1,720,992 research outputs found
Structural and Functional Characterization of Non-Canonical Ubiquitin Conjugating Enzymes
Get PDFThesis (Ph.D.)--University of Washington, 2015Thirty-five years ago, during its initial discovery, the field of ubiquitination could not have been more aptly named. In fact, as the study of ubiquitin (Ub) expands, it is becoming clear that this small post-translational modification is far more ubiquitous than the founders of this field could have originally imagined. Initially identified as a protein degradation signal, Ub is now known to regulate vastly disparate cellular functions, including the DNA-damage response and immune signaling. This small (8.5kDa) modification achieves its diverse signaling capabilities partly through the enzymes that catalyze its attachment to cellular substrates. These enzymes (E1, E2, and E3) work in succession to identify and modify substrates with varying Ub signals. The manner in which Ub is attached (monoubiquition or 8 types of polyubiquitin chains) and the site of attachment dictate a substrate's cellular fate. Furthermore, much of the diversity in Ub signaling can be attributed to the structural characteristics of the enzymes in the ubiquitin cascade. This thesis examines the structural and biochemical features of two E2 ubiquitin-conjugating enzymes, Ube2w and Ube2h. First, we show that Ube2w specifically attaches monoubiquitin to the N-terimni of disordered protein substrates. To attach this unique Ub modification, Ube2w has evolved a non-canonical domain architecture, solved by our group using nuclear magnetic resonance (NMR) techniques. We also show, that another E2, Ube2h, contains a disordered C-terminus and that it can ubiquitinate a substrate, histone H2A/H2B, in vitro, in the absence of an E3. Studies presented here build the foundations to understand the cellular impact of Ube2w and Ube2h
Molecular Insights into the Ubiquitin Transfer Mechanism of RING-in-between-RING Ubiquitin ligases
No full textThesis (Ph.D.)--University of Washington, 2016-12Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work. Ubiquitination is a posttranslational modification that regulates virtually every aspect of cellular function in eukaryotes including cell cycle progression, endocytosis, cell signaling, transcription, translation, DNA damage and even autophagy. Substrate modification with ubiquitin (Ub) requires the coordination of two types of enzymes: Ub-conjugating enzymes (E2s) and Ub ligases (E3s). While E3s are generally thought to bind substrates, substrate ubiquitination can be performed by either an E2 or an E3, depending on the type of E3. There are three classes of eukaryotic E3s: the RING (Really Interesting New Gene) E3s, do not contain an active site. They bind E2~Ub and activate Ub transfer directly onto a substrate. RBR (RING-in-between-RING) and HECT (Homologous to E6AP C-Terminus) E3s contain an active site Cys residue to which Ub is transferred from the E2~Ub to generate a covalent E3~Ub thioester that transfers Ub to a substrate. It was only six years ago that a landmark study discovered that RBR E3 are indeed not RING-type Es3 as had been assumed for many years. In 2011, when I began my studies, very little was known about how RBR E3s function. In fact, RBR E3s were originally termed RING-type E3s based on their primary sequence analysis of the two domains: RING1 (binds E2) and RING2 (contains active site). Based on structural work by other groups, we know today that RING2 does not contain a typical RING fold whereas RING1 domains are structurally similar to canonical RINGs. Nevertheless, in my graduate work I demonstrate that RING1 domains perform opposing functions to canonical RING E3s: instead of promoting closed E2~Ub conformations, RING1s actively favor open E2~Ub conformations. This strategy ensures that the transfer of Ub proceeds via the active site Cys on RING2 and therefore that the type of product generated is determined by the RBR E3 and not by the E2 (as done in the context of canonical RING domains). That RBR RING1 domains have opposing functions to canonical RING domains can be explained functionally, yet a structural explanation for this observation is not immediately apparent. I found that a two-residue extension of the second Zn2+-loop - unique to RING1 domains - is largely responsible for promoting open E2~Ubs. Three years ago, an initial study proposed that the RBR E3 HHARI is able to bind to neddylated cullins (N8-CUL) which are RING-type E3s; and that this interaction results in activation of the normally auto-inhibited HHARI. The biological significance of this complex formation was perplexing. In conjunction with others, my work shows that two types of E3s, the RBR E3 HHARI and the RING E3 N8-CUL-1, work together with their respective E2s to coordinately ubiquitinate a common substrate and that this plays an essential role in a developmental pathway in C. elegans. This work was based on the combination of structural, biochemical and organismal studies that led to a deeper understanding of how RBR E3s work
Defining Protein Interactions With Small Heat Shock Proteins
Get PDFThesis (Ph.D.)--University of Washington, 2015Small heat shock proteins (sHSPs) are a class of molecular chaperones broadly observed across organisms where they play a central role in maintaining protein homeostasis under conditions of cellular stress. sHSPs are known to make protein-protein interactions that can be defined into two classes: 1) interactions with unfolded and aggregate-prone proteins (clients) to delay protein aggregation and 2) interactions with each other, leading to the formation of sHSPs oligomers. Here, progress in characterizing details of these interactions is presented. sHSP are defined by three regions: an N-terminal region (NTR), a conserved Alpha Crystallin Domain (ACD), and a C-terminal region (CTR). All three regions are involved in sHSP oligomer formation. Here, we demonstrate the peptides mimicking CTRs of human sHSPs are able to interact with isolated ACDs. Using a strategy based on this observation, we are able to characterize the requirements for ACD/CTR binding and demonstrate this interaction can occur the context of human sHSP heterooligomers. Further, though other interactions are involved in forming sHSP oligomers, we are able to demonstrate ACD/CTR interactions plays a key role in recruiting an incoming subunit to a sHSP oligomer. These observations are relevant to understanding the details dynamic subunit exchange and heterooligomer formation in human sHSPs. Defining how sHSPs interact with clients remains one of the pressing challenges in the sHSP field. It is unclear whether sHSPs interact with clients through a conserved mechanism. Using a variety of techniques, we set to compare and contrast how the human sHSP, HSPB5, and the wheat sHSP, wHSP16.9, interact with clients. Our observations suggest these sHSPs interact with clients through distinct binding modes. Further, the techniques presented were used to characterize other client/sHSP interactions including, client interactions with the HSPB5 disease-associated mutation, R120G
Interplay of Local Structure, Dynamics, and Self-Association in HSPB1
No full textThesis (Ph.D.)--University of Washington, 2018Small heat shock proteins (sHSPs) comprise a class of ATP-independent chaperones that prevent aggregation of destabilized proteins in the cell. The structural mechanisms by which sHSPs complete their function remain enigmatic due to inherent properties that make them refractory to traditional approaches in structural biology. While sHSP monomers are small (on average 20 kDa), for certain sHSPs these monomers assemble into large homo-oligomers (~500 kDa) of variable size and shape. Structural heterogeneity is observed not only on a global scale but also at the local level. Observed heterogeneity is thought to stem from the predominantly disordered N-terminal region (NTR) of sHSPs. There is evidence for numerous sites on sHSPs for interacting with client proteins, although these interactions are often transient. The combination of transient interactions with clients, competing sHSP-client and sHSP-sHSP interactions, and presentation of different binding sites in variable states of an oligomer make it particularly challenging to understand structure-function relationships in sHSPs. The work presented in this thesis correlates local and global structural properties of the polydisperse human sHSP HSPB1. Hydrogen-deuterium exchange mass spectrometry (HDXMS) was used to identify solvent protected regions in the NTR of HSPB1 oligomers, providing the first full look at the NTR of an oligomeric human sHSP. Additionally, specific regions of local structural heterogeneity were identified in the NTR. Utilizing known mutations that perturb inter-protomer interactions in oligomers, a dimeric, full-length form of HSPB1 was obtained to represent a dispersed subunit of the system. The dimeric form of HSPB1 shows altered local structure in the NTR by HDXMS relative to WT oligomers, indicating distinct structural changes among different states of HSPB1. The dimeric construct is also amenable to traditional NMR approaches, allowing for development of a comprehensive model of full-length HSPB1 dimers, which still show structural heterogeneity. Disease-associated mutations in the NTR of HSPB1 were also characterized using HDXMS and a suite of other biophysical techniques. Specific regions in the NTR have altered protection among these disease mutants. These disease mutations were introduced to the dimeric construct to probe each mutation’s effect on oligomerization propensity. Interestingly, two of the disease mutations promote oligomerization while the other two disease mutations retain some dimer-like characteristics in the dimer-mutant context. HDXMS analysis of the disease mutants in the dimeric construct reveals distinct local structural effects for each mutant. It is most remarkable that mutations in the same region of HSPB1 have different local mechanistic effects, and that the subsequent global effects on oligomeric size or chaperone activity can appear similar for certain mutants. The methods explored in this thesis for characterizing heterogeneous structure in HSPB1 can be implemented to other sHSPs and similarly complex protein systems
Functional Interactions of E2~Ubiquitin Conjugates in and outside the Ubiquitination Pathway
No full textThesis (Ph.D.)--University of Washington, 2012Thirty years of research have implicated ubiquitin (Ub) signaling in nearly every aspect of eukaryotic cell biology. What appears to be a simple signaling molecule in fact has the ability to encode countless cellular fates that include proteasomal degradation, cell cycle control, and DNA damage repair. Although the enzymes within the Ub transfer pathway are well established (E1, E2, E3), the molecular details required for protein ubiquitination are largely unknown. To address questions regarding the final stages of Ub transfer, we began with structural characterization of the pathway intermediate, the E2~Ub conjugate (~ indicates the thioester linkage between the E2 active site cysteine and the Ub C-terminus). NMR and SAXS techniques were used to describe the array of conformations populated by the flexibly linked UbcH5c~Ub and Ubc13~Ub conjugates. Unlike the Ubc13~Ub conjugate, the UbcH5c~Ub intermediate strongly prefers extended conformations in which the Ub is positioned directly below the E2 active site. We determined that addition of RING/U-box E3 ligases induced a shift in the ensemble of UbcH5c~Ub populated states toward more "closed" conformations. A variety of biochemical assays were used to show that these closed conformations represent the activated states by which E3 ligases promote Ub transfer. This mechanism of conformational activation is utilized by diverse RING/U-box E3:E2 pairs, and is mediated by an intermolecular hydrogen bond stemming from a conserved basic residue on the E3. To identify mutations that augment the E3's ability to facilitate Ub transfer, we collaborated with the Stan Fields lab to screen nearly 100,000 E3 sequence variants for ubiquitination activity. Rare mutations were identified that increased ligase activity as much as 20-fold. NMR analysis showed that the mutations fall into two classes based on their effects; the first class of mutants enhance activity by increasing E3:E2 binding affinity while the second class does so by augmenting the ability to induce closed E2~Ub conformations. Lastly, we characterized an interaction between the E2~Ub conjugate and Shigella effector kinase OspG to find that, while OspG has little effect on ubiquitination activity, formation of the OspG:UbcH5c~Ub complex results in a pronounced increase in OspG kinase activity
BRCA1/BARD1-Dependent Ubiquitylation of Nucleosomal Histone H2A
No full textThesis (Ph.D.)--University of Washington, 2022Breast cancer type-1 susceptibility protein (BRCA1) was the first gene to be linked to heritable breast and ovarian cancer over thirty years ago. Since then, remarkable progress has been made with regards to genetic testing, prophylactic measures, and treatment options available to patients with BRCA1 mutations. However, thousands of BRCA1 variants of unknown significance still exist, leaving doctors and patients with no clear path forward. Understanding the underlying biology of the gene product of BRCA1 (the BRCA1 protein) is an important goal that may improve health outcomes. The BRCA1 protein forms an obligate heterodimeric complex with its binding partner, BARD1 (BRCA1/BARD1). Together, this large complex functions in the nucleus of cells, protecting the integrity of genome by helping to repair double-stranded DNA breaks and regulate the transcription of certain genes. These functions are mediated, in-part, by the sole known enzymatic function of BRCA1/BARD1 as a RING-type E3 ubiquitin (Ub) ligase. Recently, histone H2A in nucleosomes has emerged as a central substrate for BRCA1/BARD1-dependent Ub ligase activity. BRCA1/BARD1 targets specific lysine sites on the extreme C-terminal tail of histone H2A for Ub transfer which serves as a signal for DNA repair and transcriptional regulation. In this thesis, I first review what is known about the E3 ubiquitin ligase function of BRCA1/BARD1 in general (Chapter 1) and specifically at chromatin (Chapter 2). In Chapter 3, I present a cryo-EM structure of the BRCA1/BARD1 RING/RING heterodimer bound to a nucleosome substrate, with additional structural and biochemical data that provide key mechanistic insight into the basis for site-specific ubiquitylation of nucleosomal H2A. In Chapter 4, I reveal interactions between non-RING regions of BARD1 and nucleosome substrates that enhance chromatin binding and H2A ubiquitylation, describing BRCA1/BARD1/chromatin complexes with extensive multivalency. Overall, my work uncovers a critical role for the previously underappreciated partner of BRCA1, BARD1. Together, this body of work sets the stage for additional discoveries into the biological importance of BRCA1/BARD1 H2A-specific E3 Ub ligase activity in DNA repair and transcriptional regulation. These findings may ultimately be used to predict or experimentally measure the impacts of patient mutations of unknown clinical significance in addition to explaining the effects of known pathogenic mutations. Knowledge of the molecular effects of these mutations and their underlying biology may ultimately lead to enhanced prophylactic measures or new avenues of therapeutics for patients
Structural and Functional Characterization of Ube2H, a Ubiquitin-Conjugating Enzyme with a C-terminal Extension
