S-Nitrosylation of OTUB1 Alters Its Stability and Ubc13 Binding

ACS Chem Neurosci. 2022 May 18;13(10):1517-1525. doi: 10.1021/acschemneuro.1c00855. Epub 2022 May 2.

Abstract

S-Nitrosylation is a reversible post-translational modification that regulates protein function involving the covalent attachment of the nitric oxide (NO) moiety to sulfhydryl residues of the protein. It is an important regulator in the cell signaling process under physiological conditions. However, the release of an excess amount of NO due to dysregulated NOS machinery causes aberrant S-nitrosylation of proteins, which affects protein folding, localization, and activity. Here, we have shown that OTUB1, a deubiquitinating enzyme, undergoes S-nitrosylation under redox stress conditions in vivo and in vitro. Previously, we have shown that OTUB1 forms an amyloid-like structure that promotes phosphorylation of α-synuclein and neuronal toxicity. However, the mechanistic insight into OTUB1 aggregation remains elusive. Here, we identified that OTUB1 undergoes S-nitrosylation in SH-SY5Y neuroblastoma cells under rotenone-induced stress, as well as excitotoxic conditions, and in rotenone-treated mouse brains. The in vitro S-nitrosylation of OTUB1 followed by mass-spectrometry analysis has identified cysteine-23 and cysteine-91 as S-nitrosylation sites. S-Nitrosylated OTUB1 (SNO-OTUB1) diminished its catalytic activity, impaired its native structure, promoted amyloid-like aggregation, and compromised its binding with Ubc13. Thus, our results demonstrated that nitrosylation of OTUB1 might play a crucial role in regulating the ubiquitin signaling and Parkinson's disease pathology.

Keywords: OTUB1; Parkinson’s disease; S-nitrosylation; Ubc13; neurodegenerative disease; nitric oxide; post-translational modification; protein misfolding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amyloid / metabolism
  • Animals
  • Cysteine / metabolism
  • Cysteine Endopeptidases* / metabolism
  • Mice
  • Nitric Oxide / metabolism
  • Parkinson Disease* / metabolism
  • Protein Processing, Post-Translational
  • Rotenone / pharmacology
  • Ubiquitin-Conjugating Enzymes* / metabolism

Substances

  • Amyloid
  • Rotenone
  • Nitric Oxide
  • Ube2n protein, mouse
  • Ubiquitin-Conjugating Enzymes
  • Cysteine Endopeptidases
  • Otub1 protein, mouse
  • Cysteine