作者: Yu, Bo ; Yang, Mian ; Kong, Yingjin ; Cui, Jingxuan ; Yang, Qiannan ; Chen, Yujia ; Wu, Jian ; Chen, Jianfeng ; Mang, Ge ; Xie, Huiqi ; Ma, Ruishuang ; Yan, Xiangyu ; Du, Jingwen ; Wang, Dongni ; Zhang, Maomao ; Tong, Zhonghua ; Sun, Ping ; Jin, Jiaqi ; Wang, Xiaoqi

AbstractBackground and AimsHeart failure (HF) is a leading cause of mortality worldwide and characterized by significant co-morbidities and dismal prognosis. Neutrophil extracellular traps (NETs) aggravate inflammation in various cardiovascular diseases; however, their function and mechanism of action in HF pathogenesis remain underexplored. This study aimed to investigate the involvement of a novel VWF-SLC44A2-NET axis in HF progression.MethodsNET levels were examined in patients with HF and mouse models of transverse aortic constriction (TAC) HF. PAD4 knockout mice and NET inhibitors (GSK-484, DNase I, NEi) were used to evaluate the role of NETs in HF. RNA sequencing was used to investigate the downstream mechanisms. Recombinant human ADAMTS13 (rhADAMTS13), ADAMTS13, and SLC44A2 knockouts were used to identify novel upstream factors of NETs.ResultsElevated NET levels were observed in patients with HF and TAC mouse models of HF. PAD4 knockout and NET inhibitors improved the cardiac function. Mechanistically, NETs induced mitochondrial dysfunction in cardiomyocytes, inhibiting mitochondrial biogenesis via the NE-TLR4-mediated suppression of PGC-1α. Furthermore, VWF/ADAMTS13 regulated NET formation via SLC44A2. Additionally, sacubitril/valsartan amplifies the cardioprotective effects of the VWF-SLC44A2-NET axis blockade.ConclusionsThis study established the role of a novel VWF-SLC44A2-NET axis in regulating mitochondrial homeostasis and function, leading to cardiac apoptosis and contributing to HF pathogenesis. Targeting this axis may offer a potential therapeutic approach for HF treatment.

2024-10-01·Talanta

Immobilized PAD4 enzyme on magnetic nanoparticles for screening natural inhibitors from traditional Chinese medicines

Article

作者: Bian, Yangyang ; Li, Yanfeng ; Zhang, Shengxiang ; Zhao, Juanjuan ; Deng, Nan ; Zuo, Haiyue ; Zhao, Zeyuan ; Ou, Junjie ; Wang, Chenyang ; Dong, Jianhui

Dysregulation of peptidyl arginine deiminase 4 (PAD4) is involved in a variety of diseases including rheumatoid arthritis (RA) and Alzheimer's disease (AD), and it has emerged as potential and promising therapeutic target. However, no PAD4 inhibitor is ready for clinical use. Immobilized enzyme screening technology has gained increasing attention due to its low cost, reusability, easy separation from the reaction mixture, and resistance to changes in environmental conditions. In this study, PAD4 was immobilized on the magnetic nanoparticles (MNP) to prolong its activity stability, and a simple and rapid screening strategy of traditional Chinese medicine inhibitors based on immobilized PAD4 was established. The PAD4 enzyme was immobilized on magnetic nanoparticles (MNP) via Schiff base reaction using glutaraldehyde (GA) as crosslinking agent. Compared with free PAD4, the resulting MNP@GA@PAD4 exhibited an enhanced tolerance to temperature and storage stability, and its reusability was greatly improved with 66 % of initial enzyme activity after being recycled 10 times. The inhibitory activity of the immobilized PAD4 was assessed using two known PAD4 inhibitors GSK484 and BB-Cl-amidine. The semi-maximum inhibitory concentrations (IC₅₀) of GSK484 and BB-Cl-amidine for MNP@GA@PAD4 were 1.00 and 0.97 μM, respectively, for free PAD4 were 0.64 and 0.85 μM, respectively. Finally, the MNP@GA@PAD4 was employed to rapid screen of natural PAD4 inhibitors from forty traditional Chinese medicines (TCMs). Under the same conditions, the controlled experiment was conducted with free PAD4. The screening results of TCMs inhibitors on MNP@GA@PAD4 and free PAD4 were similar, the alcohol extracts of Cinnamomi Cortex and Caryophylli Flos had significant inhibitory effects on PAD4 enzyme activity. The IC₅₀ values of Cinnamomi Cortex extract for MNP@GA@PAD4 and free PAD4 were determined as 27 and 48 μg/mL, respectively. The IC₅₀ values of Caryophylli Flos extracts for MNP@GA@PAD4 and free PAD4 were determined as 48 and 32 μg/mL, respectively. For the first time, this study proposed a method to immobilize PAD4 on magnetic materials, and developed a rapid, reusable and feasible strategy to screening natural PAD4 inhibitors from TCMs.

2024-08-01·International Journal of Biological Macromolecules

Citrullinating enzyme PADI4 and transcriptional repressor RING1B bind in cancer cells

Article

作者: Soto-Conde, Lourdes ; Araujo-Abad, Salome ; de Juan Romero, Camino ; Neira, José L ; Velazquez-Campoy, Adrian ; Vidal, Miguel ; Abian, Olga ; Rizzuti, Bruno

Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1-221) and C-terminal (residues 228-336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2-10 μM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.

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适应症	最高研发状态	国家/地区	公司	日期
自身免疫性疾病	临床前	德国	Cellzome GmbH	2015-03-01
心血管疾病	临床前	德国	Cellzome GmbH	2015-03-01
肿瘤	临床前	德国	Cellzome GmbH	2015-03-01