Chemistry & Biochemistry Theses and Dissertations
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Item SITE-SPECIFIC INVESTIGATIONS OF UBIQUITIN ACTIVATION, CARBAMYLATION, AND INTERACTIONS WITH UBIQUITIN-BINDING DOMAINS(2023) Pawloski, Westley; Fushman, David; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The post-translational modification of proteins with ubiquitin (Ub) can induce a multitude of cellular signaling processes. Ubiquitination involves the attachment of the C-terminus of Ub to lysines on the substrate protein through an isopeptide linkage. This process is facilitated by a multitude of enzymes which work in concert to write and erase these linkages. The power of Ub signaling is that Ub itself can be modified by additional Ub units to generate polyubiquitin chains through any of the seven lysines or N-terminal amine, and each of these attachment points produces polyubiquitin (polyUb) chains with unique orientations of the internal Ub. This allows for K48 polyUb chains to mark a substrate for proteasomal degradation or K63 polyUb chains to trigger DNA repair and maintenance processes. The Ub signaling system is an amalgamation of post-translational modifications, enzymatic activity, and carefully curated protein-protein binding interactions for this small 76 amino acid protein. My work presented in this disseration involves harnessing the power of nuclear magnetic resonance (NMR) experimentation to observe interactions of multiple components of the Ub system with site-specific resolution and selective kinetics. To this end, I have implemented some standard and atypical NMR experiments to observe the potential for carbon dioxide carbamates to modulate the Ub signaling system. I have determined the kinetics of the enzymatic Ub-activating process, and this was extrapolated to understand how ubiquitiun-like proteins, which share a similar fold to Ub, are discriminated from erroneously taking the place of Ub. I have solved the solution structure of an unusual ubiquitin-like domain and explored how it interacts with Ub. Lastly, I will report on the implementation of an unnatural amino acid that is a photosensitive cross-linker and demonstrate that this technology can be used to detect novel ubiquitin-binding proteins.Item LYSINE-48 LINKED UBIQUITN CHAIN BINDING CYCLIC PEPTIDES AS POTENTIAL CANCER THERAPEUTICS(2022) Lemma, Betsegaw Ephraim; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin (Ub) is a protein that serves several important functions in all eukaryotes. Its role involves post-translational modification of target proteins which results in signaling for cellular activities of various kinds. Given several malignancies and neurodegenerative diseases caused by defects that arise in ubiquitin mediated pathways, understanding all the variations in ubiquitin based signaling and the selectivity and specificity involved in the pathways is imperative in potentially targeting and treating various ailments that are caused by the disruption within said pathways. For this purpose, this study examined possible therapeutic methods that could target the ubiquitin-proteasome system at the ubiquitin chain level. Binding studies utilizing NMR in addition to various SDS PAGE gel assays and crystallography were important in understanding how select therapeutic molecules interacted with ubiquitin chains. A secondary study involved Rub1, a protein that is very similar to ubiquitin in terms of sequence and structure. Comparative mutation-based gel assays, NMR analysis and mass spectrometry methods were explored with the goal of incorporating Rub1 within ubiquitin conjugation pathways and making polyRub1 chains. In addition, NMR, DSC, and CD based temperature studies were performed to further understand the structural differences between these closely related proteins and hopefully gain more insight into the seemingly special functional nature of ubiquitin.Item BIOPHYSICAL STUDIES OF UBIQUITIN: FROM FOLDING TO PROTEIN ENGINEERING(2021) Camara, Christina M; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The signaling protein ubiquitin is known for its ubiquity — existing in nearly all cel- lular compartments, holding a prominent role in major cellular signaling pathways and serving as a model system for protein folding. Herein, we honor this stature by exploring several aspects of the ubiquitin system form biophysical, structural, and computational per- spectives. Our efforts begin from the standpoint of protein engineering, where we extend ubiquitin’s