Ubiquitin-Like protein UBL1 orchestrates monocyte recruitment and pro-inflammatory macrophage activation in critical limb ischemia: Involving SUMOylation and stabilization of PER1.

Critical limb ischemia (CLI), a severe manifestation of peripheral artery disease (PAD), is marked by accumulation of pro-inflammatory monocytes (Mos) and macrophages (Mφs) in ischemic skeletal muscles. Ubiquitin-Like Protein UBL1, an effector of small ubiquitin-like modifier mediated modification (SUMOylation), has been implicated in Mo/Mφ-associated tissue inflammation. However, the role of UBL1 in CLI has not been fully investigated. In this study, UBL1 expression was downregulated in peripheral blood Mos from CLI patients, and from hind limb ischemia (HLI) mice induced by femoral artery ligation (FAL). Adeno-associated virus (AAV)-mediated UBL1 knockdown (KD) in HIL mice hindered blood flow recovery, aggravated tissue injury, and induced pro-inflammatory Mφ activation. Moreover, UBL1 KD reduced period circadian regulator 1 (PER1) expression in peripheral blood Mos isolated from HLI mice. UBL1 promoted PER1 SUMOylation at lysine 146 (K146) site, thereby enhancing its protein stability. Hypoxia significantly inhibited this process, with ubiquitin conjugating enzyme E2 I (UBE2I) being critically involved. Additionally, UBL1 KD promoted hypoxia-induced Mo migration via C-C Chemokine receptor 2 (CCR2) signaling and advanced pro-inflammatory cytokine release from hypoxic THP-1 derived Mφs. More importantly, rescue assays demonstrated that PER1 mediated UBL1 KD-induced Mo migration and pro-inflammatory Mφ activation. In summary, this study highlights the significance of UBL1 in HLI via regulating PER1 SUMOylation.