Cathepsin B S-nitrosylation promotes ADAR1-mediated editing of its own mRNA transcript via an ADD1/MATR3 regulatory axis.

Genetic information is generally transferred from RNA to protein according to the classic "Central Dogma". Here, we made a striking discovery that post-translational modification of a protein specifically regulates the editing of its own mRNA. We show that S-nitrosylation of cathepsin B (CTSB) exclusively alters the adenosine-to-inosine (A-to-I) editing of its own mRNA. Mechanistically, CTSB S-nitrosylation promotes the dephosphorylation and nuclear translocation of ADD1, leading to the recruitment of MATR3 and ADAR1 to CTSB mRNA. ADAR1-mediated A-to-I RNA editing enables the binding of HuR to CTSB mRNA, resulting in increased CTSB mRNA stability and subsequently higher steady-state levels of CTSB protein. Together, we uncovered a unique feedforward mechanism of protein expression regulation mediated by the ADD1/MATR3/ADAR1 regulatory axis. Our study demonstrates a novel reverse flow of information from the post-translational modification of a protein back to the post-transcriptional regulation of its own mRNA precursor. We coined this process as "Protein-directed EDiting of its Own mRNA by ADAR1 (PEDORA)" and suggest that this constitutes an additional layer of protein expression control. "PEDORA" could represent a currently hidden mechanism in eukaryotic gene expression regulation.

Hsp90 S-nitrosylation at Cys521, as a conformational switch, modulates cycling of Hsp90-AHA1-CDC37 chaperone machine to aggravate atherosclerosis.

Endothelial dysfunction is the initial process of atherosclerosis. Heat shock protein 90 (Hsp90), as a molecular chaperone, plays a crucial role in various cardiovascular diseases. Hsp90 function is regulated by S-nitrosylation (SNO). However, the precise role of SNO-Hsp90 in endothelial dysfunction during atherosclerosis remains unclear. We here identified Hsp90 as a highly S-nitrosylated target in endothelial cells (ECs) by biotin switch assay combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The elevation of SNO-Hsp90 was observed in atherosclerotic human and rodent aortas as well as in oxidized LDL (oxLDL)-treated ECs. Inhibition of inducible nitric oxide synthase (iNOS) or transfection with Hsp90 cysteine 521 (Cys521) mutation plasmid decreased the level of SNO-Hsp90 in oxLDL-cultured ECs. Coimmunoprecipitation and proximity ligation assay demonstrated that SNO-Hsp90 at Cys521 suppressed the interaction between Hsp90 and activator of Hsp90 ATPase activity 1 (AHA1), but promoted the association of Hsp90 and cell division cycle 37 (CDC37). Hsp90 Cys521 mutation increased endothelial nitric oxide synthase (eNOS) activity and inhibited nuclear factor kappa-B (NF-κB) signaling, thereby increasing nitric oxide (NO) bioavailability and alleviating endothelial adhesion, inflammation and oxidative stress in oxLDL-treated ECs. Also, administration of endothelial-specific adeno-associated viruses of Cys521-mutated Hsp90 significantly mitigated vascular oxidative stress, macrophage infiltration and atherosclerosis lesion areas in high fat diet-fed ApoE-/- mice. In conclusion, SNO-Hsp90 at Cys521, that serves as a conformational switch, disrupts Hsp90/AHA1 interaction but promotes recruitment of CDC37 to exacerbate atherosclerosis.

P16 I NK 4a Deletion Ameliorates Damage of Intestinal Epithelial Barrier and Microbial Dysbiosis in a Stress-Induced Premature Senescence Model of Bmi-1 Deficiency.

This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16 INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1-deficient (Bmi-1-/- ), Bmi-1 and p16 INK4a double-knockout (Bmi-1-/-p16 INK4a-/- ), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16 INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16 INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)-dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1-/- and Bmi-1-/-p16 INK4a-/- mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1-/- mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α-dependent barrier permeability and aging. Accumulated p16 INK4a combined with occludin at the 1st-160th residue in cytoplasm of intestinal epithelium cells from Bmi-1-/- mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16 INK4a deletion could maintain barrier function and microbiota balance in Bmi-1-/- mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16 INK4a accumulation. The clearance of p16 INK4a -positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1-160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16 INK4a with occludin for protecting TJ.