1. Structural and functional characterization of the triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 and its dimerization-dependent inhibitory activity
Beata Prabucka, Marcin Mielecki, Magdalena Chojnacka, Wiesław Bielawski, Mariusz Czarnocki-Cieciura, Sławomir Orzechowski Phytochemistry. 2017 Oct;142:1-10. doi: 10.1016/j.phytochem.2017.06.008. Epub 2017 Jun 24.
Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin.
2. Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors
Tian Zhao, Zhipeng Li, Zhouliang Guo, Ali Wang, Zhenxing Liu, Qing Zhao, Yuyin Li, Edward A McKenzie, Aipo Diao Protein Expr Purif. 2018 Oct;150:12-16. doi: 10.1016/j.pep.2018.05.003. Epub 2018 May 7.
Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris. The pPICZα-LGMN expression plasmid was constructed and transformed into Pichia pastoris strain and positive recombinants were identified. Fermentation conditions were optimized and it was found that Legumain was most highly expressed under pH 6 culture conditions. In addition, the enzyme activity of the purified Legumain was tested using a fluorogenic substrate (Z-Ala-Ala-Asn-AMC) assay and the optimum pH for the autocatalytic activation of recombinant Legumain was very acidic at a pH value of 3. The recombinant protein was then used to screen a library of compounds and small molecule 1773 (Terramycin) was shown to effectively inhibit Legumain enzyme activity. These results indicate that the Pichia pastoris expression system can produce highly active recombinant Legumain protein allowing it to be used for High-throughput screening (HTS) applications.
