Goals and Projects
- The long-term goal of our uveal melanoma project is the development of a multi-antibody immunological assay for UM metastasis that will complement current cytogenetic and genetic prognostic methods and establish for the first time methods to detect and quantify circulating uveal melanoma tumor cells.
- The long-term goals of our glaucoma projects are to better understand the molecular mechanisms of glaucomatous vision loss, identify therapeutic targets and develop a panel of blood-borne glaucoma biomarkers.
- The long-term goal of our age-related macular degeneration (AMD) project is the development of molecular technology for assessing AMD risk and monitoring AMD therapeutics.
Uveal Melanoma(UM) is the most common primary malignancy of the eye and has a high mortality rate (40%). Survival rates have not improved in part because the pathobiology is poorly understood and primary UM tumors (pUM) can metastasize before diagnosis and UM micrometastases can lie dormant for years. Earlier detection of metastatic pUM is critical for earlier interventions. About 95% of primary uveal melanoma (pUM) originate in the capillary-rich uveal tract (i.e., the iris, ciliary body, and choroid), which facilitates hematogenous dissemination. The most common site of metastases is the liver (93%), but the majority of patients exhibit multiple sites. Our laboratory has completed the most extensive proteomic characterization of primary uveal melanoma tumors (pUM) on record as of 2019. Using LC MS/MS iTRAQ technology, we have analyzed 100 pUM specimens (45 that metastasized and 55 that were non-metastasizing). The pUM were collected from enucleated eyes in collaboration with ophthalmic pathologist Sarah Coupland, MBBS, PhD (Director, North West Cancer Research Centre, University of Liverpool, UK) and Cleveland Clinic ophthalmic oncologist Arun Singh, MD. The metastatic status of pUM was determined by cytogenetic and gene expression analyses, and clinical survival data. Thirteen choroid specimens excised from UM eyes distant from the pUM were utilized as a pooled control. Tryptic digests were labeled with unique iTRAQ tags, fractionated by RPHPLC at pH10 and subjected to LC MS/MS on an Orbitrap Fusion Lumos Tribrid mass spectrometer. The study resulted in the quantification of 3952 tumor proteins and the identification of a large number of differentially expressed (DE) proteins with Met/NoMet ratios exhibiting p values less than or equal to 0.05 (adjusted test), including 119 DE proteins that are predicted to be plasma membrane/cell surface proteins. Prediction modeling of the 100 proteomic datasets using DE protein predictors has provided discriminatory accuracies (C-statistics) and metastatic status prediction success rates greater than 90% with as little as 12-16 cell surface DE proteins. We are now developing an immunoassay for UM metastasis using multiple antibodies to select DE proteins as a prognostic tool for characterizing UM tumor biopsies and for the detection and quantification of pUM circulating in the blood.
Glaucomais a multifactorial optic neuropathy and a leading cause of blindness worldwide. Glaucomatous damage to the visual system can occur at normal and elevated levels of intraocular pressure (IOP). Age and IOP are risk factors for the neuropathy, but the identification of molecular risk factors for IOP elevation and glaucomatous vision loss are a high priority. Previous proteomic analyses in our laboratory have demonstrated that cochlin, a protein associated with deafness, is abnormally expressed in human trabecular meshwork (TM) from primary open angle glaucoma donors, suggesting that this protein may contribute to obstruction of the aqueous humor (AH) outflow pathway through the TM and elevated IOP. Other proteomic studies in the laboratory have demonstrated peptidyl arginine deiminase 2 (PAD2) in human POAG optic nerve and in monkey experimental glaucoma optic nerve head (ONH) and retina. Still other proteomic studies in the laboratory have shown that treating cultured human TM cells with either transforming growth factor beta 2 (TGFb2) or dexamethasone significantly altered the abundance of TM proteins and identified many proteins not previously associated with TGFb2-signaling or glucocorticoid-signaling in the eye. We have completed a preliminary quantitative proteomic study of experimental glaucoma (EG) versus control eye differences within the ONH and retina from 3 monkeys with unilateral, laser-induced high IOP (IOP Max >28 mm Hg). Glaucoma-altered proteins in the ONH strongly support the connective tissue deformation and remodeling evident in the EG monkey and strongly implicated myelin-associated neurodegeneration. Glaucoma-altered proteins in the retina implicated dysfunction in the mitochondria, oxidative stress response, cytoskeletal/connective tissue organization, and transport and regulatory processes. Up to 1819 proteins were quantified in the same tissues from one mild IOP EG monkey (IOP Max < 20 mm Hg). Both the direction and character of glaucoma-induced proteomic alterations were vastly different in mild IOP versus high IOP monkey EG eyes. Site-specific deimination differences in the ONH and retina were also detected between mild and high IOP as well as quantitative differences in PAD2. Deimination has been linked to cancer, immune and neurodegenerative disorders and to the regulation of multiple cellular processes. We hypothesize that the proteomic changes we observed at mild IOP are representative of real differences between those at high IOP and that they contribute to molecular mechanisms of early EG pathology. Efforts are now focused on using additional animals to rigorously compare glaucoma-induced proteomic alterations in monkey ocular tissues, including deimination, at mild IOP versus those at high IOP.
