibited. In conclusion, we established a system based on the cleavage site-directed antibodies to monitor the dynamics of caspase-8 processing and activation during apoptosis. Using this system, we found that Fas-susceptibility changes during U937 differentiation occur upstream of caspase-8 processing/activation.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0021-924X
pubmed:author
pubmed:issnType
Print
pubmed:volume
132
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
53-62
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed-meshheading:12097160-Amino Acid Sequence, pubmed-meshheading:12097160-Antibodies, Monoclonal, pubmed-meshheading:12097160-Antigens, CD95, pubmed-meshheading:12097160-Apoptosis, pubmed-meshheading:12097160-CASP8 and FADD-Like Apoptosis Regulating Protein, pubmed-meshheading:12097160-Carrier Proteins, pubmed-meshheading:12097160-Caspases, pubmed-meshheading:12097160-Cell Differentiation, pubmed-meshheading:12097160-Enzyme Activation, pubmed-meshheading:12097160-Flow Cytometry, pubmed-meshheading:12097160-Humans, pubmed-meshheading:12097160-Hydroxycholecalciferols, pubmed-meshheading:12097160-Interferon-gamma, pubmed-meshheading:12097160-Intracellular Signaling Peptides and Proteins, pubmed-meshheading:12097160-Jurkat Cells, pubmed-meshheading:12097160-Microscopy, Confocal, pubmed-meshheading:12097160-Molecular Sequence Data, pubmed-meshheading:12097160-Protein Structure, Tertiary, pubmed-meshheading:12097160-Subcellular Fractions, pubmed-meshheading:12097160-Substrate Specificity, pubmed-meshheading:12097160-Tretinoin, pubmed-meshheading:12097160-U937 Cells
pubmed:year
2002
pubmed:articleTitle
Monitoring of caspase-8/FLICE processing and activation upon Fas stimulation with novel antibodies directed against a cleavage site for caspase-8 and its substrate, FLICE-like inhibitory protein (FLIP).
pubmed:affiliation
Division of Cell Biology and Biochemistry, Department of Basic Medical Science, Institute of Medical Science, The University of Tokyo (IMSUT), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't