title: Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis.
abstract: A major theme of TBI (traumatic brain injury) pathology is the over-activation of multiple proteases. We have previously shown that calpain-1 and -2, and caspase-3 simultaneously produced alphaII-spectrin BDPs (breakdown products) following TBI. In the present study, we attempted to identify a comprehensive set of protease substrates (degradome) for calpains and caspase-3. We further hypothesized that the TBI differential proteome is likely to overlap significantly with the calpain- and caspase-3-degradomes. Using a novel HTPI (high throughput immunoblotting) approach and 1000 monoclonal antibodies (PowerBlottrade mark), we compared rat hippocampal lysates from 4 treatment groups: (i) naïve, (ii) TBI (48 h after controlled cortical impact), (iii) in vitro calpain-2 digestion and (iv) in vitro caspase-3 digestion. In total, we identified 54 and 38 proteins that were vulnerable to calpain-2 and caspase-3 proteolysis respectively. In addition, the expression of 48 proteins was down-regulated following TBI, whereas that of only 9 was up-regulated. Among the proteins down-regulated in TBI, 42 of them overlapped with the calpain-2 and/or caspase-3 degradomes, suggesting that they might be proteolytic targets after TBI. We further confirmed several novel TBI-linked proteolytic substrates, including betaII-spectrin, striatin, synaptotagmin-1, synaptojanin-1 and NSF (N-ethylmaleimide-sensitive fusion protein) by traditional immunoblotting. In summary, we demonstrated that HTPI is a novel and powerful method for studying proteolytic pathways in vivo and in vitro.
authors.affiliation: Center for Neuroproteomics and Biomarkers Research, Department of Psychiatry, McKnight Brain Institute, University of Florida, P.O. Box 100256, Gainesville, FL 32610, USA.
authors.forename: Ming Cheng
authors.initials: MC
authors.lastname: Liu
authors.forename: Veronica
authors.initials: V
authors.lastname: Akle
authors.forename: Wenrong
authors.initials: W
authors.lastname: Zheng
authors.forename: Jitendra R
authors.initials: JR
authors.lastname: Dave
authors.forename: Frank C
authors.initials: FC
authors.lastname: Tortella
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Hayes
authors.forename: Kevin K W
authors.initials: KK
authors.lastname: Wang
mesh_terms: D000818:Animals; D001930:Brain Injuries / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D005786:Gene Expression Regulation; D006624:Hippocampus / Q000378:metabolism; D006801:Humans; D008297:Male; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D020543:Proteome; C505407:CASP3 protein, human; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases