Ligand selectivity: binding at the protein-protein interface of Keap1 and NEMO

This dissertation comprises identifying the structural determinants of binding selectivity as demonstrated in three systems. The first system involves the structure determination of Keap1-small molecule fragment complexes to locate binding surfaces. The second system involves the structural determination of a NEMO/IKKbeta complex to serve as a platform for future fragment binding validation studies. The third system involves the structural investigation of a bacterial phosphoglycosyltransferase found in Campylobacter concisus to find the active site. Keap1 binding of Nrf2 is a regulatory mechanism to inhibit the transcription factor activity of Nrf2 to upregulate Nucleoporin p62 (p62). Nucleoporin p62 is a regulator of tau protein aggregates in Alzheimer's disease. The determination of binding hot spots in the Keap1 active site could serve as a starting point for the development of inhibitors as a treatment method for Alzheimer’s disease. To achieve this, I have developed a crystal form of Keap1 that allows for fragment-based study of binding in the active site via small molecule fragment screening and X-ray crystallography. Analysis of collected data has resulted in the solution of four structures, one containing a peptide fragment and three containing small molecule fragments that occupy a region of binding within the Keap1 active site, demonstrating the utility of the crystal form and affording information on binding hot spots. Nuclear factor κ-light-chain enhancer of activated B cells (NF-κB) is a transcription factor and has been linked to cancer, inflammation, and immune dysfunction. The enzyme complex IκB kinase (IKK) is a regulator of NF-κB and consists of three subunits: IKK-α, IKK-β, and NEMO. If NEMO activity is abrogated, IKK is unable to activate NF-κB, making it a promising therapeutic target. My research has found crystallization conditions and performed trials of phase determination on an N terminal IKKβ-binding construct of NEMO containing previously uncharacterized regions of this protein. Glycosylation is a commonly occurring post-translational modification that affects a number of processes including protein folding, trafficking, cell-cell interactions and host immune response. The phosphoglycosyl transferase PglC is an essential part of the Campylobacter glycosylation pathway and a possible antibacterial target. My research determined the crystallization conditions and has developed complexes and protein constructs for phase determination of this single-pass transmembrane protein and will in the future provide a platform for structure-based inhibition of this protein

PROTEOMIC DISSECTION OF KEAP1/NRF2 SIGNALING TO DETERMINE NEW PATHWAY INTERACTORS IN CANCER

KEAP1/NRF2 signaling regulates intracellular reactive oxygen species and protects cells from reactive oxygen-induced damage. KEAP1 serves as the substrate adaptor for a CULLIN3-based E3 ubiquitin ligase (KEAP1-CUL3-RBX1). Under homeostatic conditions, the KEAP1-CUL3-RBX1 ligase targets its well-established substrate NFE2L2/NRF2 for rapid proteasomal degradation. During oxidative stress conditions, KEAP1 is inactivated, and NRF2 protein levels increase. NRF2 then drives the transcription of a battery of cytoprotective genes that ultimately mitigate the cellular stress that was sensed by KEAP1. This elegant signaling pathway has long been thought to be the primary function of the redox-sensitive KEAP1 E3 ligase complex. KEAP1/NRF2 signaling is the cell’s primary defense against reactive oxygen stress. Therefore, perturbations in this pathway are associated with a number of human pathologies, including cancer. The KEAP1/NRF2 pathway is frequently mutated in cancer, where NRF2-activating mutations correlate with disease progression and poor patient outcomes. In addition to somatic gene mutations in KEAP1, NRF2 or CUL3, we have demonstrated that NRF2 is activated at the protein level in tumors by a competitive binding method, underscoring the importance of understanding the protein-protein interactions within this pathway. Utilizing mass spectrometry-based approaches, we identified the KEAP1 protein interaction network under basal and proteasome-inhibited conditions. Coupling this screening with a candidate-based approach, MCM3 and NRF1 were identified as putative, novel KEAP1-CUL3-RBX1 substrates for ubiquitylation. MCM3, a subunit of the essential DNA replicative helicase, was validated as a KEAP1-CUL3-RBX1 substrate for ubiquitylation. We have characterized the binding and ubiquitylation of MCM3 by KEAP1 and determined that KEAP1 does not regulate MCM3 protein stability. Rather, we propose a model where KEAP1 ubiquitylates MCM3 to regulate its function within the replicative helicase. We demonstrate that KEAP1 associates with chromatin in a cell cycle-dependent fashion with kinetics similar to MCM3 and is thus poised to affect MCM3 function. We also demonstrate that loss of KEAP1 affects cell cycle progression and proliferation in normal cells. Therefore, we have found previously unappreciated roles for KEAP1 in cell cycle progression and chromatin dynamics.Doctor of Philosoph

