Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

Abstract

The Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

Genome-wide screening identified SEC61A1 as an essential factor for mycolactone-dependent apoptosis in human premonocytic THP-1 cells

“…Therefore, it is possible to infer that thapsigargin induces eIF2α phosphorylation independent of SEC61A1, whereas ATF6 expression was dependent on SEC61A1. It was reported that the SEC61A1 inhibitor ipomoeassin F also suppressed ATF6 protein expression in HepG2 cells [ 9 ]. Morel et al .…”

“…In a clinical study, de novo missense mutations in SEC61A1 were reported to be the cause of common variable immunodeficiency and glomerulocystic kidney disease, a rare hereditary disorder characterized by the cystic dilation of Bowman's capsule due to protein instability and functional impairment with dysregulated calcium homeostasis [37]. In addition, other inhibitors that directly target SEC61A1, such as CT7, CT8, and ipomoeassin F, have similar effects to mycolactone [8][9][10][11][12]. Mycolactone physically binds to SEC61A1, maintains the Sec61 translocon conformation, and prevents the access of signaling peptides to the binding site of SEC61A1 [13].…”

“…In vitro studies showed that mycolactone blocks the translocation of nascent proteins across the ER membrane, and similar inhibitors such as CT7, CT8, and ipomoeassin F directly target the ER Sec61 translocon [7][8][9][10][11][12]. The SEC61α amino acid mutation R66G partly rescued mycolactone-induced cell death and protein translocation in HEK293 cells [11].…”

Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F

“…Linear DNA of the short form of human HLA class II histocompatibility antigen gamma chain (Ii; P04232, isoform 2, residues 17–232) or human glycophorin C (GypC; P04921) were generated by PCR and transcribed into RNA using T7 RNA polymerase (Promega). Membrane insertion assays (20 µL, 1 h at 30 °C, containing 6.5% ( v / v ) nuclease-treated ER microsomes (from stock with OD280 = 44/mL)), endoglycosidase H f (New England Biolabs) treatment, sample resolution by SDS-PAGE (16% polyacrylamide gels) and gel drying were performed as previously described [ 13 , 14 , 37 , 38 , 42 ]. Following exposure to a phosphorimaging plate for 24–72 h, radiolabeled products were visualized using a Typhoon FLA-7000 (GE Healthcare, Tokyo, Japan) and the ratio of the signal intensity for the N-glycosylated (XGly) and non-glycosylated (0Gly) forms obtained using AIDA v.5.0 (Raytest Isotopenmeβgeräte).…”

Inhibitors of the Sec61 Complex and Novel High Throughput Screening Strategies to Target the Protein Translocation Pathway

“…Ipomoeassin F (IpoF) is a natural plant derived resin glycoside cytotoxin that showed a high anticancer potency in different cell lines [ 149 , 150 , 151 ]. Later, IpoF was shown to be a non-selective inhibitor of protein secretion via the co-translational translocation process [ 151 , 152 , 153 ]. In vitro translocation assays showed that IpoF is specific for the inhibition of Sec61 dependent protein translocation as tail-anchored proteins, but also type III single pass membrane proteins were resistant to IpoF activity [ 151 ].…”