Abstract
BackgroundWhereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process.MethodsWe performed proteomics, Western Blot, real-time PCR, ELISA and pharmacological experiments in conjunction with a clinically relevant experimental endotoxemia model of brain death based on intravenous administration of Escherichia coli lipopolysaccharide in adult male Sprague–Dawley rats.ResultsProteomics, Western blot and enzyme activity analyses demonstrated that polyubiquitination was preserved and de-ubiquitination by ubiquitin C-terminal hydrolase isozyme-L1 (UCH-L1) was sustained, alongside increased monoubiquitin availability or proteasome activity in RVLM over the course of experimental endotoxemia. However, real-time PCR revealed no significant alteration in proteasome subunit alpha type-1, ubiquitin or UCH-L1 at mRNA level. Functionally, whereas microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) potentiated survival, an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or an UCH-L1 inhibitor exacerbated mortality.ConclusionsWe proposed previously that the progression towards brain death entails a tug-of-war between pro-death and pro-life programs in RVLM. It is conceivable that ubiquitination or de-ubiquitination in RVLM participate in brain death by regulating the degradation of the proteins involved in those programs.
Highlights
Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive
Based on a computer algorithm that our laboratory developed for online and real-time spectral analysis of systemic arterial blood pressure (SAP) signals [2], we identified previously in a series of clinical studies that a common denominator exists in comatose patients who succumbed to systemic inflammatory response syndrome [3], organophosphate poisoning [4] or brain injury [5]
Based on MALDITOF mass spectrometric analysis on protein spots in the Coomassie blue-stained 2-D electrophoresis gels from randomly selected samples (n = 8), in conjunction with search results using the MASCOT program (Figures 1, 2, 3 and 4) we found that members of the ubiquitin-proteasome system (UPS), including proteasome subunit alpha type-1, ubiquitin and ubiquitin C-terminal hydrolases (UCHs) isozyme-L1 (UCH-L1) are present in rostral ventrolateral medulla (RVLM) of rats that were subject to experimental brain death and the sham-controls
Summary
Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process. Most proteins in the cytosol and nucleus of eukaryotic cells are degraded via the ubiquitin-proteasome system (UPS), in a process that is energy-dependent. The highly conserved 76 amino acid protein ubiquitin is best known for its role in targeting proteins for proteasomal degradation. The ubiquitin chain is released from the target protein remnant after the degradative process and is disassembled by de-ubiquitinating enzymes that include the ubiquitin C-terminal hydrolases (UCHs) [10]. Of the three known mammalian members of the UCH family, UCH isozyme-L1 (UCH-L1) is among the most abundantly present proteins in brain [12]
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