No full textThesis (Master's)--University of Washington, 2019The human proteome contains over forty ubiquitin-conjugating enzymes, each harboring a conserved core domain. Despite their structural similarities, E2s must discriminate between hundreds of E3 ligases to interact specifically with their cognate partners. Structural and functional characterization of E2s in the last few decades has revealed unique mechanistic details for each E2 studied thus far, but no universal rule appears to govern how E2 enzymes specifically recognize their cognate binding partners. Many E2s remain uncharacterized and promise to reveal novel strategies by which these apparently simple enzymes achieve specificity in both E3 binding and Ub transfer. Ube2H is a human E2 enzyme with an uncharacterized C-terminal extension of 32 residues. The extension is phylogenetically conserved, but its function is unknown. In this thesis, I employ biophysical and biochemical techniques to investigate the structural and biochemical function of Ube2H’s C-terminal extension, both alone and in the presence of putative E3 ligases and binding partners
Structural Studies of RNF146-Mediated PARylation-Dependent Ubiquitylation
No full textThesis (Ph.D.)--University of Washington, 2017-06The ubiquitylation of proteins is involved in nearly every cellular process. Hence, precise control of the ligases that facilitate the attachment ubiquitin (Ub) to target substrates is of the utmost importance to eukaryotic biology. My thesis work on the RING E3 ubiquitin ligase RNF146 has defined a new mechanism by which a ligase can be allosterically activated. I have provided the first example of a RING domain (E3 domain) that has a conformational change, with my discovery that RNF146 is conformationally “switched on” by binding the post translational modification poly(ADP-ribose) (PAR). My work on RNF146 (and other proteins) builds on a growing theme of RING E3 ligase regulation, upending the notion that all single subunit RING E3 domains are constitutively able to bind and activate an E2~Ub conjugate to transfer ubiquitin to substrates. Protein poly(ADP-ribosyl)ation (PARylation) is involved in many cellular processes including DNA repair, cell division, and cell death. It was recently shown that PARylation catalyzed by the PARPs tankyrase-1 and -2 can mark proteins for ubiquitylation and subsequent degradation via the proteasome. The E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is the only E3 identified thus far that participates in PARylation-dependent ubiquitylation (PARdU) with tankyrases (TNKSs). Through PARdU, RNF146 controls the levels of important regulatory proteins including Axin and 3BP2. We provide a structural basis for the role of RNF146 in PARdU and how RNF146 achieves substrate specificity. First, we show that the smallest internal poly(ADP-ribose) (PAR) structural unit, iso-ADPr, binds between the WWE and RING domains of RNF146 acting as an allosteric signal to switch the RING domain from an auto-inhibited conformation to an active one. In the absence of PAR/iso-ADPr, RNF146 only weakly binds an E2 and cannot activate it for ubiquitin transfer. In this unliganded state, a loop of the RING domain blocks the E2-E3 interaction. PAR binding causes the incorporation of this loop into the main helix of the RING domain, generating a productive RING E3 ligase. Second, we demonstrate that RNF146 forms a complex with tankyrases via its disordered c-terminus. This complex appears to be mediated by at least four elongated tankyrase-binding motifs (TBMs). We have solved a structure of one of these motifs in complex with a fragment of tankyrase-1 confirming that these non-canonical TBMs are bona fide binding motifs. Mutants that disrupt either the allosteric activation of the RING domain or the RNF146-TNKS interaction inhibit Axin turnover in vivo. Third, we show through structural modeling that tankyrases oligomerize through their sterile alpha motif domains in a head-to-tail manner. We can disrupt this interaction with site-directed mutagenesis and oligomerization-deficient mutants cannot promote tankyrase- mediated Wnt signaling enhancement. Hence, we show that RNF146 represents a new class of E3 ligases in which binding of ligands causes conformational changes in the RING domain, that PARdU substrate specificity is likely facilitated by the TNKS-substrate interaction, and that tankyrases oligomerize via a head-to-tail SAM-SAM interaction which likely affects the tankyrase-substrate interaction and the role tankyrases play in Wnt signaling
Chaperone effects on tau amyloid formation
No full textThesis (Ph.D.)--University of Washington, 2019Tau is a microtubule-associated protein that forms insoluble amyloid fibrils in a set of neurodegenerative disorders termed tauopathies, which include Alzheimer’s disease, frontotemporal dementia, and chronic traumatic encephalopathy. Molecular chaperones are proteins that act to prevent aberrant protein aggregation such as tau fibril formation. This dissertation characterizes the mechanisms by which chaperones interact with tau to counteract its aggregation, with particular focus on the small heat shock protein HspB1. Chapter 2 compares the effects of HspB1 on tau aggregation with the effects of another structurally-unrelated chaperone, Hsc70. Chapter 3 describes interactions between the disordered N-terminal region (NTR) and the structured alpha-crystallin domain (ACD) of HspB1. Both domains are involved in chaperone activity toward tau. Understanding how they interact with each other enables further structural characterization of interactions between HspB1 and tau in Chapter 4. Chapter 4 also identifies a way by which interactions between the NTR and ACD can be perturbed to improve HspB1 chaperone activity against tau
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