A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin

AbstractThe SCF (Skp1–Cullin–F-box) E3 ubiquitin ligase family was discovered through genetic requirements for cell cycle progression in budding yeast. In these multisubunit enzymes, an invariant core complex, composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein, engages one of a suite of substrate adaptors called F-box proteins that in turn recruit substrates for ubiquitination by an associated E2 enzyme. The cullin–RING domain–adaptor architecture has diversified through evolution, such that in total many hundreds of distinct SCF and SCF-like complexes enable degradation of myriad substrates. Substrate recognition by adaptors often depends on posttranslational modification of the substrate, which thus places substrate stability under dynamic regulation by intracellular signaling events. SCF complexes control cell proliferation through degradation of critical regulators such as cyclins, CDK inhibitors and transcription factors. A plethora of other processes in development and disease are controlled by other SCF-like complexes, including those based on Cul2–SOCS-box adaptor protein and Cul3–BTB domain adaptor protein combinations. Recent structural insights into SCF-like complexes have begun to illuminate aspects of substrate recognition and catalytic reaction mechanisms

Mechanisms controlling the KEAP1-NRF2 signaling pathway in lung cancer

An ability to effectively regulate intracellular reactive oxygen species is imperative to prevent conditions of oxidative stress, and ultimately aberrant cell death. The primary means by which cells control reactive species is through the KEAP1-NRF2 signaling pathway. NRF2 is a transcription factor that is constitutively degraded by the E3 ubiquitin ligase adaptor KEAP1 under homeostatic conditions. When intracellular levels of reactive oxygen species rise, these reactive molecules inactivate KEAP1, thus inhibiting degradation of NRF2. NRF2 then translocates to the nucleus where it drives transcription of several genes including reactive oxygen species-scavenging genes, drug efflux genes, and cell survival genes. NRF2 interacts with KEAP1 via two amino acid motifs, the ETGE and DLG, which position NRF2 in a sterically favorable position for ubiquitination. An emerging alternative mechanism for activation of NRF2, referred to as the Competitive Binding Mechanism, has been proposed. This mode of NRF2 activation relies on KEAP1 interacting proteins that contain a motif similar to the ETGE motif of NRF2, which compete with NRF2 for binding to KEAP1. We have identified several KEAP1 interacting proteins that bind to KEAP1 in an ETGE-dependent manner, including the dipeptidase DPP3. Identification of these interacting proteins not only validate the Competitive Binding Model, but also introduce DPP3 as a protein relevant in NRF2 activation in cancer. In addition to competitive binding, somatic mutations in KEAP1 have also been shown to activate NRF2. Unlike activating mutations in NRF2, which cluster to the ETGE and DLG motifs, mutations in KEAP1 are present throughout the entirety of the protein. How these somatic mutations affect KEAP1 function is currently not known. We have characterized 18 mutations in KEAP1 derived from The Cancer Genome Atlas lung squamous cell carcinoma cohort. In addition to determining that the majority of KEAP1 mutations are hypomorphic, we also identify a novel class of KEAP1 mutations that bind NRF2 and facilitate its ubiquitination, but cannot degrade NRF2.Doctor of Philosoph

