Protein arginine deiminases (PADs) are calcium-dependent enzymes that mediate the posttranslational conversion of arginine into citrulline. Dysregulated PAD activity is associated with numerous autoimmune disorders and cancers. In breast cancer, PAD2 citrullinates histone H3R26 and activates the transcription of estrogen receptor (ER) target genes. However, PAD2 lacks a canonical Nuclear Localization Sequence (NLS), and it is unclear how this enzyme is transported into the nucleus. Here, we show for the first time that PAD2 translocates into the nucleus in response to calcium signaling. Using BioID2, a proximity-dependent biotinylation method for identifying interacting proteins, we found that PAD2 preferentially associates with ANXA5 in the cytoplasm. Calcium binding to PAD2 weakens this cytoplasmic interaction, which generating a pool of calcium bound PAD2 that can interact with Ran. We hypothesize that this latter interaction promotes the translocation of PAD2 into nucleus. These findings highlight a critical role for ANXA5 in regulating PAD2 and identify an unusual mechanism whereby proteins translocate between the cytosol and nucleus.

Nicotinamide N-methytransferase (NNMT) catalyzes the S-adenosyl-L-methionine (SAM)dependent methylation of nicotinamide (NAM) to form N-methylnicotinamide (Me-NAM). This enzyme detoxifies xenobiotics and regulates NAD + biosynthesis. Additionally, NNMT is overexpressed in various cancers. Herein, we describe the first NNMT-targeted suicide substrates. These compounds, which include 4-chloropyridine and 4-chloronicotinamide, exploit the broad substrate scope of NNMT; methylation of the pyridine nitrogen enhances the electrophilicity of the C4 position, thereby promoting an aromatic nucleophilic substitution by C159, a non-catalytic cysteine. Based on this activity, we developed a suicide inhibition-based protein labeling (SIBLing) strategy using an alkyne-substituted 4-chloropyridine that selectively labels NNMT in vitro and in cells. In total, this study describes the first NNMT-directed activity-based probes.Nicotinamide N-methyltransferase (NNMT) is a cytosolic S-adenosyl-L-methionine (SAM)dependent methyltransferase that catalyzes the methylation of nicotinamide (NAM) to form N-methylnicotinamide (Me-NAM) and S-adenosyl-L-homocysteine (SAH) as a by-product (Figure 1A). 1,2 NNMT also methylates numerous other pyridine-containing compounds. 3 Although widely expressed, NNMT is predominantly found in the liver, where it detoxifies xenobiotics by producing N-methylated metabolites. [1][2][3][4][5][6][7][8][9] Additionally, NNMT regulates NAD + biosynthesis. 8, 10 Overexpression of NNMT is linked with several diseases, including

Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Cytoplasmic male sterility (CMS) plays an important role in the application of heterosis in wheat (Triticum aestivum L.). However, the molecular mechanism underlying CMS remains unknown. This study provides a comprehensive morphological and proteomic analysis of the anthers of a P-type CMS wheat line (P) and its maintainer line, Yanshi 9 hao (Y). Cytological observations indicated that the P-type CMS line shows binucleate microspore abortion. In this line, the tapetum degraded early, leading to anther cuticle defects, which could not provide the nutrition needed for microspore development in a timely manner, thus preventing the development of the microspore to the normal binucleate stage. Proteomic analysis revealed novel proteins involved in P-type CMS. Up to 2576 differentially expressed proteins (DEPs) were quantified in all anthers, and these proteins were significantly enriched in oxidative phosphorylation, glycolysis/gluconeogenesis, citrate cycle (TCA cycle), starch and sucrose metabolism, phenylpropanoid biosynthesis, and pyruvate metabolism pathways. These proteins may comprise a network that regulates male sterility in wheat. Based on the function analysis of DEPs involved in the complex network, we concluded that the P-type CMS line may be due to cellular dysfunction caused by disturbed carbohydrate metabolism, inadequate energy supply, and disturbed protein synthesis. These results provide insights into the molecular mechanism underlying male sterility and serve as a valuable resource for researchers in plant biology, in general, and plant sexual reproduction, in particular.

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