HERC5, a HECT E3 ubiquitin ligase tightly regulated in LPS activated endothelial cells

Abstract

By differential screening we isolated genes upregulated in inflammatory cytokine-stimulated human skin microvascular endothelial cells. One of these cDNAs encoded RCC1 (regulator of chromosome condensation 1)-like repeats and a HECT (homologous to E6-AP C-terminus) domain, representing a member of the HERC (HECT and RCC1 domain protein) family of ubiquitin ligases. The mRNA level of this member, HERC5, is specifically upregulated in endothelial cells by the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β, and by lipopolysaccharide (LPS), but is hardly expressed in other cells of the vascular wall such as primary smooth muscle cells and fibroblasts. Regulation of HERC5 gene expression suggests a critical role for the transcription factor NF-κB. In contrast to mRNA expression HERC5 protein is subject of enhanced degradation upon LPS stimulation of endothelial cells. The time course of LPS-induced changes in HERC5 protein and mRNA levels suggests that the initial drop in HERC5 protein is balanced by increased protein synthesis due to upregulation of HERC5 mRNA. This leads to recovery of HERC5 protein levels within 12 hours of LPS stimulation and points at a tight control of HERC5 protein. To analyze functional activity of this putative member of the ubiquitin-conjugating pathway we performed in vitro assays with different ubiquitin-conjugating enzymes. We found that HERC5 possesses ubiquitin ligase activity and requires the presence of the ubiquitin-conjugating enzyme UbcH5a for its activity. These data show for the first time that a functionally active HECT ubiquitin ligase exhibits a tightly controlled cytosolic level under inflammatory conditions in endothelial cells.

Herc5 Attenuates Influenza A Virus by Catalyzing ISGylation of Viral NS1 Protein

“…In addition, bacteria and virus infection could directly induce the ISG15 modification system, possibly via IRF3/IRF7 transcription factors (39,59,60). Because IAV-NS1 is a strong inhibitor of type I IFN induction during IAV infection, it would be difficult to reveal the functional difference of ISG15 modification toward IAV in IFNR 2/2 and WT mice, because both types of mice would induce ISG15 modification system to almost the same extent during IAV infection (61)(62)(63).…”

“…The expression of Herc5 is especially high in testis and fetal brain but low in most tissues (38). Interestingly, Herc5 is induced by the proinflammatory cytokines TNF-a and the b form of pro-IL-1 (39). Herc5 is also markedly induced by type I IFNs or viruses and functions as an ISG15 E3 ligase to promote overall ISGylation in host cells (29,30).…”

TGF-β-induced apoptosis in endothelial cells mediated by M6P/IGFII-R and mini-plasminogen

“…Cells, antibodies and reagents Both human umbilical-vein endothelial cells (HUVECs) and human umbilical-vein smooth-muscle cells (HUVSMCs) were isolated by collagenase digestion from human umbilical cords as described (Kroismayr et al, 2004). Cells were grown on gelatin-coated cell culture flasks at 37°C and 5% CO 2 .…”

Positive Regulation of Interferon Regulatory Factor 3 Activation by Herc5 via ISG15 Modification

“…In particular, HERC3 has been shown to be a ubiquitin binding protein (5). The expression of HERC5 is especially high in testis and fetal brain but low in most tissues (22). Interestingly, HERC5 is induced by the proinflammatory cytokines TNF-␣ and interleukin-1␤ (IL-1␤) (22).…”