Life Sciences Commons^™

Two Dot1 Enzymes Cooperatively Mediate Efficient Ubiquitin-Independent Histone H3 Lysine 76 Tri-Methylation In Kinetoplastids, Victoria Frisbie, Hideharu Hashimoto, Yixuan Xie, Francisca De Luna Vitorino, Josue Baeza, Tam Nguyen, Zhangerjiao Yuan, Janna Kiselar, Benjamin Garcia, Erik Debler

Department of Biochemistry and Molecular Biology Faculty Papers

In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 through histone H2B mono-ubiquitin interaction, while the kinetoplastid Trypanosoma brucei di-methyltransferase DOT1A and tri-methyltransferase DOT1B efficiently methylate the homologous H3K76 without H2B mono-ubiquitination. Based on structural and biochemical analyses of DOT1A, we identify key residues in the methyltransferase motifs VI and X for efficient ubiquitin-independent H3K76 methylation in kinetoplastids. Substitution of a basic to an acidic residue within motif VI (Gx₆K) is essential to stabilize the DOT1A enzyme-substrate complex, while substitution of the motif X sequence VYGE by CAKS renders a rigid active-site …

Emerging Roles Of Cullin-Ring Ubiquitin Ligases In Cardiac Development, Josue Zambrano-Carrasco, Jianqiu Zou, Wenjuan Wang, Xinghui Sun, Jie Li, Huabo Su

Department of Biochemistry: Faculty Publications

Heart development is a spatiotemporally regulated process that extends from the embryonic phase to postnatal stages. Disruption of this highly orchestrated process can lead to congenital heart disease or predispose the heart to cardiomyopathy or heart failure. Consequently, gaining an in-depth understanding of the molecular mechanisms governing cardiac development holds considerable promise for the development of innovative therapies for various cardiac ailments. While significant progress in uncovering novel transcriptional and epigenetic regulators of heart development has been made, the exploration of post-translational mechanisms that influence this process has lagged. Culling-RING E3 ubiquitin ligases (CRLs), the largest family of ubiquitin ligases, …

Investigating The Role Of Er-Associated Degradation In An Olfactory Neuron In The Nematode, Caenorhabditis Elegans, Heino Hulsey-Vincent

WWU Graduate School Collection

The breakdown of misfolded proteins is key for cellular health. For proteins at the endoplasmic reticulum (ER), this breakdown is performed through endoplasmic reticulum associated degradation (ERAD). ERAD uses a cascade of E1, E2, and E3 class enzymes to ubiquitylate un- or mis-folded proteins, signaling their destruction. We use the microscopic roundworm Caenorhabditis elegans (C. elegans) as a model organism for studying ERAD. This project investigated the regulation of a neuronal receptor, ODR-10. Our findings suggest that the E2, UBC-6, and the E3, HRDL-1, are required for normal ODR-10 abundance. We worked to uncover whether this regulation is …

Tmem27 Suppresses Tumor Development By Promoting Ret Ubiquitination, Positioning, And Degradation, Qianjin Guo, Zi-Ming Cheng, Hector Gonzalez-Cantú, Matthew Rotondi, Gabriela Huelgas-Morales, Purushoth Ethiraj, Zhijun Qiu, Jonathan Lefkowitz, Wan Song, Bethany N Landry, Hector Lopez, Cynthia M Estrada-Zuniga, Shivi Goyal, Mohammad Aasif Khan, Timothy J Walker, Exing Wang, Faqian Li, Yanli Ding, Lois M Mulligan, Ricardo C T Aguiar, Patricia L M Dahia

Student and Faculty Publications

The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell …

The Involvement Of Ubiquitin In Med13 Cyclin C Degradation Following Cellular Stress, Ayesha Gurnani, Brittany Friedson, Katrina Cooper

Rowan-Virtua Research Day

The Cdk8 Kinase Module is a dissociable regulator of cellular stress response genes, with degradation of its components Med13 and cyclin C eventually determining cell fate decisions such as engaging cell survival or cell death mechanisms. We aimed to explore the roles of ubiquitin in degradation of the Cdk8 Kinase Module following nitrogen starvation, with respect to the potential involvement of deubiquitinating enzyme Doa4, lysine linkage at position K63, and E2 ubiquitin conjugating enzymes Ubc4 and Ubc5. We utilized Western blot analysis to observe nitrogen starvation-induced degradation of Med13-HA in wild-type, doa4 mutant, and K63R yeast strains; degradation of cyclin …

