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Toward drugging the translocon: sequence determinants and cellular consequences Sec61 inhibition


Secretory and membrane proteins (secretory proteins) are the conduits through which cells communicate. Membrane proteins make up 35% of the human genome and, though greater than 60% of drug targets are secretory proteins, they remain difficult to inhibit, as many lack traditional small molecule binding sites. In eukaryotes, the biogenesis of most secretory proteins begins with cotranslational translocation into the endoplasmic reticulum (ER). Cotransins are small molecule inhibitors of cotranslational translocation and therefore inhibitors of secretory protein expression. Cotransins inhibit translocation by binding directly to the Sec61 translocon and perturbing the interaction between Sec61α and the targeting sequence of the nascent chain (either an N-terminal signal peptide or the first transmembrane domain). Remarkably, cotransins inhibit translocation of only a small subset of secretory proteins, dependent on the sequence of the targeting signal.

Here, we describe work toward both identifying the sequence determinants of cotransin selectivity and the broader cellular consequences of cotransin inhibition, including the discovery of Sec61α as a novel target in multiple myeloma. Using a quantitative membrane proteomics appraoch, we directly identify targets of a selective cotransin analog, CT8, in an unbiased manner. We show that CT8 inhibits only 25% of the identified secreted proteome. CT8-sensitivity of signal peptides correlates with the free energy of membrane integration suggesting that 1) signal peptides and transmembrane domains gate Sec61&apha; via a similar mechanism and 2) cotransins interfere with this signal-partitioning step. Finally, we use this metric to predict the sensitivity of novel CT8-sensitive proteins based solely on primary amino acid sequence.

To solidify Sec61α as a novel target for myeloma, we show that cotransin potently induces apoptosis in patient-derived CD138+ cells while leaving CD138- cells virtually unaffected. We begin to explore the myeloma-specific mechanism of CT8-induced apoptosis by showing that the CT8-sensitive ER-resident chaperone p58ipk is an essential protein in multiple myeloma cells.

This study furnishes a method for predicting new cotransin-sensitive proteins, thereby allowing for the rapid identification of clinically relevant arenas in which to begin testing these compounds. Within the context of multiple myeloma, we show that Sec61α is a viable therapeutic target and that cotransins represent a promising lead for further development. Using the information uncovered here, it will be possible to design new analogs to more specifically target proteins relevant to the anti-cancer properties of cotransins.

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