Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel

Abstract

Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit

“…This path to incorporation of branched-chain starter units was identified recently in the annotation of gene clusters for biosynthesis of gephyronic acid (Gph) 18 and apratoxin A (Apr) 20, 21 , a Sec61 inhibitor 22, 23 . Based on the natural product structures, the AprA loading module installs a pivaloyl group, whereas GphF introduces an isobutyryl group into gephyronic acid (Figure 1a, 1b).…”

Mycolactone reveals the substrate-driven complexity of Sec61-dependent transmembrane protein biogenesis

“…For the majority of small molecules known to modulate ER translocation, they appear to do so by acting directly at the Sec61 complex (Kalies and Römisch, 2015). Compounds such as eeyarestatin and apratoxin inhibit the Sec61-dependent translocation of a broad range of substrates (Cross et al, 2009a; Paatero et al, 2016). In contrast, and despite their common Sec61 target, HUN-7293-derived compounds such as CAM741 and the cotransin family display clear substrate specificity (Besemer et al, 2005; Garrison et al, 2005; Maifeld et al, 2011).…”

The signal peptide as a new target for drug design

“…A number of small-molecule exotoxins, virulence factors, and plant secondary metabolites have been identified that inhibit protein cotranslational translocation nonselectively by binding and blocking the translocon. [45][46][47] Compounds including apratoxin A, 48 eeyerestatin I, 49 exotoxin A, 50 mycolactone, 51 ipomoeassin F, 52,53 and coibamide A 54 inhibit expression of many proteins, generally producing toxicity. A pharmacokinetic evaluation of mycolactone as a subcutaneous analgesic was recently reported.…”

“…Resistance mutations occur in a similar area of Sec61α as for cotransin and decatransin, so it has been proposed that all of these compounds bind and act in a similar, but not identical, manner. 48 The macrocyclic triamine cyclotriazadisulfonamide (CADA) is a small molecule that inhibits replication of various strains of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) by selectively down-modulating expression of cell-surface cluster of differentiation 4 (CD4). 27,46,[64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] This type 1 integral transmembrane glycoprotein is expressed on immune cells and is the primary receptor required by HIV to enter host cells.…”