br Restenosis after angioplasty remains a
Restenosis after angioplasty remains a remarkable challenge, although drug-eluting stents have reduced the incidence of restenosis considerably . Vascular smooth muscle HS-173 (VSMCs) play a pivotal role in the development of intimal thickening and restenosis. VSMCs proliferate and migrate from the media to the intima , . Thus, finding new targets against VSMCs is important. The ubiquitin proteasome system is the main intracellular protein degradation route by which cells get rid of excess and mis-folded proteins. A growing body of evidence has implicated the ubiquitin proteasome system (UPS) as being involved in the regulation of the complex cell signaling processes that are fundamental to atherosclerotic diseases. Recent studies have identified the contribution that the UPS makes to the initiation and complication of atherosclerosis via the regulation of vascular inflammation, apoptosis, oxidative stress and cholesterol metabolism . An increased activity in ubiquitination-proteasome in neointimal areas and the role of ubiquitin gene expression in these settings have been mentioned . Furthermore, the effects of proteasome inhibition on neointima formation have been well characterized, and the proteosome is considered as a therapeutic target , . However, the consequences of blocking protein degradation by inhibiting the apex of protein ubiquitination remain largely unknown. Here, we used chemical and genetic approaches to investigate the inhibition of protein ubiquitination in VSMCs both in vitro and in vivo. The ubiquitin moiety is generally attached via an E1-E2-E3 multi-enzyme cascade. In the first step, the ubiquitin-activating enzyme E1, UBA1 (E1), binds ATP·Mg and ubiquitin and catalyses C-terminal ubiquitin acyl-adenylation and the binding of a molecule . This ubiquitin is then available to be transferred to one of the E2 ubiquitin conjugating enzymes. E2 enzymes then interact with one of the hundreds of ubiquitin E3 ligases to transfer the ubiquitin to the ε-amino group of a lysine residue in the target protein. After several cycles, four or more ubiquitins linked via lysine-48 of ubiquitin (K48) are attached to the target protein. The K48-linked polyubiquitination chain is the canonical ubiquitin chain that targets the ubiquitinated protein for degradation by the proteasome enzyme complex . Monoubiquitination with a single ubiquitin conjugated to a protein regulates DNA repair, nuclear export and histone regulation rather than protein degradation , . To date, dozens of E2 enzymes and hundreds of E3 enzymes have been identified, whereas only two ubiquitin E1 enzymes have been discovered, of which E1 is the predominant isoform in the UPS pathway. Because the inhibition of the proteasome effectively reduces neointima formation in vivo, we hypothesized that inhibition of UBA1, the apex of the UPS, would also effectively block the UPS pathway (A), as do proteasome inhibition, and may thus attenuate neointimal hyperplasia.
Introduction The ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent cascade modification of target proteins with ubiquitin (Ub). Ubiquitin itself, discovered less than 50 years ago, tags thousands of diseased proteins for destruction , . It is small (only 76 amino acids), and is found unchanged in mammals, birds, fish and even worms. Because of its universality, Ub is a valuable proving ground for universal biophysical theories discussing protein amino acid sequences, structure and function . Indeed key features of Ub functionality (hydropathic waves) were identified using critical point thermodynamic scaling theory . The general biochemical logistics of Ub activation, conjugation and ligation are orchestrated sequentially by the Ub conjugation cascade of E1, E2 and E3 enzymes. Humans are known to harbor two E1, ∼30 E2 and ∼600 E3 enzymes in the Ub conjugation cascade . While Ub is “perfect”, Uba1 (E1) has evolved only modestly from slime mold to humans. The details of this evolution express several leading features of enzyme functionality. Uba1 (E1) is a large protein (>1000 amino acids), but it is readily treated by critical point thermodynamic scaling theory, with its firm foundations in statistical mechanics and its bioinformatically determined universal parameters . It turns out that hydropathic waves are also useful for Uba1 (E1), which is >14 times larger than Ub.