Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1

Abstract

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61beta. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.

The Role of p58IPK in Protecting the Stressed Endoplasmic Reticulum

“…Reagents for in vitro transcription, translation, and translocation assays were prepared and used as described previously (Fons et al, 2003, and references therein). Hun-7293 was obtained from D. Boger (The Scripps Research Institute), and it was tested in translocation assays of vascular cell adhesion molecule (VCAM)1 (our unpublished data) to have the same properties as those previously published for Cotransin and CAM741 (Besemer et al, 2005;Garrison et al, 2005). The p58 IPK sequence was analyzed using SignalP 3.0 (Bendtsen et al, 2004).…”

The Translocation Inhibitor CAM741 Interferes with Vascular Cell Adhesion Molecule 1 Signal Peptide Insertion at the Translocon

“…The compound does not inhibit targeting of the VCAM1 nascent chains to the translocon, but although a salt-resistant tight complex is formed, translocation to the luminal side of the ER is prevented (35). Concurrently, Garrison et al (36) reported that the synthetic compound "cotransin," which has a structure very similar to CAM741, inhibits VCAM1 translocation and demonstrated that the inhibition involves the Sec61 channel but not accessory factors, such as TRAM, TRAP, and the Sec62/63 complex.…”

“…The SEAP fusions of the mouse VCAM1 SP plus additional four amino acid residues of the mature region, all VCAM1 SP mutants and constructs encoding fusions of the N-terminal tag to the SP were generated by PCR and subcloned into pcDNA3.1. The truncated VCAM1 cDNA, encoding the 112 amino acid residues subcloned into pcDNA3.1, has been described (35). The truncated VCAM1 cDNA encoding 182 amino acid residues was generated by PCR and subcloned into pcDNA3.1.…”

“…Plasmid Constructions-The wild-type (wt) VCAM1 construct has been described (35). The SP-secreted alkaline phosphatase (SEAP) fusion constructs were generated by PCR and subcloned into the pcDNA3.1 vector (Invitrogen).…”

“…Then, 2 l of Triton X-100 and 3 l of N-glycosidase F (Roche Applied Science) were added and samples incubated at 37°C for 60 min. Chemical cross-linking was carried out as described (35). Immunoprecipitations were then performed with a polyclonal Sec61␤ or Sec61␣ antiserum (Upstate).…”