Characterization of the Role for the N‐Terminal Domain on Sil1 Activities as a Nucleotide Exchange Factor and Reductase

Abstract

The accumulation of reactive oxygen species (ROS) can lead to oxidative stress and play a role in human diseases such as cancer and neurodegenerative diseases. We are interested in studying how cells use ROS as signaling molecules to maintain a balanced redox environment. Using yeast as our model system, we have previously shown that the Hsp70 in the endoplasmic reticulum (ER), BiP, acts as a direct sensor of peroxide, a type of ROS produced during protein folding. BiP has two major domains that regulate its function as a chaperone: an ATPase domain and a substrate‐binding domain. Under ER oxidative stress, yeast BiP's conserved cysteine gets modified/oxidized by peroxide. Modification of BiP's cysteine causes a conformational change, which enhances BiP's ability to bind to substrates, leading to protection of cells under ER oxidative stress. When cells go back to non‐stress conditions, reversibility of BiP's cysteine modification is important for normal cellular function and survival. We have evidence that the protein Sil1, a known nucleotide exchange factor (NEF) of BiP, can remove the modification on BiP's cysteine. We have shown that in addition to acting as a NEF, Sil1 has an unexpected reductase activity that depends on two cysteines, Cys‐52 and Cys‐57, located at the N terminus. While the N‐terminal domain is required for Sil1 activity as a reductase, Sil1 is competent as a NEF even in the absence of the N‐terminal domain. Our goal is to further elucidate the molecular details relating to how the N‐terminal region of Sil1 impacts both Sil1 NEF and reductase activity by utilizing a variety of biochemical and biophysical approaches. Our preliminary data suggest that NEF activity is influenced by the presence of the N terminus. Our current data also suggest the N‐terminal domain may adopt multiple conformations, allowing for regulation of Sil1 NEF activity and potentially its activity as a reductase. Of note, mutations in the Sil1 gene have been found in over half of patients with Marinesco‐Sjögren Syndrome (MSS), a rare autosomal disease characterized by early onset cataracts, cerebellar ataxia, and delayed mental development. One such mutations maps near the N‐terminal domain, and we expect that characterization of full‐length Sil1 may also lend further insight into how mutations in Sil1 can cause MSS.Support or Funding InformationNSF GRFP and SUNY Diversity Fellowship