Novel Mechanistic Insight Into Ciliary Regulation: Old Pathways Yield New Mechanisms, Larissa L. Dougherty

Dartmouth College Ph.D Dissertations

Cilia are structures present on most eukaryotic cells which provide important signaling and motile components to cells from early development to fully differentiated and matured cells. Regulation of these structures is critical to proper functioning of the cell and is known to be tied to the cell cycle. Preparation for ciliary assembly following cell cycle exit and ciliary disassembly following cell cycle reentry requires components throughout the cell body and within the cilium to facilitate this process. Here I identify how the cell adapts to ensure modifications to cilia occur for assembly or disassembly using the model organism Chlamydomonas reinhardtii. …

Examining The Immune Regulation Of Nlrp12 Through Novel Protein Interactions, Catherine Rippe

MSU Graduate Theses

NOD-like receptors (NLRs) are intracellular proteins that play an important role in the regulation of the innate immune response to pathogens. Since being identified, various functions for NOD-like receptor pyrin domain containing 12 (NLRP12) have been suggested. It has been shown to negatively regulate the inflammatory response through canonical and noncanonical NF-kB signaling pathways, control tumorigenesis and gut homeostasis and exacerbate inflammation through the formation of a multi-protein complex called an inflammasome. Due to the varying roles established for NLRP12, the mechanisms by which it functions remain poorly understood. In this study, I sought to confirm a novel protein-protein interaction …

A Tale Of Two-Hybrids: Investigating The Interactomes Of Cullin-Associated Proteins, Elyse Reitter

Dissertations and Theses

Cul3 is the major component of an E3 ligase in human cells. Cul3 was initially identified in the Singer lab as a protein that binds and degrades cyclin E, and subsequent studies have shown it to be part of a complex that is involved in a multitude of biological functions. The nature of this complex, its constituents, its regulation, and its dynamics is just beginning to be understood. The research presented here utilizes a series of two hybrid screens to identify families of interactomes with Cul3 at the center. This information will complement other work in the lab in which …

Kinetic And Structural Influences Of Acetylation On Ubiquitin Processing, Rachel E. Lacoursiere

Electronic Thesis and Dissertation Repository

Ubiquitin (Ub) is a small modifying protein abundant in cells where it serves numerous regulatory roles including immune signaling, transcriptional regulation, and proteostasis. To exert its function, Ub covalently interacts with a series of E1, E2, and E3 enzymes before final substrate modification. Dysregulation of Ub signaling has implications in human maladies such as cancer, autoimmune disorders, and neurodegenerative diseases. In these diseases and associated in cellulo models, modifications to Ub serve an additional role in Ub regulation. Post-translational modifications like acetylation or phosphorylation modulate protein-protein interactions and Ub signaling. To understand how acetylation of Ub alters the central E2 …

Here, There, And Everywhere: Probing Ubiquitin-Cavitand Binding Via 15n-1h Hsqc, Julia Didziulis

Scripps Senior Theses

Ubiquitination and other post-translational modifications play a major role in the proliferation of many neurodegenerative diseases, developmental disorders, and cancers, and as such are subjects of recently increased biochemical interest. Expanding upon such research, this study confirmed a robust method of ubiquitin expression and purification, then used 15N-1H HSQC to analyze ubiquitin samples containing varying concentrations of a deep cavitand with affinity for lysine and arginine side chains. In the pure ubiquitin spectrum, arginine side chain chemical shifts were detected in the nitrogen 80-85 ppm range and lysine side chain signals were undetected. However, upon introduction of the cavitand, collection …

Usp11 And Usp7 Deubiquitinases Regulate Sprtn Auto-Proteolysis And Sprtn-Mediated Dna-Protein Crosslink Repair, Megan C. Perry

Theses & Dissertations

DNA repair pathways that recognize and remove damaged DNA are vital for maintenance of genomic stability and prevention of tumorigenesis. Conversely, these pathways may be robust in tumor cells, thus diminishing the anti-cancer potential of available therapies. DNA-protein crosslinks (DPCs) are particularly deleterious DNA adducts that occur when proteins become irreversibly covalently bound to the DNA. DPCs represent a diverse group of lesions, as any protein can be crosslinked to the DNA duplex by non-specific crosslinking agents like reactive aldehydes and radiation. Additionally, functional DNA-binding proteins such as topoisomerases may become permanently crosslinked to DNA ends by abortive enzymatic processes …

Mechanisms Of Substrate Recognition By The Cul3-Based E3 Ligase, Katia Graziella De Oliveira Rebola