Molecular Mechanisms of Patient-Derived KEAP1 Superbinder Mutants

In 30% of lung cancers, mutations in KEAP1 or NRF2 result in constitutive NRF2 activity. This promotes tumor progression, resistance to radio- and chemotherapy, and predicts poor patient outcome. Over 700 somatic mutations in the KEAP1 tumor suppressor gene have been identified in cancer, yet the mechanism and functional consequences of these mutations are unknown. This dissertation focuses on determining the phenotype and molecular profiles of patient-derived KEAP1 mutations. The objectives were to assign function for patient-derived KEAP1 mutations and to investigate the molecular mechanism(s) and phenotypes of functional classes of KEAP1 mutations. Through biochemical characterization of 18 KEAP1 mutations identified in lung squamous cell carcinoma, we defined a novel class of KEAP1 ‘superbinder’ mutants. These superbinder mutants had increased association with the transcription factor NRF2, yet could not fully suppress NRF2-dependent transcription of cytoprotective genes. Cell-based and in vitro studies determined that superbinder mutants ubiquitylated NRF2 but were impaired for NRF2 proteasomal degradation. Molecular biology techniques were employed to understand the mechanism and phenotypic consequences of the KEAP1 superbinder mutants. Through these studies, five core characteristics were attributed to the superbinder mutant class. First, superbinder residues are highly conserved and are among the most frequently mutated residues across a variety of cancer types. Second, KEAP1 superbinders increase NRF2 association but are not altered in their association with other KEAP1 substrates proteasomal chaperones, or ubiquitin receptors. Third, KEAP1 superbinders may impact KEAP1 tertiary structure thus stabilizing its interaction with the NRF2 degron. Fourth, KEAP1 superbinders sequester a pool of NRF2 in p62-dependent spherical clusters that are cleared by the cell. Furthermore, these clusters are comprised of a KEAP1 core surrounded by the autophagy adapter p62, phosphorylated p62 (pS351), polyubiqutin, and occasionally NRF2. Fifth, superbinders confer resistance to the DNA-damaging agent bleomycin in lung cancer cell lines stably overexpressing KEAP1 superbinder mutants. These studies expand our mechanistic understanding of KEAP1 superbinder mutants and provide insight into the dynamics and subcellular compartmentalization of the KEAP1-NRF2 complex.Doctor of Philosoph

Mechanistic studies of GID/CTLH E3 ubiquitin ligases

The integrative structural, biochemical, and cell biology data revealed mechanistic principles of substrate targeting by evolutionarily conserved GID/CTLH E3 ubiquitin ligases. The pliable substrate receptors adapt their conformations to recognize various N-terminal peptide/degron sequences, whereas the chelator-like supramolecular assembly of a catalytically active E3 ligase core configures multi-pronged targeting of oligomeric substrates. The revealed principles provide a conceptual framework for understanding the emerging complexity of the GID E3 ligase family

The Ugly Sequestosome1:the role of p62/SQSTM1 in autophagy and multisystem proteinopathy

Multisystem proteinopathy (MSP) defines a spectrum of degenerative diseases unified by TDP-43 pathology that affect muscle, brain and bone. Mutations in several proteins (VCP, p62/SQSTM1, HNRNPA2B1, HNRNPA1) can all cause MSP via impairments in autophagic protein degradation (VCP and SQSTM1) or RNA granule dynamics (HNRNPA2B1 and HNRNPA1). Phenotypically, MSP mutations lead to variable penetrance of several phenotypes: Paget’s disease of the bone (PDB), rimmed vacuolar inclusion body myopathy (RV-IBM), amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). However, how a same mutation of a protein can develop different diseases remains unclear. Understanding of p62/SQSTM1 (SQSTM1) function is critical to answer this question. In this dissertation, we provide evidence that SQSTM1 is regulated via its UBA domain ubiquitination. We find that Keap1/Cullin3 ubiquitinates SQSTM1 at lysine 420 within its UBA domain. Substitution of lysine 420 with arginine or disease-associated mutation of SQSTM1 disrupts its ubiquitination, sequestering activity, and degradation. In contrast, overexpression of Keap1/Cullin3 in SQSTM1-WT expressing cells increases ubiquitinated inclusion formation, SQSTM1’s association with autophagosomes and rescues proteotoxicity. We also provide evidence that the oligogenic inheritance of a disease associated SQSTM1 mutation with a rare coding variant in the low-complexity domain (LCD) of the RNA-binding protein, TIA1 (p.N357S) can dictate a myodegenerative phenotypes. Deletion or mutation of SQSTM1 along with TIA1 disease mutants synergistically impairs RNA stress granules clearance and their dynamics. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin mediated autophagic degradation that defines the penetrance of a MSP phenotype

