Rationale: The Johns Hopkins University (JHU) proposal applies state-of-the-art proteomic methods and develops new approaches and techniques to investigate the biological and clinical aspects of heart failure (HF). HF is a clinical syndrome in which cardiovascular function is insufficient to support the metabolic needs of the body. On a population basis, its burden is substantial and increasing with 5-6 million people affected in the United States. Several factors contribute to the etiology and evolution of the clinical presentation including ischemia, hypertension, diabetes and genetic predisposition. Regardless of therapeutic efforts, mortality and morbidity remains high. Many gaps in understanding HF remain and it is the goal of this center to identify how HF impacts signaling cascades and several subproteomes (mitochondrial, contractile, cell surface and secretory) emphasizing post-translational modifications (PTMs) in order to discover novel ways by which protein modifications either contribute to disease or could be targeted to improve disease outcome.
Philosophy and goals: The central philosophy of our center is that innovative technologies in proteomics will be essential to pursue compelling biological and clinical questions. Building on our past successes in developing innovative technologies, we will broaden our developmental scope and focus on JHU’s clinical and research strength in HF. A high level overview is presented below.
The program: Innovative technologies in proteomics are being pursued in the context of compelling biological and clinical questions in HF.
All of these technologies are unified by the need to develop new clinical therapies and diagnostics in HF, in part due to intrinsic nature of the heart. Technological innovation comprises five projects, which feature the development of quantitative methods for detailed characterization of cardiac muscle proteins including quantification of PTMs targeting cardiac specific isoforms. The five application projects take newly developed technologies and move them into highly relevant complex biological or clinical questions. The technical projects are integrated and broadly focus on i) phosphorylation in signaling, ii) O-GlcNAc iii) Cys and Lys modifications and iv) glycosylation of the cell surface and secretory subproteomes. All of these technologies are focused on HF with biological/clinical applications in the areas of i) G-coupled signaling, ii) myofilament subproteome, iii) mitochondria, iv) HIF-1α signaling and v) biomarker development. To maximize synergy, we envision a central role for the bioinformatics core and the integrated modeling core. Figure 1 depicts the inter-relationships among the various projects and the two central cores.
Technical challenges to be addressed:
1. Exploitation and continued development of proteomic methods for the global investigation of a wide range of biologically important PTMs with the explicit goals of identifying and quantifying each modified amino acid residue. By achieving this we can explore the dynamic nature of the proteome and the crosstalk between signaling cascades and the end effector molecules.
2. Development of workflows for improved separation and enrichment of proteins and peptides, as well as, mass spectrometry methods to extend detection and quantification to the full dynamic range observed in tissue and body fluids.
3. Innovation in bioinformatics focused on automated solutions for protein name redundancy, comparisons/querying, PTM iteration for both improved protein identification and maximized protein characterization (isoforms, PTMs). Including the development of accessible databases for human cardiac proteins and their PTMs and CardioMRM, a peptide database specific for the cardiac proteome. The bioinformatics core is designed to improve integration between projects and to help to emphasize targets for further study.
4. Integration of sophisticated cell modeling with quantitative proteomic data and functional endpoints to provide mechanistic insights into the interplay between subproteomes of the human cardiac myocyte.
Technology Project 1: Phosphorylation and kinase tool development (Pandey, Cotter)
Technology Project 2: O-GlcNAc Tool Development: Crosstalk Between GlcNAcylation and Phosphorylation (Primary PI, Hart).
Technology Project 3: Tool Development for Cys and Lys Modifications. (PI, Cotter, co-PI Van Eyk)
- Part A. Modifications of Cys residues.
- Part B. Modification of Lys residues
Technology Project 4: Cell surface and Secretory Pathway Tool Development (PI, Graham, Co-PI Zhang, Van Eyk)
Application Project 1: G-coupled receptor signaling PTM in HF (PI, Kass, collaborator Graham, Van Eyk, Pandey)
Application Project 2: Myofilament PTMs in diabetic cardiomyopathy (PI, Murphy, collaborator Hart)
Application Project 3: Regulation of HIF-1 in HF (PI, Semenza, collaborator Pandey)
Application Project 4: Mitochondrial acetylation in HF (PI - O’Rourke, collaborator Cotter)
Application Project 5: Tools and approaches for plasma biomarker development: HF risk stratification (PI Van Eyk, co-PIs Miller, Coresh, Zhang)
This project is funded by the National Heart, Lung, and Blood Institute