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First published online 25 August 2004
doi: 10.1242/jcs.01338

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

Renate Kroismayr, Ulrike Baranyi^*, Christian Stehlik, Andrea Dorfleutner, Bernd R. Binder and Joachim Lipp

Department of Vascular Biology and Thrombosis Research, Medical University of Vienna and BMT, Bio-Molecular Therapeutics, Schwarzspanierstrasse 17, 1090 Vienna, Austria

View larger version (26K): [in a new window]   Fig. 1. Structure and expression of HERC5. (A) Schematic domain structure of HERC5. Amino acids in Croco different from those in Ceb1 are shown as grey bars. aa, amino acid; HECT, homologous to E6-AP C-terminus; RLD, RCC1-like domain. (B) Comparison of Croco and Ceb1. Differences in the cDNA and protein sequences are indicated in bold. Amino acids are given in the single-letter code and positions of amino acids are indicated below. GenBank accession numbers: Ceb1, AB027289; Croco, AY337518. (C) HERC5 mRNA expression in human tissues. A commercially available multiple tissue blot containing poly(A)+ RNA derived from different human tissues was probed for HERC5 transcripts. sk. muscle, skeletal muscle. (D) Quantification of HERC5 mRNA in selected tissues. Quantitative real-time RT-PCR was performed to quantify HERC5 expression in heart, placenta and testis normalized to ß2-microglobulin expression.

View larger version (55K): [in a new window]   Fig. 2. HERC5 mRNA expression in vascular cells. (A,B) Total cellular RNA from human ECs of different origin stimulated with either LPS or TNF for the indicated periods of time was probed for HERC5 mRNA by northern blotting. HAEC were stimulated for 6 hours, FB and HSMC were stimulated for 9 hours. FB, human skin fibroblasts; HAEC, human aortic endothelial cells; HM2, a microvascular endothelial cell line; HSMC, human smooth muscle cells; HSMEC, human skin microvascular endothelial cells; HUMEC, human uterus microvascular endothelial cells; HUVEC, human umbilical vein endothelial cells. (C) HERC5 expression analysis by quantitative real-time RT-PCR. Cells were stimulated as described in A and quantitative real-time RT-PCR was performed. HERC5 expression levels are normalized to ß2-microglobulin expression. Changes in HERC5 mRNA were significant over time. (D) Comparison of HERC5 expression levels as determined by quantitative real-time RT-PCR in HSMECs, HUVECs and HSMCs.

View larger version (45K): [in a new window]   Fig. 3. Regulation of HERC5 mRNA expression. (A) HERC5 gene expression in HM2 cells in response to different stimuli after 8 hours of treatment. 10 µg of total RNA were loaded per lane for analysis. Northern blots were probed for HERC5 expression and reprobed for GAPDH to confirm equal loading of RNA. CHX, cycloheximide; EGF, epidermal growth factor; IL, interleukin; LPS, lipopolysaccharide; TGFß, transforming growth factor ß; TNF, tumor necrosis factor , VEGF, vascular endothelial growth factor. (B) Northern blot analysis of rAd.IB-infected cells. HSMEC were infected with either the rAd. IB or a control adenovirus as indicated above each lane and 48 hours post infection cells were left unstimulated or stimulated with LPS for 6 hours. 10 µg of total RNA were analyzed for mRNA of HERC5, IB and GAPDH by subsequent hybridizations. (C) HUMECs were treated as described in B but stimulated with LPS for 8 hours; isolated RNA was analyzed by quantitative real-time RT-PCR. *P<0.05.

View larger version (45K): [in a new window]   Fig. 4. Expression of HERC5 protein. (A) HERC5 protein expression. Immunoprecipitation with anti-HERC5 serum from HSMECs, HUVECs and HSMCs metabolically labeled for 16 hours were analyzed by 9% SDS-PAGE. Samples are normalized to cell number. HERC5 protein is marked by an asterisk. (B) Immunoprecipitation with anti-HERC5 serum from HSMECs metabolically labeled for 16 hours in the absence or presence of LPS were resolved by 6.5% SDS-PAGE. Duplicates were immunoprecipitated with pre-immune serum as controls. Integrity of ECs was tested by IL-8 secretion upon proinflammatory cytokine treatment before assaying. Cytosolic cell extracts are normalized to counts. HERC5 signal is marked by an asterisk. pi-serum, pre-immune serum. (C) Results from pulse-chase experiments in HSMECs in the absence () or presence () of LPS during chase. The calculated regression curves were significantly different to each other (P<0.05). (D) HERC5 protein was precipitated from cell lysates of HSMECs metabolically labeled for 20 hours. Where indicated, LPS was added for 8 hours and CHX for 4 hours before cell lysis. As a control, actin was precipitated from the same protein extracts. CHX, cycloheximide.

View larger version (50K): [in a new window]   Fig. 5. In vitro thioester bond formation assay. (A) Comparison of the C-terminal amino acid sequences of E6-AP and HERC5 are shown. The catalytic site cysteine residues are boxed. Identical residues are marked by asterisks. (B) Wild-type or C833A mutant E6-AP proteins were synthesized in vitro in the presence of L-[35S]methionine and tested for thioester bond formation in the presence (+) or absence (-) of GST-ubiquitin. Reactions were stopped in non-reducing sample buffer for 20 minutes at room temperature and analyzed by 9% SDS-PAGE and autoradiography. Slower migrating proteins are marked by an asterisk. GST-ub, GST-ubiquitin; wt, wildtype. (C) HERC5-HECT or HERC5-HECT C994A proteins were synthesized and processed as in B. Crude bacterial lysates from cells overexpressing a particular E2 enzyme were added to the samples as indicated. Slower migrating proteins are marked by an asterisk.