Fu Laboratory

Research in our laboratory focuses on the role of protein oxidation and other post-translational modifications in the pathogenesis of inflammatory and thrombotic diseases by incorporating the use of mass spectrometry and analytical biochemistry. These studies encompass three primary areas:

Blood Coagulation Proteins

A major effort in our laboratory centers on the molecular mechanisms regulating blood coagulation proteins. We are currently investigating von Willebrand factor (VWF), ADAMTS13, and fibrinogen under oxidative conditions. We propose that oxidants produced by activation of inflammatory cells might modify the ability of ADAMTS13 to effectively cleave VWF. Furthermore, we are investigating the effects of fibrinogen oxidation and degradation products on its polymerization, which may play a key role in blood clotting events.  These studies are in collaboration with Dr. José López at the Bloodworks Northwest Research Institute, as well as Investigators at the University of Washington. The long-term goal is to explore the functional consequences of post-translational modifications (PTMs) in blood coagulation proteins and to discover biomarkers for related diseases.

Red Blood Cell (RBC) Oxidation and Storage Lesion

It has been reported that the RBC storage lesion is associated with oxidation of the cell during storage, but very little is known about the molecular mechanisms. We are currently applying proteomic approaches to study the role of post-translational modifications to both membrane proteins and hemoglobin in the RBC storage lesion and RBCs from patients. Our long-term goal is to understand the role of specific modifications, including oxidation, phosphorylation and glycosylation, on the viability of stored and diseased red blood cells.

Oxidative Stress Markers for Clinical Trials

Oxidative stress has increasingly been associated with the pathogenesis of a wide variety of diseases including sickle cell disease (SCD) and thrombotic thrombocytopenic purpura (TTP).  In collaboration with Drs. Konkle and López, we are evaluating the use of N-acetylcysteine (NAC) as an antioxidant treatment in patients suffering from SCD or TTP, and are focused on determining relevant biomarkers for measuring oxidative stress.  Glutathione (GSH) has long been identified as a biomarker of oxidative stress, but it is especially difficult to quantify in plasma and there are numerous related metabolites which may also be important in evaluating oxidative stress.  We have developed a liquid chromatography-mass spectrometry (LC-MS) method to quantify a panel of small-molecule thiols and disulfides, including NAC, in a variety of sample types, representing the most complete LC-MS method to date. Our method can further be used to quantify the distribution of these thiols between free and disulfide form, including those bound to protein.

