Theses and Dissertations from UMD
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Item INTERROGATING PROTEIN CARGOES OF MDSC-DERIVED EXOSOMES ON THE BASIS OF POST-TRANSLATIONAL MODIFICATIONS(2017) Chauhan, Sitara; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Myeloid-derived suppressor cells (MDSC) are immature myeloid cells which accumulate in cancer patients and tumor-bearing mice. Their function in the tumor microenvironment is to inactivate the immune response to cancer by suppressing both the adaptive and innate immune system. Therefore, MDSC are a major obstacle in immunotherapeutic approaches designed to cure cancer. MDSC-derived from tumor bearing mice have been found to shed exosomes. Exosomes are nano-sized vesicles that carry biologically active molecules and play a role in intercellular communication. MDSC-derived exosomes have been reported to mediate the immunosuppressive functions of the parental cells by stimulating the accumulation of MDSC and also by converting macrophages to a tumor-promoting phenotype. Recent developments in government policy have launched a goal of curing cancer using immune-based therapies (Cancer MoonShot 2020). The understanding of the mechanisms and functions of MDSC immune suppression will be crucial in the success of these therapeutic endeavors. Our current study focuses on interrogating the protein cargo carried by MDSC-derived exosomes based on differential post-translational modifications (PTMs). Post-translational modifications have important roles in functions, signaling, location and interactions of proteins. Selecting proteins based on a specific post-translational modification can aid in the identification of low-abundance proteins which may not be identified in a shotgun proteomics approach. The first aim of this work was to successfully modify an existing surface chemistry method to use on exosomes. We then used a proteomic strategy to identify glycoproteins on the surface of MDSC-derived exosomes, and then test if selected glycoproteins contribute to exosome-mediated chemotaxis and migration of MDSCs. Furthermore, we also aimed at examining the ubiquitome of the MDSC-derived exosomes, using top-down and bottom-up proteomics. Since inflammation has been reported to enhance the tumor promoting activity of the MDSC, the bottom-up analysis focused on the effects of increased inflammation on the ubiquitination of the protein cargo of MDSC-derived exosomes. Spectral counting was used to estimate differences in abundance of proteins found with ubiquitinated proteoforms in high and basal levels of inflammation. The top-down analysis aimed at characterizing the length and topology of ubiquitin linkages on substrate proteins in MDSC-exosomes.Item INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS(2016) Adams, Katherine R.; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.Item PROTEOMIC CHARACTERIZATION OF EXOSOMES SHED BY MYELOID-DERIVED SUPPRESSOR CELLS(2015) Burke, Meghan Catherine; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes are a class of extracellular vesicles that have been shown to contribute to metastasis when derived from tumor cells. Myeloid-derived suppressor cells (MDSC) are an immature population of myeloid cells that accumulate in the tumor microenvironment and inhibit anti-tumor immunity. Given the role of the source cells, it is our hypothesis that MDSC-derived exosomes may contribute to or mediate the effects of MDSC in the tumor microenvironment. The goal of this work is to use mass-spectrometry based proteomics to characterize exosomes produced by MDSC that are induced by 4T1 mammary carcinoma. The protein content of the exosomes will be analyzed to determine if the exosomal proteome is representative of the parental cells or if it reflects active protein sorting. Increased inflammation in the tumor microenvironment is associated with an increased population of MDSC, which further increases the level of immune suppression. Here, the relative change in abundance of exosomal proteins under a heightened level of inflammation in the tumor microenvironment will be performed using the spectral count method. While it is known that exosomes first form through invagination at the plasma membrane, the mechanism(s) through which the protein cargo is sorted into exosomes remains poorly understood. Given the role of ubiquitination in protein localization and trafficking, immunoaffinity enrichment coupled to mass spectrometry has been employed to identify exosomal proteins that carry this modification. Identification of the substrate proteins in MDSC-derived exosomes may provide insight into exosome formation and/or function.