Myeloid derived suppressor cells (MDSC) are present in virtually all cancer patients, where they are induced and activated by inflammation. Because they are a major obstacle to immunotherapy of cancer, it is imperative to understand how these cells mediate their effects and communicate within the tumor microenvironment. The long-term objective of this collaborative program is to identify the molecules that MDSCs use to hijack the immune system and to use this information to re-program or neutralize MDSC. Preliminary evidence indicates that this intercellular communication is facilitated by exosomes, nanoscale membrane-bound vesicles shed by MDSC. We have identified several proteins in MDSC exosomes that have significant physiologic effects on other MDSC, and on macrophages, another population of myeloid cells that promote tumor growth. Preliminary experiments also indicate that proteins conjugated with ubiquitin chains are readily enriched from exosomes shed by MDSC. Protein modification by ubiquitination is a powerful signal, which is known to direct proteins to diverse cellular fates according to structural variations in te massive polyubiquitin tags. This multi-PI study will test the hypothesis that ubiquitinated protein in exosomes shed by MDSC and macrophages contribute to immune suppression. We will ask how ubiquitination marks cellular proteins for export as exosome cargo, and if ubiquitination is altered by inflammatory stimulation. Mass spectrometry has provided most of the information presently available about sites of ubiquitin conjugation on targeted proteins;however characterization of the linkages within ubiquitin chains remains a significant analytical challenge We will use novel synthetic methods to prepare polyubiquitins and proteins conjugated with polyubiquitins as standard reference compounds. We will use these standards to develop and test a proteomic strategy to characterize connection sites and their sequence in ubiquitin chains. This novel customized strategy, incorporating Asp-selective proteolysis, linkage-specific antibodies, LC-MS/MS and bioinformatics, will then be applied to characterize ubiquitin conjugates from exosomes shed by MDSC and macrophages. We will focus initially on chains with isopeptide bonds at K63, K48 and K11. These are considered to occur most frequently, have known functional correlations, and can be addressed by linkage-specific antibodies. Biological activity of selected ubiquitinated and other exosomal proteins will be validated by determining their ability to enhance pro-tumor activity and immune suppressive potency.

Public Health Relevance

Recently a family of cells has been shown to suppress the body's natural immune-response to tumor growth, and also to prevent the effectiveness of cancer immunotherapy. This project will characterize novel chemical signals by which suppressive myeloid cells control the immune- response, and provide new targets for intervention.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM021248-36
Application #
8630511
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Edmonds, Charles G
Project Start
1987-07-01
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
36
Fiscal Year
2014
Total Cost
$375,608
Indirect Cost
$99,536
Name
University of Maryland College Park
Department
Chemistry
Type
Schools of Earth Sciences/Natur
DUNS #
790934285
City
College Park
State
MD
Country
United States
Zip Code
20742
Lee, Amanda E; Geis-Asteggiante, Lucia; Dixon, Emma K et al. (2016) Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top-down mass spectrometry. J Mass Spectrom 51:315-21
Beury, Daniel W; Carter, Kayla A; Nelson, Cassandra et al. (2016) Myeloid-Derived Suppressor Cell Survival and Function Are Regulated by the Transcription Factor Nrf2. J Immunol 196:3470-8
Kim, Yeji; Edwards, Nathan; Fenselau, Catherine (2016) Extracellular vesicle proteomes reflect developmental phases of Bacillus subtilis. Clin Proteomics 13:6
Ostrand-Rosenberg, Suzanne (2016) Tolerance and immune suppression in the tumor microenvironment. Cell Immunol 299:23-9
Castañeda, Carlos A; Dixon, Emma K; Walker, Olivier et al. (2016) Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins. Structure 24:423-36
Bronte, Vincenzo; Brandau, Sven; Chen, Shu-Hsia et al. (2016) Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun 7:12150
Parker, Katherine H; Horn, Lucas A; Ostrand-Rosenberg, Suzanne (2016) High-mobility group box protein 1 promotes the survival of myeloid-derived suppressor cells by inducing autophagy. J Leukoc Biol 100:463-70
Burke, Meghan C; Wang, Yan; Lee, Amanda E et al. (2015) Unexpected trypsin cleavage at ubiquitinated lysines. Anal Chem 87:8144-8
Shah, Swati; Cannon, Joe R; Fenselau, Catherine et al. (2015) A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria. Infect Immun 83:4349-61
Parker, Katherine H; Beury, Daniel W; Ostrand-Rosenberg, Suzanne (2015) Myeloid-Derived Suppressor Cells: Critical Cells Driving Immune Suppression in the Tumor Microenvironment. Adv Cancer Res 128:95-139

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