SET7 methylates the deubiquitinase OTUB1 at Lys 122 to impair its binding to E2 enzyme UBC13 and relieve its suppressive role on ferroptosis.

The deubiquitinating enzyme OTUB1 possesses canonical deubiquitinase (DUB) activity and noncanonical, catalytic-independent activity, which has been identified as an essential regulator of diverse physiological processes. Posttranslational modifications of OTUB1 affect both its DUB activity and its noncanonical activity of binding to the E2 ubiquitin-conjugation enzyme UBC13, but further investigation is needed to characterize the full inventory of modifications to OTUB1. Here, we demonstrate that SET7, a lysine monomethylase, directly interacts with OTUB1 to catalyze OTUB1 methylation at lysine 122. This modification does not affect DUB activity of OTUB1 but impairs its noncanonical activity, binding to UBC13. Moreover, we found using cell viability analysis and intracellular reactive oxygen species assay that SET7-mediated methylation of OTUB1 relieves its suppressive role on ferroptosis. Notably, the methylation-mimic mutant of OTUB1 not only loses the ability to bind to UBC13 but also relieves its suppressive role on Tert-Butyl hydroperoxide-induced cell death and Cystine starvation/Erastin-induced cellular reactive oxygen species. Collectively, our data identify a novel modification of OTUB1 that is critical for inhibiting its noncanonical activity.

Methyltransferase SMYD3 impairs hypoxia tolerance by augmenting hypoxia signaling independent of its enzymatic activity.

Hypoxia-inducible factor (HIF)1α, a main transcriptional regulator of the cellular response to hypoxia, also plays important roles in oxygen homeostasis of aerobic organisms, which is regulated by multiple mechanisms. However, the full cellular response to hypoxia has not been elucidated. In this study, we found that expression of SMYD3, a methyltransferase, augments hypoxia signaling independent of its enzymatic activity. We demonstrated SMYD3 binds to and stabilizes HIF1α via co-immunoprecipitation and Western blot assays, leading to the enhancement of HIF1α transcriptional activity under hypoxia conditions. In addition, the stabilization of HIF1α by SMYD3 is independent of HIF1α hydroxylation by prolyl hydroxylases and the intactness of the von Hippel-Lindau ubiquitin ligase complex. Furthermore, we showed SMYD3 induces reactive oxygen species accumulation and promotes hypoxia-induced cell apoptosis. Consistent with these results, we found smyd3-null zebrafish exhibit higher hypoxia tolerance compared to their wildtype siblings. Together, these findings define a novel role of SMYD3 in affecting hypoxia signaling and demonstrate that SMYD3-mediated HIF1α stabilization augments hypoxia signaling, leading to the impairment of hypoxia tolerance.

Repression of p53 function by SIRT5-mediated desuccinylation at Lysine 120 in response to DNA damage.

p53 is a classic tumor suppressor that functions in maintaining genome stability by inducing either cell arrest for damage repair or cell apoptosis to eliminate damaged cells in response to different types of stress. Posttranslational modifications (PTMs) of p53 are thought to be the most effective way for modulating of p53 activation. Here, we show that SIRT5 interacts with p53 and suppresses its transcriptional activity. Using mass spectrometric analysis, we identify a previously unknown PTM of p53, namely, succinylation of p53 at Lysine 120 (K120). SIRT5 mediates desuccinylation of p53 at K120, resulting in the suppression of p53 activation. Moreover, using double knockout mice (p53-/-Sirt5-/-), we validate that the suppression of p53 target gene expression and cell apoptosis upon DNA damage is dependent on cellular p53. Our study identifies a novel PTM of p53 that regulates its activation as well as reveals a new target of SIRT5 acting as a desuccinylase.