Age-Related Macular Degeneration(AMD) is a complex disease and a major cause of vision loss in the elderly. Of those with early AMD, clinicians cannot predict who will progress to advanced disease and severe visual loss. Only a fraction of early/mid-stage AMD patients progress to advanced AMD, with neovascular or “wet” AMD being more prevalent than advanced dry AMD (also known as geographic atrophy). Effective molecular biomarkers would facilitate early clinical assessment of AMD progression, the monitoring of AMD therapeutics and help prevent or slow severe visual loss. Growing evidence supports AMD as an inflammatory disease involving oxidative stress. A host of oxidative protein modifications have been associated with AMD, including adducts derived from docosahexaenoate-lipids such as carboxyethylpyrrole (CEP). Our laboratory participated in the initial immunodetection of elevated CEP in AMD ocular tissues and AMD plasma. CEP-protein stimulates neovascularization in vivo, mice immunized with CEP-protein develop a dry AMD-like phenotype, and anti-CEP antibodies have utility in monitoring the efficacy of select pharmacological interventions. However, no significant mass spectrometric evidence supports the presence of CEP-protein adducts in vivo despite the strong CEP immunoreactivity demonstrated in AMD plasma and ocular tissues. We hypothesize that CEP adducts are metabolically altered in vivo to structures that remain recognized by anti-CEP antibodies. This raises questions like “What is the molecular identity of the protein modification(s) responsible for anti-CEP immunoreactivity and do they have bioactivities?” Toward answers to such questions, we are structurally and functionally characterizing a peptide modification we have identified on 43 plasma peptides captured by anti-CEP immunoaffinity chromatography. This lysine modification exhibits a mass addition of 120.0206, rather than the 122.0362 expected for CEP and has been detected in 16 proteins and at 6 lysine residues in human serum albumin that correspond to CEP modification sites in our authentic CEP-bovine serum albumin standard. Preliminary analyses suggest it is more abundant in AMD than control plasma.
Crabb, JW, M Miyagi, X Gu, K Shadrach, KA West, H Sakaguchi, M Kamei, A Hasan, L Yan, ME Rayborn, RG Salomon, JG Hollyfield (2002) Drusen Proteome Analysis: an Approach to the Etiology of Age-Related Macular Degeneration Proc Natl Acad Sci USA 99:14682-14687.
Miyagi M, H Sakaguchi, RM Darrow, L Yan, KA West, KS Aulak, DJ Stuehr, JG Hollyfield, DT Organisciak and JW Crabb (2002) Evidence that Light Modulates Protein Nitration in Rat Retina. Molecular and Cellular Proteomics 1: 293-303.
Golovleva I, S Bhattacharya, Z.Wu, N Shaw, Y Yang, K Andrabi, KA West , MS Burstedt, K Forsman, G Holmgren, O Sandgren, N Noy , J Qin and JW.Crabb (2003) Disease Causing Mutations in the Cellular Retinaldehyde-binding Protein Tighten as well as Abolish Retinoid Interactions. J Biol Chem 278: 12397-12402.
