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Characterization of the Arabidopsis At5-MMP Matrix Metalloproteinase
Tang Guozhu,Wu Shuchi,Zhao Bingyu,Flinn Barry S. 한국식물학회 2022 Journal of Plant Biology Vol.65 No.6
The plant extracellular matrix (ECM) represents the continuum between the plasma membrane and the apoplast, and undergoes significant modifications during plant growth, development and stress responses. While matrix metalloproteinases (MMPs) are significant proteolytic modulators of the vertebrate ECM, they remain poorly understood in plant biology. Of the five Arabidopsis thaliana MMPs, At5-MMP is the most constitutively expressed and was chosen for further characterization. Autoactivation of recombinant At5-MMP generated a functional metalloproteinase which exhibited maximum proteolytic activity at pH 7.5, and a broad level of activity into the basic range. Furthermore, an E to A mutation within the HEIGH portion of the At5-MMP catalytic domain abolished proteolytic activity. Using GFP-tagged At5-MMP variants expressed in Nicotiana benthamiana, the predicted signal peptide was required for targeting to the cell periphery, and deletion of this peptide resulted in intracellular, localized At5-MMP accumulation. Using a generic MMP cleavage PXG(L/I) peptide sequence, the predicted Arabidopsis extracellular proteome was screened for potential candidate MMP substrates. The extracellular domain of one candidate, WAK1, was expressed as a recombinant protein and shown to be cleaved internally at specific sites by At5-MMP in vitro. These same cleavage sites were identified using mammalian MMP cleavage prediction software.
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( Liangzhi Li ),( Tianyi Yang ),( Weiqiang Guo ),( Xin Ju ),( Cuiying Hu ),( Bingyu Tang ),( Jiaolong Fu ),( Jingsheng Gu ),( Haiyang Zhang ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.4
The mitogen-activated protein kinase HOG1 (high-osmolarity glycerol response pathway) plays a crucial role in the response of yeast to hyperosmotic shock. Trichosporonoides oedocephalis produces large amounts of polyols (e.g., erythritol and glycerol) in a culture medium. However, the effects of HOG1 gene knockout and environmental stress on the production of these polyols have not yet been studied. In this study, a To-HOG1 null mutation was constructed in T. oedocephalis using the loxP-Kan-loxP/Cre system as replacement of the targeted genes, and the resultant mutants showed much smaller colonies than the wild-type controls. Interestingly, compared with the wild-type strains, the results of shake-flask culture showed that To-HOG1 null mutation increased erythritol production by 1.44-fold while decreasing glycerol production by 71.23%. In addition, this study investigated the effects of citric acid stress on the T. oedocephalis HOG1 null mutants and the wild-type strain. When the supplementation of citric acid in the fermentation medium was controlled at 0.3% (w/v), the concentration of erythritol produced from the wild-type and To-HOG1 knockout mutant strains improved by 18.21% and 21.65%, respectively.
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