Structural and Biochemical Characterization of Suppressor of T Cell Receptor Signaling (Sts) Proteins

Abstract

T cell receptors (TCR) recognize foreign antigens and convey the message to the nucleus to induce a response. The TCR complex activate cytoplasmic tyrosine kinases (such as Lck and Zap-70), which act in conjunction with a myriad of downstream adaptor proteins to initiate signals that bring about T cell activation. Sts are supposed to negatively regulate the T cell signaling pathway by dephosphorylating Zap-70. There are two homologous Sts proteins: Sts-1 and Sts-2, they are highly conserved both in their active sites and overall structure, but their specific functional mechanisms are still unknown. C. albicans is the fourth leading cause of bloodstream infections, with ~50,000 cases reported yearly in US, and global mortality rates have not decreased in recent 20 years1. Functional inactivation of both Sts enzymes leads to profound resistance to systemic infection by C.albicans. 80% of mice lacking Sts-1 and −2 survive in a dose of C.albicans (2.5*105 CFU/mouse) while less than 10% wild-type mice survive after 10 days of infection2. Even the mechanism of the enhanced resistance to C.albicans infection is still not clear yet, we assume Sts proteins as druggable targets to prevent or treat systemic candidiasis infection. So we are aimed at: Structural and functional characterization of human Sts proteins to figure out their functional mechanisms. Identify and biochemical characterize small molecules that can target human Sts as potent inhibitors. The methods we used are: Crystallography: to characterize Sts-2 protein structure. Enzyme activity assays: to measure their phosphatase activity. We combine the results of high throughput inhibitor screening and test the Ki of some top inhibitor hits. In addition, we use Surface Plasmon Resonance (SPR) to measure the binding affinity of the inhibitors towards Sts-1 in real time. We solved the protein structure of Sts-2 and compare with the structure of Sts-1, the non-active site residues are different which may be responsible for the noticeable difference in their phosphatase activity. We also find two groups of inhibitors that have intermediate to low Ki value toward Sts protein which may become precursors of potent drugs towards Sts proteins in the future. Support or Funding Information This work is supported by Stony Brook University, the National Heart, Lung, and Blood Institute of the National Institutes of Health and the Office of the Assistant Secretary of Defense for Health Affairs. Thanks for the help from Dr. French's group and Dr. Carpino's lab (cooperator) at Stony Brook University. Phosphatase activity of Sts-2HP and Sts-1 full-length towards 3 different substrate analogues Substrate/Enzyme Km (mM) kcat (S−1) kcat/Km (M−1S−1) pNPP Human STS-2HP 5.61 ± 1.69 1.23 ± 0.15 2.19 × 102 Full-length STS-1 3.31 ± 0.27 158.6 ± 6.48 4.77 × 104 OMFP Human STS-2HP 1.07 ± 0.24 5.86 ± 0.78 5.48 × 103 Full-length STS-1 ND ND ND DiFMUP Human STS-2HP 0.85 ± 0.23 4.92 ± 0.76 5.79 × 103 Full-length STS-1 ND ND ND Azol dyes phosphatase activity inhibition towards Sts-1, Sts-2 and other phosphatases (PTP1B/SHP-1) Ki (uM) Congo Red Evans Blue Chicago Blue Tetracycline STS-1HP 0.0928 0.326 ND 20–100* STS-2HP 6.60 5.26 61.92 No inhibition PTP1B 10.28 1.89 ND ND SHP-1 5.30 0.994 ND ND This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.