Small molecule modulation of protein secretion
- Author(s): Garrison, Jennifer Lynn
- Advisor(s): Taunton, Jack
- et al.
Specific small molecule inhibitors can be used as molecular tools to dissect complex cellular processes and illuminate basic biological questions, such as protein secretion. A random screen for inhibitors of cell adhesion molecule expression led to the identification of HUN-7293, a fungal natural product that potently inhibits the expression of vascular cell adhesion molecule 1 (VCAM1) in activated endothelial cells. The mechanism of action and molecular target of HUN-7293 were unknown prior to the work described here. To understand its cellular role, we designed and synthesized a simplified novel analogue of HUN-7293, which we named "cotransin".
We discovered that cotransin selectively modulates protein translocation into the ER. Further, we have shown that cotransin blocks cotranslational translocation by interacting directly with the Sec61 translocation channel. Although cotransin binds to the same channel utilized by all secreted and membrane proteins, it is highly selective for a small subset of these proteins. Moreover, we demonstrated sensitivity to cotransin is controlled entirely by a protein's N-terminal signal sequence. This is an unprecedented mechanism of action for any biologically active small molecule described to date, and makes cotransin the first tool for probing the interactions between different signal sequences and their corresponding binding site(s) within the channel. Sensitivity to cotransin can be modulated by changes in a protein's N-terminal signal sequence, implying that sequence variations among signals from different proteins can be selectively exploited to modulate their functional expression. In this paradigm, secretory and membrane protein expression could be selectively down-regulated by small molecule inhibitors at their point of entry into the secretory pathway. Further, cotransin is the only specific small molecule tool available to investigate cotranslational translocation in living cells.
We sought to identify the precise features of the signal sequence that confer sensitivity to cotransin. We synthesized variants of cotransin that contain a purification handle and either a photoaffinity label or a site that is amenable to derivatization with a radioactive label. Using these analogs, we found that that cotransin's inhibitory activity is highly sensitive to both compound and signal sequence structures. To identify the precise features in the signal sequence that confer sensitivity (and resistance) to cotransin we screened a large portion of the human secreted proteome (~550 proteins) for sensitivity to CT8 and CT9, potent cotransin analogs.
Thus, it is possible to modulate the translocation, and hence functional expression of a subset of secretory proteins, despite utilization of common translocation machinery. This implies that sequence variations among signals from different substrates can be selectively exploited to modulate their functional expression. Deciphering the molecular details of how cotransin influences the signal sequence interaction with the Sec61 channel is likely to shed light on the mechanism by which unrelated signal sequences gate a communal translocation channel.