Purification and structural characterization of lectin with antibacterial and anticancer properties from grubs of hide beetle, Dermestes frischii. Arokiyaraj Charles,Tamilarasan Kamalanathan,Manikandan Ramar,Janarthanan Sundaram International journal of biological macromolecules Lectins or haemagglutinins are diverse classes of non-immune proteins; they bind to carbohydrates and are abundant in nature. In the present study, a coleopteran lectin from grubs of hide beetle, Dermestes frischii called DFL, was purified by glutaraldehyde (fixative-agent) fixed hen erythrocytes and characterized further for its functional properties. The purified DFL was stable between pH range 5 to 9 and heat-stable up to 50 °C. It was insensitive to EDTA and did not require any divalent cations. DFL native molecular mass was approximately 69 kDa with three different polypeptide subunits of 33 (pI ~4.4), 22 (pI ~6) and 14 (pI ~4.4) kDa. Haemagglutinating activity of DFL was highly inhibited by N-acetyl-D-glucosamine. DFL partial peptide sequences obtained from peptide mass fingerprinting experiments matched with amino acid sequences of lectins from different organisms confirmed its nature. Biological properties of purified DFL namely antibacterial and bacterial agglutination experiments revealed that DFL have both the effects against laboratory cultures of Aeromonas hydrophila, Enterococcus faecalis, Escherichia coli and habitat bacterial isolates of Staphylococcus cohnii and Bacillus cereus. In addition, the DFL exhibited substantial anticancer properties against HeLa cells. These results concluded that purified DFL could serve as a potent therapeutic agent for various biomedical applications. 10.1016/j.ijbiomac.2022.01.099

Increased hemoglobin and heme in MALDI-TOF MS analysis induce ferroptosis and promote degeneration of herniated human nucleus pulposus. Shan Liang,Xu Ximing,Zhang Jing,Cai Peng,Gao Han,Lu Yingjie,Shi Jiangang,Guo Yinlong,Su Yue Molecular medicine (Cambridge, Mass.) BACKGROUND:Neovasculogenesis is characteristic of herniated lumbar discs, in which extruded nucleus pulposus is prone to heme iron-induced cytotoxicity (increased oxidative stress causing ferroptosis). However, recent analyses of neovascularization are very complicated, and the mechanism of action is rarely reported. METHODS:Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was performed to analyze human herniated and nonherniated nucleus pulposus. Then, the clinical relevance of the MALDI-TOF MS results and Pfirrmann classification of the degenerative nucleus pulposus were analyzed. To explore the mechanism, the heme-induced ferroptosis effect was evaluated at both the tissue and cell levels using high-resolution MALDI-TOF MS and molecular biology methods. RESULTS:The spectra revealed that hemoglobin (Hb) and heme signals were greatly increased, thus serving as predictors of vasculogenesis in herniated nucleus pulposus. The clinical relevance analysis demonstrated that the intensity of Hb and heme peaks was closely related to the Pfirrmann classification of degenerative nucleus pulposus. Mechanistically, increased heme catabolism and downregulation of glutathione peroxidase 4 (GPX4) levels were detected in herniated nucleus pulposus, reflecting iron-dependent cell death or ferroptosis. Iron levels was also increased in herniated nucleus pulposus compared with that in nonherniated nucleus pulposus. Furthermore, accuracy mass measurements confirmed that the levels of ferroptosis-related metabolites, such as glutathione, arachidonic acid (AA), sphinganine, polyunsaturated fatty acid (PUFA), and tricarboxylic acid (TCA) cycle metabolites, were significantly different between herniated and nonherniated tissues, indicating that the interior of the herniated tissues is a pro-oxidant environment. Moreover, heme-induced ferroptosis was verified in human nucleus pulposus cells (HNPCs), and the underlying mechanism might be associated with the Notch pathway. CONCLUSIONS:Neovascularization in herniated nucleus pulposus may expose tissues to high levels of heme, which can induce cytotoxicity and ferroptosis within tissues and accelerate the progressive degeneration of herniated nucleus pulposus. This study is beneficial for understanding the pathological mechanism of herniated nucleus pulposus and facilitating the development of nonoperative interventions for treating lumbar disc herniation (LDH). 10.1186/s10020-021-00368-2

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins. Phytomedicine : international journal of phytotherapy and phytopharmacology BACKGROUND:Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE:It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN:A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS:Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS:The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION:Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species. 10.1016/j.phymed.2022.154451