Signaling, Sensing, and Enzymatic Roles of Disordered Selenoproteins

PROJECT SUMMARY Selenoproteins are prominently engaged in the integrated cellular response to stress. Here we focus on the ER- bound membrane proteins selenoprotein K (selenok) and selenoprotein S (selenos), which are mostly known for their connection to ER stress resolution. However, they also contribute to other cellular processes, such as

protein quality control, protein palmitoylation, cytokine secretion, immune response, calcium signaling, protein trafficking, and differentiation. Consequently, their genetic variations are associated with heightened risks for - among others- cardiovascular diseases, diabetes, and cancer, while their expression levels are tied to cancer

prognosis. However, there is no unifying and comprehensive view that plausibly spans their involvement in multiple protein complexes and connects their seemingly disparate cellular roles. Motivated by our work, we propose that selenok and selenos are putative ER-bound transcription factors. Upon stimulation, they are processed and migrate to the nucleus to affect gene expression. To test selenok’s and

selenos’s direct involvement in gene transcription, we will determine their nuclear forms and evaluate their interactions with nucleotides and transcription factors. We also suggest that in the ER membrane, selenok and selenos contribute to protein quality control by acting as sensors of misfolded and misassembled proteins. There

are indications that the two selenoproteins act as accessory proteins of derlins, which are part of the ER- associated degradation (ERAD) pore-forming complex. In this capacity, selenok and selenos could identify “client” proteins for extraction through the ERAD’s pore, delivering them to derlins and thus preventing harmful

accumulation. Based on preliminary experiments, selenok and selenos appear able to recognize certain protein surfaces, such as hydrophobic leucine zippers, positive patches on armadillo domains, and b-propeller folds of proteins. These ‘recognition’ surfaces are usually hidden when proteins are properly folded, and complexes are

well-assembled. However, when these systems fail to take on their intended forms, these surfaces get exposed. Using a structural biology approach, we will characterize the complex of selenok, selenos, and derlin. To substantiate this sensing function, we will investigate how the presence and absence of selenos influence the

interactions between representative degradable ‘clients’ and derlins using in vivo photo-crosslinking of unnatural amino acids at site-specific locations. In addition, we will study the interactome of the less characterized selenok and identify its directly bound protein partners and enzymatic substrates.

These dual roles of selenok and selenos in gene transcription and ERAD sensing would explain how they can influence numerous signaling pathways, engage with multiple protein complexes, and respond to cellular stress. Altogether, this proposed research will provide fundamental insights into the rich diversity of selenium-based

redox biology and the specifics of cellular roles of selenok and selenos under normal and stressed conditions, thus providing a molecular-level explanation of why they exert such a significant influence on human health.