227 research outputs found
The Small Ubiquitin-related Mmodifier in the Stress Response and the Use of Mass Spectrometry/SUMmOn for Identification of Ubiquitin and Ubiquitin-like Protein Conjugation Sites
No full textUbiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that can be covalently conjugated to a variety of “target” molecules to modulate their turnover rate, localization and/or function. The full range of Ubl functions is only beginning to be understood. The Raught lab is using mass spectrometry and high throughput screening methods, along with standard cell biology and biochemistry approaches, to better understand Ubl function. Here, I describe the role of a Ubl called small ubiquitin-related modifier (SUMO) in the budding yeast alcohol stress response. We identified a regulatory mechanism of the SUMO system, involving modulation of the localization of a SUMO protease. Secondly, using mass spectrometry (MS), I assisted in identifying several yeast and mammalian Ubl “chain” linkages. Finally, I propose an integrated MS methodology designed to complement standard database software for the confident identification of Ub/Ubl conjugation sites.MAS
The Small Ubiquitin-related Mmodifier in the Stress Response and the Use of Mass Spectrometry/SUMmOn for Identification of Ubiquitin and Ubiquitin-like Protein Conjugation Sites
No full textUbiquitin (Ub) and the ubiquitin-like proteins (Ubls) are polypeptides that can be covalently conjugated to a variety of “target” molecules to modulate their turnover rate, localization and/or function. The full range of Ubl functions is only beginning to be understood. The Raught lab is using mass spectrometry and high throughput screening methods, along with standard cell biology and biochemistry approaches, to better understand Ubl function. Here, I describe the role of a Ubl called small ubiquitin-related modifier (SUMO) in the budding yeast alcohol stress response. We identified a regulatory mechanism of the SUMO system, involving modulation of the localization of a SUMO protease. Secondly, using mass spectrometry (MS), I assisted in identifying several yeast and mammalian Ubl “chain” linkages. Finally, I propose an integrated MS methodology designed to complement standard database software for the confident identification of Ub/Ubl conjugation sites.MAS
PPP1R35 is a novel centrosomal protein that regulates centriole length in concert with the microcephaly protein RTTN
No full textCentrosome structure, function, and number are finely regulated at the cellular level to ensure normal mammalian development. Here, we characterize PPP1R35 as a novel bona fide centrosomal protein and demonstrate that it is critical for centriole elongation. Using quantitative super-resolution microscopy mapping and live-cell imaging we show that PPP1R35 is a resident centrosomal protein located in the proximal lumen above the cartwheel, a region of the centriole that has eluded detailed characterization. Loss of PPP1R35 function results in decreased centrosome number and shortened centrioles that lack centriolar distal and microtubule wall associated proteins required for centriole elongation. We further demonstrate that PPP1R35 acts downstream of, and forms a complex with, RTTN, a microcephaly protein required for distal centriole elongation. Altogether, our study identifies a novel step in the centriole elongation pathway centered on PPP1R35 and elucidates downstream partners of the microcephaly protein RTTN.</p
Determining the Biological Role(s) of Ubiquitin Fold Modifier 1(UFM1)
No full textUbiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.MAS
Characterizing the Role of SUMO in Transcriptional Repression in S. cerevisiae
No full textSmall ubiquitin-like modifier, SUMO, is a member of the ubiquitin-like protein family. Similar to ubiquitin, SUMO is conjugated to target proteins as a post-translational modification via an E1, E2, and E3 enzymatic cascade, in a process known as SUMOylation. SUMOylation leads to altered target protein localization, half-life, or protein-protein interactions. Bursts of SUMOylation following stresses (SUMO Stress Response / SSR) have been documented, however what triggers the SSR, the kinetics of the response, and the cellular components necessary to mediate the response have not been well characterized. Additionally, SUMO has been widely implicated in transcriptional regulation and chromatin organization, although the nature of its role remains unclear. This thesis aims to: 1) complete a comprehensive characterization of the SSR, and 2) elucidate the true role of SUMO in transcriptional regulation and chromatin organization. To this end, I characterized the SSR following a variety of stresses and time courses. I then used mutants and inhibitors to demonstrate that the SSR is triggered by coordinated transcriptional changes (such as in response to a stress). RNA-Seq of a SUMO system mutant identified 162 transcripts which show a SUMO-dependent transcriptional de-repression following SUMO system inactivation. These transcripts are primarily inducible genes which are normally induced under specific conditions. Notably, these mutants were still able to activate and inactivate transcription in response to a stress, demonstrating that SUMO is not required for acute transcriptional activation or inactivation. Subsequently I investigated possible mechanisms for SUMO-dependent transcriptional repression. Firstly, I used pulldowns and site directed mutagenesis to demonstrate that Rlf2, a component of the histone chaperone CAF-1, is able to bind SUMO in S. cerevisiae. This raises the possibility that CAF-1 plays a role in SUMO-mediated transcriptional repression. Secondly, I used ATAC-Seq to demonstrate that SUMO system inhibition may increase chromatin openness, which could lead to the observed transcriptional de-repression. Together, these data indicate that SUMO may be involved in maintaining chromatin in a closed state; however, further research is needed to elucidate the mechanism. These experiments assist in understanding the role of SUMOylation and the consequences of using SUMO as a therapeutic target.Ph.D
Characterizing the Human Ubiquitin Conjugating Enzymes
No full textUbiquitin is a small (76 amino acid) evolutionarily conserved protein that is covalently conjugated to lysine residues (or rarely N-terminal amines) on substrate polypeptides, to alter their half-life, localization or function. Ubiquitin itself also contains seven internal lysine residues, all of which can be modified by additional ubiquitin molecules to form extended ubiquitin “chains”. Monoubiquitylation or different types of ubiquitin chain linkages can confer very different biological outcomes to a protein target.
