The function of intrinsically disordered selenoproteins
Date
2020
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
SELENOS and SELENOK are intrinsically disordered selenoproteins that take part in the endoplasmic-reticulum-associated degradation (ERAD) pathway, which degrades misfolded proteins and maintains cellular protein hemostasis. They also function in antioxidant defense and various protein complexes in the ER. SELENOS and SELENOK each have one selenocysteine (Sec) in their cytosolic disordered segment. It is unusual for intrinsically disordered proteins (IDPs) to function as enzymes, but SELENOS and SELENOK have enzymatic activity due to the presence of Sec. In this thesis, we focus on characterizing functions of SELENOS and SELENOK. ☐ SELENOS recruits the ATPase, p97 (an unfoldase) to the ER membrane to provide energy for the retro-translocation of ERAD substrates. We developed Sec mediated expressed protein ligation (EPL) for efficient preparation of selenoproteins, and semi-synthesized the cytosolic SELENOS (cSELENOS) and full-length SELENOS. We introduced a new redox potential assay and 77Se NMR and characterized the redox property of Sec incorporated SELENOS. Moreover, the interaction of SELENOS and p97 is measured in vitro with a moderate binding affinity, Kd of 10 µM. We revealed that the recruitment of p97 by SELENOS is nucleotide-dependent, ATP bound p97 binds tighter to SELENOS than ADP bound p97. SELENOS does not change the ATPase activity of p97 when they interact. ☐ SELENOK plays roles in the ERAD, protein palmitoylation, and recently was found in lipid droplets. We discovered that SELENOK has auto-proteolytic activity. Self-cleavage of SELENOK releases its C-terminal fragments that contain Sec, the first such observation for a selenoprotein. We show that the autocatalytic cleavage of SELENOK takes place both in vivo and in vitro. Strikingly, SELENOK has multiple cleavage sites around its SH3 recruitment element. Upon cleavage, SELENOK generates protein fragments that may still interact with protein partners. Furthermore, we found SELENOK can also cleave other proteins in vitro, indicating its potential role as a protease. The membrane-bound segment of SELENOK is essential for its cleavage activity. Several key mutations in SELENOK can change its cleavage sites and rate. We propose that the auto-proteolysis of SELENOK may regulate its cellular location and association with different protein complexes in response to ERAD stress and signaling.
Description
Keywords
Selenoproteins, Endoplasmic-reticulum-associated degradation, Intrinsically disordered proteins