1. Fu X, Kassim SY, Parks WC, Heinecke JW: Hypochlorous acid oxigenates the cysteine switch domain of pro-matrilysin (MMP-7): A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem 276(44):41279-41287, 2001. PMID11533038
2. Fu X, Mueller DM, Heinecke JW: Generation of intramolecular and intermolecular sulfenamides, sulfinamide, and sulfinamide by hypochlorous acid: A potential pathway for oxidative cross-linking of low-density lipoprotein by myeloperoxidase. Biochemistry 41(4):1293-1301, 2002. PMID11802729
3. Fu X, Kassim SY, Parks WC, Heinecke JW: Hypochlorous acid oxygenated by myeloperoxidase modifies adjacent tryptophan and glycine residues in catalytic domain of matrix metalloproteinase-7 (Matrilysin). J Biol Chem 278:28403-28409, 2003. PMID12759346
4. Fu X, Kao J, Bergt C, Kassim SY, Huq NP, d’Avignon A, Parks WC, Mecham RP, Heinecke JW: Oxidative cross-linking of tryptophan to glycine restrains matrix metalloproteinase activity. J Biol Chem 279:6209-6212, 2004. PMID14670964
5. Bergt C, Fu X, Huq, NP, Kao J, Heinecke JW: Lysine residues direct the chlorination of tyrosines in YXXK motifs of apolipoprotein A-I when hypochlorous acid oxidizes high density lipoprotein. J Biol Chem 279:7856-7866, 2004. PMID14660678
6. Shao B, Belaaouaj A, Verlinde CL, Fu X, Heinecke JW. Methionine sulfoxide and proteolytic cleavage contribute to the inactivation of cathepsin G by hypochlorous acid: An oxidative mechanism for regulation of serine proteinases by myeloperoxidase. J. Biol Chem. 280(32) 29311-21, 2005.PMID15967795
7. Kassim SY, Fu X, Liles WC, Shapiro SD, Parks WC, Heinecke JW: NADPH Oxidase Restrains the Matrix Metalloproteinase Activity of Macrophages. J. Biol. Chem. 280(34): 30201-5, 2005. PMID15983040
8. Shao B, Fu X, Mecdonald TO, Green PS, Uchida K, O’Brien KD, Oram JF, Heinecke JW: Acrolein Impairs ABCA1-dependent Cholesterol Export from Cells through Site-Specific Modification of Apolipoprotein A-I. J. Biol. Chem. 280(43):36386-96, 2005. PMID16126721
9. Fu X, Wang Y, Kao J, Irwin A,d’Avignon A, Mecham RP, Parks WC, Heinecke JW: Specific Sequence Motifs Direct the Oxygenation and Chlorination of Tryptophan by Myeloperoxidase. Biochemistry 459(12), 3961-71, 2006. PMID16548523
10. Wang Y, Rosen H, Madtes DK, Shao B, Martin TR, Heinecke JW, Fu X: Myeloperoxidase inactivates TIMP-1 by oxidizing its N-terminal cysteine residue: An oxidative mechanism for regulating proteolysis during inflammation. J. Biol. Chem. 282(44):31826-34, 2007. PMID17726014
11. Fu X, Gharib SA, Green PS, Aitken ML, Frazer DA, Park DR, Vaisar T, Heinecke JW: Spectral Index for Assessment of Differential Protein Expression in Shotgun Proteomics. J. Proteome Res. 7(3) 845-854, 2008. PMID18198819
12. Gharib SA, Green PS, Aitken ML, Frazer DA, Park DR, Vaisar T, Heinecke JW, Fu X: Mapping the lung proteome in Cystic Fibrosis. J. Proteome Res. 8(6):3020-8, 2009. PMID19354268
13. Yuan W, Wang Y, Heinecke JW, Fu X: Hypochlorous acid converts the γ-glutamyl group of glutathione disulfide to 5-hydroxy-butyrolactam: a potential marker for neutrophil activation. J. Biol. Chem. 284(39):26908-17, 2009. PMID19584048
14. Rosen H, Klebanoff SJ, Wang Y, Brot N, Heinecke JW, Fu X: Methionine Oxidation Contributes to Bacterial Killing by the Myeloperoxidase System of Neutrophils, Proc. Natl. Acad. Sci. USA, 106(44):18686-91, 2009. PMID19833874
15. Chen J, Fu X, Wang Y, Ling M, McMullen B, Kulman J, Chung D, López JA: Oxidative modification of von Willebrand factor by neutrophil oxidants inhibits its cleavage by ADAMTS13. Blood, 115(3) 706-12, 2010. PMID19812385
16. Chen J, Reheman A, Gushiken FC, Nolascol L, Fu X, Moake JL, Ni H, López JA: N-acetylcysteine reduces the size and activity of von Willebrand factor in human plasma and mice. J. Clin. Invest, 121(2):593-603, 2011. PMID21266777
17. Herrero R, Kajikawa O, Matute-Bello G, Wang Y, Hagimoto N, Mongovin S, Wong V, Park DR, Brot N, Heinecke JW, Rosen H, Goodman RB, Fu X, Martin TR, The biological Activity of FasL in humans and mice is determined by the structure of its stalk region. J Clin. Invest, 121(3):1174-90, 2011.PMID21285513
18. Fu X, Chen J, Gallagher R, Zheng Y, Chung DW, López JA: Shear stress-induced unfolding of von Willebrand factor accelerates oxidation of key methionine residues in the A1A2A3 region. Blood. 118(19) 5283-91, 2011. PMID21917758