ABSTRACT
BACKGROUND: NIMA (never in mitosis, gene A) serine/threonine kinase 7 (NEK7) is a regulator of mitosis spindle in mammals and is considered as a drug target of inflammasome related inflammatory diseases. However, most commercially available or reported recombinant NEK7 proteins are either inactive or have low purity. These shortcomings limit the pharmacological studies and development of NEK7 inhibitors. OBJECTIVE: To elucidate what causes the NEK7 low purity in E. coli, and optimize a protocol to improve the protein purity. METHODS: A comparative study of expression full length NEK7 with an N-terminal His-tag or a Cterminal His-tag was performed. His-affinity resin, ion exchange and gel filtration chromatography were used to purify NEK7. The protein was identified by mass spectrometry. The activity and folding of NEK7 were evaluated by chemiluminescent assay and thermal shift assay. RESULTS: Our results demonstrated that N-terminal tagged protein was toxic to E. coli, resulting in incomplete translated products. The C-terminal tagged NEK7-His6 had a much higher purity than that of an N-terminal tag. The Ni2+ resin one-step purification led to a purity of 91.7%, meeting the criteria of most kinase assays. With two-step and three-step procedures, the protein purities were 94.7% and ~100%, respectively. The NEK7 purified in this work maintained its kinase activity and correct conformation, and the compound-protein interaction ability. CONCLUSION: Our optimized protocol could produce good purity of His tagged NEK7 in E. coli, and the kinase activity and biophysical characteristics of which are preserved.
Subject(s)
NIMA-Related Kinases , Chromatography, Affinity , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Humans , Mass Spectrometry , NIMA-Related Kinases/biosynthesis , NIMA-Related Kinases/chemistry , NIMA-Related Kinases/genetics , NIMA-Related Kinases/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purificationABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Duan Teng Yimu decoction is a Chinese herbal medicine compound with proven therapeutic effects on inflammasome-related diseases, such as rheumatoid arthritis. This decoction consists of three Chinese herbal medicines, including Leonurus japonicus (L. japonicus), which promotes the blood circulation and exhibits detumescence activity, traditionally curing gynecologic and inflammasome diseases. AIM OF THE STUDY: To explore the anti-inflammasome activity and the underlying mechanisms of action of the compounds from L. japonicus. MATERIALS AND METHODS: A series of compounds were isolated from L. japonicus. Their anti-inflammasome activities were evaluated in macrophages that were co-stimulated by lipopolysaccharide (LPS) and NLRP3 inflammasome inducers. NLRP3 inflammasome formation and apoptosis speck like containing a CARD (ASC) oligomerization were evaluated by immunofluorescent microscopy and Western blot analysis. The regulation of autophagy after treatment of this compound was also evaluated. Lastly, in vivo activity of Leojaponin was analyzed in a mouse acute gouty arthritis model. RESULTS: Here we show that Leojaponin, a diterpenoid compound from L. japonicus, suppressed lactate dehydrogenase and IL-1ß release in Nigericin-stimulated macrophages in a pyroptosis model. Leojaponin inhibits NLRP3 inflammasome activation in both J774A.1 cells and bone marrow-derived macrophages in a dose dependent manner. Moreover, Leojaponin suppressed NLRP3-mediated ASC specks formation and ASC oligomerization. These activities of Leojaponin depend on restoration of autophagy via promoting RAPTOR phosphorylation. Furthermore, Leojaponin ameliorated monosodium urate (MSU)-induced acute gouty arthritis in vivo. CONCLUSION: Our findings suggest that Leojaponin inhibits NLRP3 inflammasome activation through enhancing autophagy via RAPTOR phosphorylation, thereby highlighting Leojaponin as a potent drug for inflammasome-related diseases.
Subject(s)
Arthritis, Gouty/drug therapy , Autophagy/drug effects , Diterpenes/therapeutic use , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Regulatory-Associated Protein of mTOR/metabolism , Animals , Arthritis, Gouty/metabolism , Cell Line , Diterpenes/chemistry , Gene Expression Regulation/drug effects , Inflammasomes/genetics , Inflammasomes/metabolism , Interleukin-1beta/genetics , Interleukin-1beta/metabolism , L-Lactate Dehydrogenase/antagonists & inhibitors , Leonurus/chemistry , Macrophages/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred C57BL , Molecular Structure , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Nigericin/toxicity , Phosphorylation , Regulatory-Associated Protein of mTOR/genetics , Up-RegulationABSTRACT
Four new diterpenoids, rubellawus A-D (1-4), along with three known compounds, were isolated and identified from the flowers of Callicarpa rubella. Their structures were elucidated by various spectroscopic analysis. All the compounds were screened for their anti-inflammatory activity and 14α-hydroxyisopimaric acid and isopimaric acid showed significant NLRP3 inflammasome inhibitory activity with IC50 values of 7.02 and 3.99â µM.