Bhattacharya SK, EJ Rockwood, SD Smith, VL Bonilha, JS Crabb, RW Kuchtey, NG Robertson, NS Peachey, CC Morton and JW Crabb(2005) Proteomics Reveals Cochlin Deposits Associated With Glaucomatous Trabecular Meshwork. J Biol Chem 280: 6080 – 6084.
EbrahemQ, K Renganathan, J Sears, A Vasanji, X Gu, L Lu, RG Salomon, JW Crabb, B Anand-Apte (2006) Carboxyethylpyrrole Oxidative Protein Modifications Stimulate Neovascularization: Implications for Age-Related Macular Degeneration Proc Nal Acad Sci USA 103: 13480-13484
Gu J, GJT Pauer, X Yue, U Narendra, GM Sturgill, J Bena, X Gu, NS Peachey, RG Salomon, SA Hagstrom, W Crabb and the Clinical Genomic and Proteomic AMD Study Group (2009) Assessing Susceptibility To Age-Related Macular Degeneration With Proteomic And Genomic Biomarkers. Mol & Cell Proteomics 8:1338-49.
Ni J, X Yuan, J Gu, X Yue, X Gu, RH Nagaraj, JW Crabb and The Clinical Genomic and Proteomic AMD Study Group (2009) Plasma Protein Pentosidine And Carboxymethyllysine, Biomarkers For Age-Related Macular Degeneration. Mol & Cell Proteomics 8: 1921-33.
Yuan X, X Gu, JS Crabb, X Yue, K Shadrach, JG Hollyfield and JW Crabb (2010) Quantitative Proteomics: Comparison of the Macular Bruch’s Membrane/Choroid Complex from Age-related Macular Degeneration and Normal Eyes. Mol & Cell Proteomics 9: 1031-1046.
Bollinger KE, Crabb JS, Yuan X, Putliwala T, Clark AF, Crabb JW (2011) Quantitative Proteomics: TGFβ2-Signaling in Trabecular Meshwork Cells. Invest Ophthal Visual Sci 52, 8287-8291.
Bollinger KE, Crabb JS, Yuan X, Putliwala T, Clark AF, Crabb JW (2012) Proteomic Similarities in Steroid Responsiveness In Normal and Glaucomatous Trabecular Meshwork Cells. Mol Vision 18: 2001-2011.
Renganathan K, J Gu, ME Rayborn, JS Crabb, RG Salomon, RJ Collier, MA Kapin, C Romano, JG Hollyfield, and JW Crabb (2013) CEP Biomarkers As Potential Tools for Monitoring Therapeutics, PLoS One 8:e76325.
Gu X, Hu Z, Ebrahem Q, Crabb JS, Mahfouz R, Radivoyevitch T, Crabb JW, Saunthararajah Y. (2014) Runx1 Regulation of Pu.1 Corepressor/Coactivator Exchange Identifies Specific Molecular Targets for Leukemia Differentiation Therapy. J Biol Chem 289: 14881-95.
Kim YW, Yakubenko VP, West XZ, Gugiu GB, Kutralanathan R, Biswas S, Gao D, Crabb JW, Salomon RG, Podrez E, Byzova T (2015) Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products. Circ Res 117: 321-332.
Crabb JW, Hu B, Crabb JS, Triozzi P, Saunthararajah Y, Tubbs R, Singh AD (2015) iTRAQ Quantitative Proteomic Comparison of Metastatic and Non-Metastatic Uveal Melanoma Tumors. PLoS ONE 10: e0135543.
Tayou J, Wang Q, Jang GF, Pronin AN, Orlandi C, Martemyanov KA, Crabb JW, Slepak VZ (2016) Regulator of G-protein Signaling 7 (RGS7) can exist in a homo-oligomeric form that is regulated by Gαo and R7-binding protein. J Biol Chem 291, 9133-47
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