Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis.
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.
Liu
Ming Cheng
MC
Center for Neuroproteomics and Biomarkers Research, Department of Psychiatry, McKnight Brain Institute, University of Florida, P.O. Box 100256, Gainesville, FL 32610, USA.
Akle
Veronica
V
Zheng
Wenrong
W
Dave
Jitendra R
JR
Tortella
Frank C
FC
Hayes
Ronald L
RL
Wang
Kevin K W
KK
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
D020543:Proteome; C505407:CASP3 protein, human; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases