Preparation of Oxidized and Reduced PTP4A1 for Structural and Functional Studies

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2023 Dec 26

PMID 38147218

Authors

Ganesan Senthil Kumar

Affiliations

Soon will be listed here.

Abstract

The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase-phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.

References

Hunter T, Karin M . The regulation of transcription by phosphorylation. Cell. 1992; 70(3):375-87. DOI: 10.1016/0092-8674(92)90162-6. View

Humphrey S, James D, Mann M . Protein Phosphorylation: A Major Switch Mechanism for Metabolic Regulation. Trends Endocrinol Metab. 2015; 26(12):676-687. DOI: 10.1016/j.tem.2015.09.013. View

Barford D, Das A, Egloff M . The structure and mechanism of protein phosphatases: insights into catalysis and regulation. Annu Rev Biophys Biomol Struct. 1998; 27:133-64. DOI: 10.1146/annurev.biophys.27.1.133. View

Alonso A, Sasin J, Bottini N, Friedberg I, Friedberg I, Osterman A . Protein tyrosine phosphatases in the human genome. Cell. 2004; 117(6):699-711. DOI: 10.1016/j.cell.2004.05.018. View

Tonks N . Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 2006; 7(11):833-46. DOI: 10.1038/nrm2039. View

Netto L, Machado L . Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity. FEBS J. 2022; 289(18):5480-5504. DOI: 10.1111/febs.16466. View

Caselli A, Marzocchini R, Camici G, Manao G, Moneti G, Pieraccini G . The inactivation mechanism of low molecular weight phosphotyrosine-protein phosphatase by H2O2. J Biol Chem. 1998; 273(49):32554-60. DOI: 10.1074/jbc.273.49.32554. View

Lee S, Yang K, Kwon J, Lee C, Jeong W, Rhee S . Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem. 2002; 277(23):20336-42. DOI: 10.1074/jbc.M111899200. View

Savitsky P, Finkel T . Redox regulation of Cdc25C. J Biol Chem. 2002; 277(23):20535-40. DOI: 10.1074/jbc.M201589200. View

10.

Zhang R, Kumar G, Hansen U, Zoccheddu M, Sacchetti C, Holmes Z . Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex. JCI Insight. 2022; 7(8). PMC: 9089796. DOI: 10.1172/jci.insight.155761. View

11.

Gabrielli A, Svegliati S, Moroncini G, Amico D . New insights into the role of oxidative stress in scleroderma fibrosis. Open Rheumatol J. 2012; 6:87-95. PMC: 3395898. DOI: 10.2174/1874312901206010087. View

12.

Karisch R, Fernandez M, Taylor P, Virtanen C, St-Germain J, Jin L . Global proteomic assessment of the classical protein-tyrosine phosphatome and "Redoxome". Cell. 2011; 146(5):826-40. PMC: 3176638. DOI: 10.1016/j.cell.2011.07.020. View

13.

Sacchetti C, Bai Y, Stanford S, Di Benedetto P, Cipriani P, Santelli E . PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis. Nat Commun. 2017; 8(1):1060. PMC: 5651906. DOI: 10.1038/s41467-017-01168-1. View

14.

Gulerez I, Funato Y, Wu H, Yang M, Kozlov G, Miki H . Phosphocysteine in the PRL-CNNM pathway mediates magnesium homeostasis. EMBO Rep. 2016; 17(12):1890-1900. PMC: 5283600. DOI: 10.15252/embr.201643393. View

15.

Kozlov G, Cheng J, Ziomek E, Banville D, Gehring K, Ekiel I . Structural insights into molecular function of the metastasis-associated phosphatase PRL-3. J Biol Chem. 2004; 279(12):11882-9. DOI: 10.1074/jbc.M312905200. View

16.

Sun J, Wang W, Yang H, Liu S, Liang F, Fedorov A . Structure and biochemical properties of PRL-1, a phosphatase implicated in cell growth, differentiation, and tumor invasion. Biochemistry. 2005; 44(36):12009-21. DOI: 10.1021/bi0509191. View

17.

Peti W, Page R . Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif. 2006; 51(1):1-10. DOI: 10.1016/j.pep.2006.06.024. View

18.

Kapust R, Tozser J, Fox J, Anderson D, Cherry S, Copeland T . Tobacco etch virus protease: mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency. Protein Eng. 2002; 14(12):993-1000. DOI: 10.1093/protein/14.12.993. View