Ubiquitin and Autophagy

This book is a collection of articles from the Cells Special Issue on “Ubiquitin and Autophagy”. It contains an Editorial and 13 articles at the intersection of ubiquitin- and autophagy-related processes. Ubiquitin is a small protein modifier that is widely used to tag proteins, organelles, and pathogens for their degradation by the ubiquitin–proteasome system and/or autophagy–lysosomal pathway. Interestingly, several ubiquitin-like proteins are at a core of the autophagy mechanism. This book dedicates a lot of attention to the crosstalk between the ubiquitin–proteasome system and autophagy and serves as a good starting point for the readers interested in the current state of the knowledge on ubiquitin and autophagy

Nrf2 regulation by Hsp90, oxidation, and in breast cancer

To cope with the dynamic range of stressful stimuli that a cell experiences within its lifetime, a host of adaptive cell survival and cell stress response pathways have evolved. The antioxidant and heat shock responses are two key cell stress response pathways primarily involved in the detoxification and elimination of oxidative stress and the maintenance of protein integrity, respectively. Traditionally, these responses are regarded and studied as two independent pathways. In this exploratory work, we hypothesize that oxidative damage to Nrf2 and Keap1 and their interactions with Hsp90 alter their function within the cellular antioxidant stress response. By establishing and characterizing a novel yeast model for human Nrf2, the transcriptional master regulator of the antioxidant response, a previously unexplored interaction was found between Nrf2 and the major heat shock response protein, Hsp90. Further investigation into this interaction using mammalian and breast cancer cells reveals the co-involvement of these proteins in key aspects of protein oxidation, protein misfolding, and cellular responses to cancer therapy. Additionally, Nrf2 and its regulating protein Keap1 were observed to misfold and form protein inclusions upon exposure to oxidative stress, which might implicate a previously unknown mechanism of Nrf2 regulation by inclusion formation. These findings suggest that investigating the antioxidant and heat shock responses in parallel may provide an additional layer of knowledge that is relevant to both basic science and clinical research

ER-associated Degradation and Cadmium Dependent Rescue of PCA1

Protein synthesis and proper folding is an essential process for all organisms. In eukaryotes proteins of the secretory pathway are synthesized and inserted into the lumen or membrane of the endoplasmic reticulum. Eukaryotic cells maintain a mechanism for removal of proteins unable to fold properly. This process is known as ER-associated degradation (ERAD). A poorly functioning ERAD can lead to a build-up of misfolded proteins which has been implicated in several degenerative diseases such as Alzheimer’s, Amyotrophic lateral sclerosis, and Parkinson’s. Thus, the study of how proteins are recognized, extracted from the ER, and degraded is essential for determining methods for maintaining protein solubility and stability, and prevention of toxic accumulation of protein aggregates. Our lab has previously identified Pca1, a cadmium exporting P1B-type ATPase in Saccharomyces cerevisiae. A genetic knockout screen led to the discovery that Pca1 expression is controlled post-translationally through the ERAD pathway. Specifically, the ERAD-Cytoplasm (ERAD-C, indicating the location of the misfolding) pathway utilizes the E3 ubiquitin ligase Doa10 to ubiquitinylate substrates. We further tested the mechanism by which Pca1 an eight transmembrane domain containing protein was extracted from the ER membrane for degradation in the cytoplasm. Surprisingly, we determined that the proteasome itself is essential for this process. Finally, we sought to determine the requirements of cadmium sensing and rescue from ERAD as well as the molecular factors involved in recognition of the degron of Pca1. Biophysical characterization revealed cadmium specific binding. A random-mutagenesis screen identified residues required for degradation of Pca1. Bioinformatical study of the Pca1 degron structure identified a hydrophobic patch that when broken with amino acid substitution stabilized the protein. It was also determined that interaction with a known recognition factor of ERAD, Ssa1, was much weaker in the presence of a hydrophilic substitution or cadmium supplementation. Collectively, our results revealed a mechanism in which Pca1 is regulated post-translationally through the ubiquitin proteasome system. We were also able to apply our findings of Pca1 to another ERAD-C substrate. Pca1 is an excellent model for the study of the ERAD-C pathway as it is short-lived and rapidly stabilized by the supplementation of cadmium. Adviser: Jaekwon Le