The ubiquitin conjugation process is carried out via a three-step enzymatic cascade consisting of an activating enzyme (E1), a conjugating enzyme (E2), and a ligase (E3). More than 600 genes coding for predicted ubiquitin E3 ligases have been identified in the human genome, along with just 35 active E2 genes and two E1s. Importantly, the specific subset of E3s that interacts with each E2, and the specific biological functions of most of the E2 enzymes, have not been elucidated.
To better understand E2 functions, I conducted a series of interactome and gene essentiality screens. Proximity-dependent biotinylation (BioID) was performed in Flp-In T-REx 293 cells to identify interacting partners for 31 different E2 proteins. 2296 high confidence protein-protein interactions were identified amongst 964 different polypeptides. To identify E2 genes (and other genes linked to protein homeostasis) essential for cell fitness, CRISPR/Cas9 dropout screens were also performed in HEK293 cells, along with two different lung cancer lines, NCI-H1975 and SW1573. Several different context-specific essential E2 genes (and many other genes linked to protein homeostasis) were identified in this screen, indicating that E2s may represent viable therapeutic targets. Finally, two common methods have been used to identify the types of ubiquitin linkages that can be generated by specific E2-E3 pairs: in vitro autoubiquitylation reactions using recombinant ubiquitin proteins bearing a single lysine-to-arginine mutation, or direct identification using mass spectrometry. We compared these two methods by characterizing the linkage specificity of 12 different E2-E3 combinations, and demonstrated that mass spectrometry-based methods yield far superior results. Together, my work has thus provided important biological context to a poorly understood component of the ubiquitin system, the E2 family.Ph.D
Functional and Structural Characterization Reveals Novel FBXW7 Biology
No full textThis thesis aims to examine aspects of FBXW7 biology, a protein that is frequently mutated in a variety of cancers. The first part of this thesis describes the characterization of FBXW7 isoform and mutant substrate profiles using a proximity-dependent biotinylation assay. Isoform-specific substrates were validated, revealing the involvement of FBXW7 in the regulation of several protein complexes. Characterization of FBXW7 mutants also revealed site- and residue-specific consequences on the binding of substrates and, surprisingly, possible neo-substrates.
In the second part of this thesis, we utilize high-throughput peptide binding assays and statistical modelling to discover novel features of the FBXW7-binding phosphodegron. In contrast to the canonical motif, a possible preference of FBXW7 for arginine residues at the +4 position was discovered. I then attempted to validate this feature in vivo and in vitro on a novel substrate discovered through BioID.M.Sc
Determining the Biological Role(s) of Ubiquitin Fold Modifier 1(UFM1)
No full textUbiquitin fold modifier 1 (Ufm1) is a member of the ubiquitin like protein (UBL) family. Like other UBLs, Ufm1 can be conjugated to protein substrates via specific E1 (Uba5), E2 (Ufc1) and E3 (Ufl1) enzymes, and removed from these substrates via the action of Ufm1-specific proteases. While Ufm1 has been implicated in endoplasmic reticulum (ER) function, its biological roles remain poorly understood. By identifying; (a) Ufm1 binding proteins, (b) protein interactors of the Ufm1 conjugation/deconjugation system, (c) Ufm1 conjugates, as well as (d) the intracellular localization of Ufm1 and its main interactors, I aimed to better characterize the biological role(s) of this poorly understood UBL.MAS
Bio-functional Analysis of Ubiquitin Ligase UBE3D
No full textThe ubiquitin system is comprised of a reversible three step process: E1 activating enzyme, E2 conjugating enzyme and an E3 ligase, leading to ubiquitin molecules being post-translationally modified onto substrate proteins leading to a plethora of downstream effects (localization, function and half-life). UBE3D, a HECT (homologous to E6-AP carboxylic terminus) E3 ligase, has a relatively elusive regulatory role within the cell. Here, we systematically analyze and characterize UBE3D as well as its highest confidence interactor, Dynein axonemal assembly factor (DNAAF2) through: Autoubiquitylation assay; intracellular localization with immunofluorescence; interaction network using proximity-dependent biotin identification (BioID) to better understand the relationship of these two proteins. DNAAF2 protein interaction mapping allowed for insight into PIH domain. In summary, I have used multiple approaches to gain novel knowledge and insight into the potential functional role of UBE3D within the cell, and its putative partner protein, DNAAF2.M.Sc
Optimization of an Affinity Purification-mass Spectrometry Pipeline and Characterization of the Rub1p and Smt3p Interactomes
No full textThe ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.MAS
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