Lysine decrotonylation of glutathione peroxidase at lysine 220 site increases glutathione peroxidase activity to resist cold stress in chrysanthemum.

Lysine crotonylation is a protein post-translational modification that has been newly discovered in recent years. There are few studies on the lysine crotonylation of proteins in plants, and their functions in response to cold stress are still unclear. In this study, the chrysanthemum (Chrysanthemum morifolium Ramat.) glutathione peroxidase (GPX) gene was selected and named DgGPX1, and was found to be responsive to low temperature. Overexpression of DgGPX1 improved the cold resistance of transgenic chrysanthemum by increasing GPX activity to reduce the accumulation of reactive oxygen species (ROS) under low-temperature conditions. Furthermore, the level of DgGPX1 lysine crotonylation at lysine (K) 220 decreased under low temperature in chrysanthemum. Lysine decrotonylation of DgGPX1 at K220 further increased GPX activity to reduce ROS accumulation under cold stress, and thereby enhanced the cold resistance of chrysanthemum. The above results show that lysine decrotonylation of DgGPX1 at K220 increases GPX activity to resist cold stress in chrysanthemum.

The Brucella Effector Protein BspF Regulates Apoptosis through the Crotonylation of p53.

The Brucella type IV secretion system (T4SS) can promote the intracellular survival and reproduction of Brucella. T4SS secretes effector proteins to act on cellular signaling pathways to inhibit the host's innate immune response and cause a chronic, persistent Brucella infection. Brucella can survive in host cells for a long time by inhibiting macrophage apoptosis and avoiding immune recognition. The effector protein, BspF, secreted by T4SS, can regulate host secretory transport and accelerate the intracellular replication of Brucella. BspF has an acetyltransferase domain of the GNAT (GCN5-related N-acetyltransferases) family, and in our previous crotonylation proteomics data, we have found that BspF has crotonyl transferase activity and crotonylation regulation of host cell protein in the proteomics data. Here, we found that BspF attenuates the crotonylation modification of the interacting protein p53, which reduces the p53 expression through the GNAT domain. BspF can inhibit the transcription and protein expression of downstream apoptotic genes, thereby inhibiting host cell apoptosis. Additionally, the Brucella ΔbspF mutant stain promotes apoptosis and reduces the survival rate of Brucella in the cells. In conclusion, we identified that the T4SS effector protein BspF can regulate host cell apoptosis to assist Brucella in its long-term survival by attenuating crotonylation modification of p53 and decreasing p53 expression. Our findings reveal a unique mechanism of elucidating how Brucella regulates host cell apoptosis and promotes its proliferation through the secretion of effector proteins.

Single-step fluorescent probes to detect decrotonylation activity of HDACs through intramolecular reactions.

Lysine crotonylation plays vital roles in gene transcription and cellular metabolism. Nevertheless, methods for dissecting the molecular mechanisms of decrotonyaltion remains limited. So far, there is no single-step fluorescent method developed for enzymatic decrotonylation activity detection. The major difficulty is that the aliphatic crotonylated lysine doesn't allow π-conjugation to a fluorophore and decrotonylation can not modulate the electronic state directly. Herein, we have designed and synthesized two activity-based single-step fluorogenic probes KTcr-I and KTcr-II for detecting enzymatic decrotonylation activity. These two probes can be recognized by histone deacetylases and undergo intramolecular nucleophilic exchange reaction to generate fluorescence signal. Notably, peptide sequence-dependent effect was observed. KTcr-I can be recognized by Sirt2 more effectively, while KTcr-II with LGKcr peptide sequence preferentially reacted with HDAC3. Compared to other methods of studying enzymatic decrotonylation activity, our single-step fluorescent method has a number of advantages, such as facileness, high sensitivity, cheap facility and little material consumed. We envision that the probes developed in this study will provide useful tools to screen inhibitors which suppress the decrotonylation activity of HDACs. Such probes will be useful for further delineating the roles of decrotonylation enzyme and aid in biomarker identification and drug discovery.