Delineating Anopheles gambiae coactivator associated arginine methyltransferase 1 automethylation using top–down high resolution tandem mass spectrometry

Coactivator‐associated arginine methyltransferase 1 (CARM1), originally defined as a coactivator for steroid receptors, is a member of the protein arginine methyltransferases. Here, we report the discovery and characterization of an automethylation event by AgCARM1, a CARM1 homologue in the mosquito Anopheles gambiae, using top–down high resolution tandem mass spectrometry, which allows fine mapping of modifications in the intact protein accurately and quantitatively without priori knowledge. Unexpectedly, we found that AgCARM1 has already been predominantly dimethylated during its expression in Escherichia coli. A single arginine methylation site, R485, was identified which is conserved among CARM1 in insects. No methylation was observed in the intact AgCARM1^R485K mutant where R485 is mutated to lysine, which confirms that R485 is the only detectable methylation site. Using AgCARM1 methyltransferase defective mutants, we confirmed that this is an automethylation event and show the automethylation of AgCARM1 occurs intermolecularly. This study represents the first comprehensive characterization of an automethylation event by top–down mass spectrometry. The unexpected high percentage of automethylated recombinant AgCARM1 expressed in E. coli may shed light on other bacterially expressed post‐translational modifying enzymes, which could be modified but overlooked in biochemical and structural studies. Top–down high resolution tandem mass spectrometry thus provides unique opportunities for revealing unexpected protein modification, localizing specific modification to one amino acid, and delineating molecular mechanism of an enzyme.     

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.     

哮喘豚鼠肺组织中的CARM1和NF-κB表达及地塞米松的干预作用

目的：探讨豚鼠支气管哮喘模型中共激活因子相关的精氨酸甲基转移酶1（coactivator-associated arginine methyltransferase1,CARM1）和核因子-B（NF-B）在气道和肺组织的表达变化及地塞米松的干预作用。方法：36只白色雄性豚鼠随机分为正常对照组、哮喘组和地塞米松治疗组。卵清蛋白致敏并激发后采用间接免疫荧光法检测气道和肺组织中CARM1和NF-B（P65）的表达,探讨其在哮喘中可能的作用机制。结果：CARM1和NF-κB（P65）在对照组、哮喘组及地塞米松治疗组均有阳性表达,主要在支气管-终末细支气管上皮细胞和肺组织细胞胞核表达。CARM1和NF-κB（P65）在哮喘组表达水平为（[123.75±41.55）和（126.92±46.74）],在地塞米松治疗组表达水平为（[84.33±27.70）和（85.00±29.22）],均高于对照组的（[51.67±8.29）和（52.75±9.07）个/400倍视野],地塞米松治疗组表达较哮喘组低。结论：CARM1和NF-B（P65）在哮喘豚鼠气道上皮及肺组织细胞胞核高表达,提示CARM1可能通过增强募集NF-B到相关位点激活NF-B信号转导通路并启动了多种前炎性基因和免疫调节基因的转录激活、诱发哮喘炎症反应。地塞米松可下调CARM1和NF-κB的表达而抑制哮喘炎症反应。     

Design and synthesis of novel PRMT1 inhibitors and investigation of their binding preferences using molecular modelling

Protein arginine methyltransferase 1 (PRMT1) catalyses the methylation of substrate arginine by transferring the methyl group from SAM (S-adenosyl-l-methionine), which leads to the formation of S-adenosyl homocysteine (SAH) and methylated arginine. We have shown previously that the Asp84 on PRMT1 could be a potential inhibitor binding site. In the current study, 28 compounds were designed and synthesized that were predicted to bind the Asp84 and substrate arginine sites together. Among them, 6 compounds were identified as potential PRMT1 inhibitors, and showed strong inhibitory effects on cancer cell lines, especially HepG2. The most potent PRMT1 inhibitor, compound 13d, was selected for molecular dynamic simulations to investigate binding poses. Based on the free energy calculations and structural analysis, we predicted that the ethylenediamine group would tightly bind to Asp84, and the trifluoromethyl group should occupy part of substrate arginine binding site, which is consistent with our original goal. Our results show for the first time that PRMT1 inhibitors can target the Asp84 binding site, which will be helpful for future drug discovery studies.     

Optimization of pyrazole inhibitors of Coactivator Associated Arginine Methyltransferase 1 (CARM1)

Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyrazole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).     

Expression and purification of full-length mouse CARM1 from transiently transfected HEK293T cells using HaloTag technology

Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in Escherichia coli forms insoluble inclusion bodies that are difficult to denature and refold. The presented results demonstrate the use of a novel HaloTag? technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10-50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1's methyltransferase activity.     

Epigenetic and transcriptional regulation of autophagy

Macroautophagy (hereafter referred to as autophagy) is an essential self-digestion process to maintain homeostasis and promote survival in response to starvation. Although the components of autophagy in the cytoplasm have been well studied, little has been known about the fine-tuning mechanism of autophagy through epigenetic regulations. Recently, we identified the histone arginine methyltransferase CARM1 as a new component and followed histone H3R17 dimethylation as a critical epigenetic mark in starvation-induced autophagy. Upon nutrient starvation, CARM1 is stabilized in the nucleus, but not in the cytoplasm, whereas it is constantly degraded under nutrient-rich conditions by the SKP2-containing SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase. We further showed that nutrient starvation induces the protein levels and activity of AMPK in the nucleus. Activated AMPK then phosphorylates FOXO3, leading to SKP2 downregulation and increased CARM1 protein levels in the nucleus. Stabilized CARM1 in turn functions as an essential co-activator of TFEB and regulates the expression of autophagy and lysosomal genes. Our findings provide a conceptual advance that activation of specific epigenetic programs is indispensable for a sustained autophagic response, and shed light on a potential therapeutic targeting of the newly identified AMPK-SKP2-CARM1 signaling axis in autophagy-related diseases.     

Role of PRMTs in cancer: Could minor isoforms be leaving a mark?

Protein arginine methyltransferases(PRMTs) catalyze the methylation of a variety of protein substrates, many of which have been linked to the development, progression and aggressiveness of different types of cancer. Moreover, aberrant expression of PRMTs has been observed in several cancer types. While the link between PRMTs and cancer is a relatively new area of interest, the functional implications documented thus far warrant further investigations into its therapeutic potential. However, the expression of these enzymes and the regulation of their activity in cancer are still significantly understudied. Currently there are nine main members of the PRMT family. Further, the existence of alternatively spliced isoforms for several of these family members provides an additional layer of complexity. Specifically, PRMT1, PRMT2, CARM1 and PRMT7 have been shown to have alternative isoforms and others may be currently unrealized. Our knowledge with respect to the relative expression and the specific functions of these isoforms is largely lacking and needs attention. Here we present a review of the current knowledge of theknown alternative PRMT isoforms and provide a rationale for how they may impact on cancer and represent potentially useful targets for the development of novel therapeutic strategies.