function by installing a transition–metal binding motif and elevate it to the sta- tus of a metalloprotein. In doing so, we introduce novel spectroscopic behaviors, reactive propensities, and the capability to form non–canonical polyubiquitin chains — with appli- cations that span from molecular nanotechnology to synthetic biology. We then shift to foundational investigations of ubiquitin’s fold. By characterizing local degrees of freedom, we demonstrate how conformational motions of ubiquitin’s C–terminus can be controlled by the cellular microenvironment. This response, in turn, can regulate molecular recog- nition within the ubiquitination cascade. Finally, we approach global aspects of ubiquitin folding — exploring how a motif containing the C–terminus and the β5 strand might assem- ble into ubiquitin’s β –grasp architecture — with general lessons for ubiquitin–like proteins and other systems with an apparent two–state folding mechanism.Item CHARACTERIZATION OF THE STRUCTURE AND BINDING OF BRANCHED K6/K48-LINKED AND BRANCHED K6/63-LINKED POLYUBIQUITIN CHAINS(2021) Abeykoon, Dulith Maduwantha Bandara; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin (Ub) is an important post-translational protein modifier in eukaryotes. Post-translational modification with Ub is an essential process for eukaryotic cellular signaling including protein degradation, DNA repair, antigen-peptide generation and endocytosis. This post-translational modification with Ub occurs through ubiquitination where Ub attach as monoUb or polyUb chains. Ub form polyUb chains by forming covalent linkages between the C-terminus of one Ub and any of seven lysines or the N-terminus of other Ubs. Polyubiquitin chains can form homogeneous, heterogeneous, linear or branched chains, leading to diversity in polyubiquitin chain signaling outcomes. This diversity in signaling is due to the variety of conformations that arise based on the linkage specificity of the polyUb chains. Recently, branched K6/K48-linked polyubiquitins were shown to enhance deubiquitinating activity of UCH37 in the presence of Rpn13. To better understand the underlying structural mechanisms, here we determined the NMR structures of branched K6/K48-linked triubiquitin (Ub3) and discovered a previously unobserved interdomain interface between each of the distal ubiquitins and the proximal domain. We performed NMR binding assays to study the interactions of branched K6/K48-linked Ub3 with hHR23a UBA2, Rap80 tUIM and UCH37/Rpn13 complex. Binding studies of branched K6/K48-linked Ub3 to the UBA2 domain of the proteasomal shuttle protein hHR23A resulted in negligible differences between branched K6/K48-linked Ub3 and related dimers (K6-Ub2 and K48-Ub2). Interestingly, introducing hydrophobic patch surface residue mutations led to stronger affinity with both distal domains suggesting a change in the binding mode. Stronger binding affinity for K6/K48-linked branched Ub3 was observed with Rap80 tUIM. Moreover, deubiquitinating enzyme UCH37 (with Rpn13) showed strong affinity for both K6-linked and K48-linked distal domains, thereby suggesting a functional impact of this interdomain interface towards enhanced deubiquitinating activity of UCH37. Moreover, mutation studies of the hydrophobic patch residues of the proximal ubiquitin have shown the importance of the hydrophobic patch surface to maintain the interdomain interface of this branched trimer and for interactions with binding partners. Finally, initial studies done with the regulatory domain of the DNA repair protein p53 (p53c) have shown that p53c is a promising candidate for ubiquitination via non-enzymatic ubiquitination method introduced by our lab.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.Item Microwave-Supported Acid Hydrolysis for Proteomics(2012) Cannon, Joe; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our goal is to develop, optimize and demonstrate workflows that incorporate rapid Asp-selective chemical proteolysis into proteomic studies of complex mixtures. This can be further divided into several specific aims. The first aim is to develop and optimize the sample preparation, mass spectrometric, and bioinformatic methods required for complex mixture analysis of peptides resulting from acid digestion both in solution and in polyacrylamide gels. Second, the optimized methods will be applied to three model systems. In the first application, the large peptides derived from microwave-supported acid hydrolysis of human ribosomes isolated from MCF-7 breast cancer