No abstract

The molecular mechanism underlying bilirubin neurotoxicity remains obscure. Ubiquitin–proteasome system-mediated proteolysis is pivotal to virtually all cellular processes and cell survival. Here we report for the first time that bilirubin at a clinically relevant elevated level impairs proteasomal function via inhibiting both the 19S proteasome-associated deubiquitinases (USP14 and UCHL5) and the chymotrypsin-like (CT-like) peptidase activity of 20S proteasomes, thereby contributing to bilirubin neurotoxicity. This is supported by multiple lines of evidence. First, sera from patients with hyperbilirubinemia were able to inhibit the peptidase activity of purified 20S proteasome in vitro in a bilirubin concentration-dependent manner; meanwhile, the blood cells of these patients showed significantly increased levels of ubiquitinated proteins (Ub-prs), consistent with proteasome inhibition. Second, intracerebroventricular injection to adult rats or intraperitoneal injections to neonatal rats of bilirubin-induced neural accumulation of Ub-prs, concurrent with other neural pathology; and brain malfunction and pathology induced by neonatal exposure to hyperbilirubinemia were detectable in the rats during their adulthood. Third, in primary cultures of hippocampal neurons, bilirubin strikingly induced Ub-pr accumulation before the activation of cell death pathway becomes discernible. Finally, bilirubin in vitro directly inhibited both the deubiquitination activity of proteasome-associated USP14 and UCHL5 and the CT-like peptidase activity of purified 20S proteasomes, in a dose-dependent manner. Hence, this study has discovered that increased bilirubin at a clinically achievable level can act as a proteasome inhibitor via targeting the 19S proteasome-associated deubiquitinases (DUBs) and, perhaps to a less extent, the 20S proteasome, identifying a novel mechanism for bilirubin neurotoxicity.

Background: Ischemic heart disease is a major global public health challenge, and its functional outcomes remain poor. Lysine crotonylation (Kcr) was recently identified as a post-translational histone modification that robustly indicates active promoters. However, the role of Kcr in myocardial injury is unknown. In this study, we aimed to clarify the pathophysiological significance of Kcr in cardiac injury and explore the underlying mechanism. Methods: We investigated the dynamic change of both the Kcr sites and protein level in left ventricular tissues at 2 time points following sham or cardiac ischemia-reperfusion injury, followed by liquid chromatography-coupled tandem mass tag mass spectrometry. After validation of the enriched protein Kcr by immunoprecipitation and Western blot, the function and mechanism of specific Kcr sites were further investigated in vitro and in vivo by gain- or loss-of-function mutations targeting Kcr sites of selected proteins. Results: We found that cardiac ischemia-reperfusion injury triggers preferential Kcr of proteins required for cardiomyocyte contractility, including mitochondrial and cytoskeleton proteins, which occurs largely independently of protein-level changes in the same proteins. Those exhibiting Kcr changes were associated not only with disruption of cardiomyocyte mitochondrial, sarcomere architecture, and gap junction but also with cardiomyocyte autophagy and apoptosis. Modulating site-specific Kcr of selected mitochondrial protein IDH3a (isocitrate dehydrogenase 3 [NAD+] alpha) at K199 and cytoskeletal protein TPM1 (tropomyosin alpha-1 chain) at K28/29 or enhancing general Kcr via sodium crotonate provision not only protects cardiomyocyte from apoptosis by inhibiting BNIP3 (Bcl-2 adenovirus E18 19-kDa–interacting protein 3)-mediated mitophagy or cytoskeleton structure rearrangement but also preserves postinjury myocardial function by inhibiting fibrosis and apoptosis. Conclusions: Our results indicate that Kcr modulation is a key response of cardiomyocytes to ischemia-reperfusion injury and may represent a novel therapeutic target in the context of ischemic heart disease.