A ROLE FOR THE N-TERMINAL DOMAIN IN MODULATING THE ACTIVITIES OF THE NUCLEOTIDE EXCHANGE FACTOR SIL1

Excessive cellular reactive oxygen species (ROS), or oxidative stress, can lead to cell damage and is implicated in diseases such as cancer, aging and neurodegenerative disorders. Yet, although these highly reactive molecules can be damaging, ROS can also elicit beneficial effects in cells as signaling molecules. In the endoplasmic reticulum (ER), where ROS are produced from protein folding, the Hsp70 BiP plays a role in cell protection during ER oxidative stress. BiP is a chaperone that binds and release substrates, and these activities are regulated by its ATPase cycle. We have previously discovered that yeast BiP’s conserved cysteine gets modified by the ROS peroxide during oxidative stress conditions. Our lab discovered that the protein Sil1 functions in cells as a reductant that can remove the modification on BiP’s cysteine. The reductant activity of Sil1 is mediated through two N-terminal cysteines. Sil1 has been well- characterized as BiP’s nucleotide exchange factor (NEF) that facilitates ADP release. Sil1 is active as a NEF even in the absence of its N-terminal region. A molecular picture as to how the N terminus, which contains the redox-active cysteines, influences interaction with BiP remains unclear. Our biochemical characterization of the full-length Sil1 has revealed that the N-terminal domain plays a role in modulating Sil1 activities. We uncovered that the presence of the N-terminal domain decreases Sil1 NEF activity and impacts the structural conformation adopted by full-length Sil1. We have mapped the region of the N terminus that accounts for the auto-inhibition of Sil1 NEF activity to a stretch of 36 amino acids of highly conserved sequence. Mutations in human SIL1 have been found in patients with Marinesco-Sjogren syndrome (MSS). One such mutation, R92W, is in the conserved N-terminal region. Through the characterization of the yeast version, Sil1-R84W, we have demonstrated that the mutation increases NEF activity, impacts reductant activity, and changes the apparent conformation of Sil1. Our overall findings suggest that the N-terminal domain’s conserved region is important in regulating Sil1 activities. We predict that this region may undergo post-translational modifications in cells to modulate NEF and reductant functions of Sil1 under specific physiological conditions