NMR structure note: human esophageal cancer-related gene 2

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Esophageal cancer is one of the most common fetal cancers worldwide and occurs at very high frequencies in developing countries (Parkin et al. 1993). Esophageal cancerrelated gene 2 (ECRG2), also named serine peptidase inhibitor Kazal-type 7 (SPINK7), was identified as a growth-regulating gene by comparing normal esophageal epithelia and primary squamous cell carcinomas tissues from high incidence families in Lin-Xian County of China using differentially displayed PCR (Su et al. 1998; Cui et al. 2010). It has been shown that ECRG2 is a tumor suppressor gene involved in the regulation of cell proliferation, induction of apoptosis, and inhibition of cancer cell migration, invasion and metastasis (Cui et al. 2003; Huang et al. 2007). ECRG2 also participates in centrosome amplification in a p53-dependent manner and has a role in maintaining chromosome stability (Cheng et al. 2008). Recently, it was found that ECRG2 regulates cell migration/invasion through urokinase-type plasmin activator (uPA)/urokinase-type plasmin activator receptor (uPAR) pathway by directly inhibiting uPA/plasmin activity, and may represent a novel therapeutic target for cancer (Huang et al. 2007; Cheng et al. 2009, 2010; Cui et al. 2010). The full length ECRG2 protein contains 85 residues, in which the N-terminal 19 residues form a signal peptide and the C-terminal 56 residues (residues 30–85) contain a typical x(8)-C-x(12)-C-x(7)-C-x(6)-Y-x(3)-C-x(2)-C-x(17)-C conserved region, coding a Kazal-type serine protease inhibitors domain (Figs. 1, 2) (Cui et al. 2010). The Kazal-type protease inhibitors with various physiological functions are conserved across species ranging from human to invertebrates (Laskowski and Kato 1980; Rimphanitchayakit and Tassanakajon 2010) while ECRG2 is classified as a serine peptidase inhibitor Kazal-type 7 and conserved in mammalians (Fig. 1). Moreover, the Kazal-type protease inhibitors have a scissile peptide bond that is potentially cleaved by the protease with a very low reaction rate (Laskowski and Kato 1980; Rimphanitchayakit and Tassanakajon 2010). The scissile peptide bond of ECRG2 is between P1 residue (Ile47) and P01 residue (Thr48) (In inhibitors, the number after ‘‘P’’ indicates the position of the residue towards the N-terminus of the scissile bond, while after ‘‘P0’’ indicates residues towards the C-terminus of the scissile bond). In addition, ECRG2 contains more residues between the first two cysteines besides its N-terminal signal peptide as compared with the other Kazal-type protease inhibitors (Geng et al. 2008). The backbone and partial side chain resonance assignments for ECRG2 were determined previously by NMR technique (Geng et al. 2008). Utilizing a homology modeling and the NMR titration experiments, a putative reactive site of ECRG2 as the uPA-binding site was identified, suggesting that ECRG2 can inhibit uPA activity through a manner similar to the other Kazal-type inhibitors of serine proteases (Geng et al. 2008). In the present study, the complete NMR resonance assignments of ECRG2 were Yingang Feng and Yong Geng contributed equally to this work.