Rabbit Anti-Human UFM1 (a.a. 1-85) Polyclonal antibody

Endothelial cell dysfunction and inflammatory responses are important early contributors to the occurrence and development of atherosclerosis (AS), which still remains to be decoded. Ubiquitin-fold modifier 1 (Ufm1) is a new member of the ubiquitin-like protein family, and its biological function remains largely unknown, particularly in endothelial cell injury and inflammatory responses. In the present study, we showed that Ufm1 was highly expressed in both the nucleus and cytoplasm of human umbilical vein endothelial cells (HUVECs). We also demonstrated that the Ufm1 expression level was increased following lipopolysaccharide (LPS) -induced inflammation in HUVECs. Moreover, overexpression of Ufm1 in HUVECs alleviated the inflammatory responses induced by LPS treatment. Additionally, we found that Ufm1 overexpression inhibited the nuclear translocation of nuclear factor-kappa B (NF-kappa B) after LPS treatment, suggesting its implication in the LPS/Toll-like receptor 4 (TLR4)/NF-kappa B pathway. Taken together, in addition to decoding its expression pattern in endothelial cells, we showed for the first time that Ufm1 is upregulated in LPS-induced inflammation and Ufm1 plays an inhibitory role in inflammatory responses by targeting NF-kappa B nuclear translocation. Thus, Ufm1 may be a novel gene that protects against inflammatory responses.

E1 enzymes activate ubiquitin or ubiquitin-like proteins (Ubl) via an adenylate intermediate and initiate the enzymatic cascade of Ubl conjugation to target proteins or lipids. Ubiquitin-fold modifier 1 (Ufm1) is activated by the E1 enzyme Uba5, and this pathway is proposed to play an important role in the endoplasmic reticulum (ER) stress response. However, the mechanisms of Ufm1 activation by Uba5 and subsequent transfer to the conjugating enzyme (E2), Ufc1, have not been studied in detail. In this work, we found that Uba5 activated Ufm1 via a two-step mechanism and formed a binary covalent complex of Uba5 similar to Ufm1 thioester. This feature contrasts with the three-step mechanism and ternary complex formation in ubiquitin-activating enzyme Uba1. Uba5 displayed random ordered binding with Ufm1 and ATP, and its ATP-pyrophosphate (PPi) exchange activity was inhibited by both AMP and PPi. Ufm1 activation and Uba5 similar to Ufm1 thioester formation were stimulated in the presence of Ufc1. Furthermore, binding of ATP to Uba5 similar to Ufm1 thioester was required for efficient transfer of Ufm1 from Uba5 to Ufc1 via transthiolation. Consistent with the two-step activation mechanism, the mechanism-based pan-E1 inhibitor, adenosine 5'-sulfamate (ADS), reacted with the Uba5 similar to Ufm1 thioester and formed a covalent, tight-binding Ufm1-ADS adduct in the active site of Uba5, which prevented further substrate binding or catalysis. ADS was also shown to inhibit the Uba5 conjugation pathway in the HCT116 cells through formation of the Ufm1-ADS adduct. This suggests that further development of more selective Uba5 inhibitors could be useful in interrogating the roles of the Uba5 pathway in cells.