cells are analyzed. Secondly, acid hydrolysis will be applied to characterize Lys63 linkages in polyubiquitins. Finally, all the above methods will be combined for the analysis of extracellular vesicles shed by myeloid derived suppressor cells from a murine mammary carcinoma model. After optimizing the mass spectrometric and bioinformatic methods required for analysis of peptides resulting from acid hydrolysis, the most comprehensive analysis using this digestion technique to date was achieved both for in gel and in solution analysis. In gel digestion resulted in identification of over twelve hundred peptides representing 642 proteins, and in solution digestion via mass biased partitioning allowed identification of over 300 proteins. Mass biased partitioning also resulted in two distinct peptide populations from the high and low mass analyses implemented. Nearly 90% of the predicted human ribosomal proteins were identified after acid hydrolysis. High resolution analysis of both precursor and product ions resulted in an average sequence coverage of 46% among identified proteins. It was also demonstrated that microwave-supported acid hydrolysis facilitates a more informative method for analysis of Lys63 linked polyubiquitin. After acid hydrolysis, ~629 Da mass shifts were found to be indicative of isopeptides. These isopeptides were easily identified from complex mixtures using tandem mass spectrometry and diagnostic b ions. Extracellular vesicles from a murine carcinoma model were then analyzed using in gel microwave-supported acid hydrolysis, mass biased partitioning after in solution digestion, and the sample was interrogated for the presence of ubiquitinated peptides.Item Molecular Interactions of Ubiquitin and Polyubiquitin with Ubiquitin Binding Domains(2007-10-22) Haririnia, Aydin; Fushman, David; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ubiquitin is a small protein that is covalently attached to proteins, either as a single ubiquitin moiety or as polyubiquitin chains. A cascade of enzymatic reactions is required for the isopeptide linkage between the C-terminus of ubiquitin and a lysine residue on a substrate protein or another ubiquitin. Attachment of ubiquitin or polyubiquitin, termed ubiquitination, mediates numerous cellular processes by acting as a versatile signal. The signal transmitted by the tag depends on the nature of the modification, which defines the specificity of the tag for different cellular machinery. This versatility is conferred by the variations in polyubiquitin tags, both in terms of length and lysine-linkage. Polyubiquitin chains can adopt a variety of different conformations based on these variations. The conformational and dynamic properties of the tag may optimize its binding to specific ubiquitin binding domains, therefore committing the target protein to distinct cellular outcomes. A combination of NMR methods are used to study the interaction of several ubiquitin binding domains with Lys48- and Lys63-linked di-ubiquitin, the simplest model of a polyubiquitin chain, to gain insights into polyubiquitin recognition. The di-ubiquitin binding interface with ubiquitin-interacting motifs (UIMs) and ubiquitin-associated domains (UBAs) are mapped. Structural models of the complexes are also presented. The results provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interface. The results also show that the UBA domain of Ede1 preferentially binds to Lys63-linked di-ubiquitin. Structural models of the UBA in complex with Lys48- and Lys63-linked di-ubiquitin are shown. Although ubiquitin is highly conserved in eukaryotes, it is promiscuous with regard to its binding partners, ranging from small molecules to UIM and UBA domains. This study examines the effects of point core leucine to serine mutations on UIM and UBA binding specificity. The results show that these mutations bestow ubiquitin with the ability to discriminate between ubiquitin-receptor proteins. Here, we solved the three-dimensional structure of the L69S Ub mutant in solution by NMR. These mutations have a profound effect on binding specificity while causing subtle changes in the protein's three-dimensional fold and reducing its stability. Modification of a specific lysine located on Ub's hydrophobic surface has been reported to inhibit proteasomal degradation and endocytosis. Here, the effects of mutation to tryptophan at this position are investigated within the context of binding to a proteasomal receptor protein, hHR23A, and an endocytic receptor protein, Ede1.