Dissertations and Theses

Cul3-based E3 ligase is responsible for regulating a variety of cellular pathways, many of which are known to have profound effects on the proper function of multicellular organisms. Although progress over the past years has been truly impressive, our understanding of the mechanisms of E2 recruitment and selection by the BCR complex and all the roles that Cul3 plays on kidneys remains in its infancy. To explore these aspects, this dissertation aims to analyze the Cul3 complex using two different approaches: (1) We used the powerful tool of chimeric analysis to map the essential domain binding characteristics of Cul3 taking …

Understanding The Pathogenesis Of Renal Medullary Carcinoma, Melinda Soeung

Dissertations & Theses (Open Access)

Renal medullary carcinoma (RMC) is a lethal cancer that predominantly affects young individuals with sickle cell trait (SCT). It is not currently understood why RMC only affects certain individuals with SCT. We found that patients with RMC more frequently participated in high-intensity exercise than matched controls. Using mouse models of SCT, we demonstrated the significant increase of renal hypoxia in the right kidney following high- but not moderate-intensity exercise. We also demonstrated in cell culture studies that SMARCB1 is ubiquitinated for proteasome-mediated degradation in hypoxia, and the re-expression of SMARCB1 leads to compromised proliferation in renal cells specifically in the …

New Perspectives On Phosphorylation State In The Parkin Ubiquitination Cascade, Karen Dunkerley

Electronic Thesis and Dissertation Repository

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. The objective of this thesis was to identify the molecular consequences of parkin phosphorylation, interaction with pUb and how this promotes ubiquitination activity of known Ub-acceptor proteins and parkin itself.

The three-dimensional structure of …

Degradation Of Transcriptional Repressor Atf4 During Late-Phase Long-Term Potentiation, Spencer Smith

Biology Theses

Maintenance of long-term synaptic plasticity requires gene expression mediated by cAMP-responsive element binding protein (CREB). Gene expression driven by CREB can commence only if the inhibition by a transcriptional repressor ATF4 (activating transcription factor 4; aka CREB2) is relieved. Previous research showed that the removal of ATF4 occurs through ubiquitin-proteasome-mediated proteolysis. Using chemically induced hippocampal long-term potentiation (cLTP) as a model system, we investigated the mechanisms that control ATF4 degradation. We observed that ATF4 phosphorylated at Serine-219 increases upon induction of cLTP and decreases by about 30 min thereafter. Proteasome inhibitor β-lactone prevents the decrease in ATF4. We found that …

Investigation Of Potentially Catalytic Residues Of Uba5 Through Mutagenesis, Purification, And Structural Characterization, Grant Bradley

Senior Honors Projects, 2020-current

Ubiquitin-fold modifier 1 (Ufm1) is a member of the Ubiquitin (Ub) family of proteins whose primary function is degradation of proteins through a sequential mechanism of chemical reactions. Though Ufm1’s specific roles are largely unknown, this family of proteins has shown to play a part in a wide variety of processes, including regulation of the cell cycle¹, secretory functions of cells^2,3, and blood clotting⁴. Ufm1’s mechanism of action proceeds with the aid of three enzymes: an E1, E2, and E3. Uba5 is the E1 activating enzyme that is specific to Ufm1, and its mechanism of …

A Novel Uv Resistance In Rad23-Depleted Tetrahymena Thermophila, Emily M. Schmoll

MSU Graduate Theses

Rad23 is a highly conserved cellular scaffold protein which participates in the nucleotide excision repair pathway and ubiquitin proteasome system. It is hypothesized that the contradictory roles of Rad23 within these two systems, acting to enhance stability or facilitate degradation respectively, could be regulated via post-translational modification of the ubiquitin-like domain of the protein. To this end, a Rad23 somatic knockout cell line was established in Tetrahymena thermophila, with the eventual goal of knocking in a mutant Rad23 protein lacking potential for UbL ubiquitylation. In contrast to the UV-sensitive phenotype observed in similar models, Rad23-depleted Tetrahymena cell lines displayed significantly …

Structural Study Of The Complex Between Dna Polymerase Iota And Ub-Pcna, Harrison Taylor

Electronic Thesis and Dissertation Repository

DNA polymerase iota (polι) is a member of the Y-family, polymerases which are key components in translesion synthesis (TLS). As part of the DNA damage response, TLS allows cells to bypass damaged template DNA. Each member of the Y-family is capable of accurately replicating across from certain lesions. All Y-family polymerases are recruited by ubiquitination of the DNA sliding clamp, PCNA, by direct interaction with PCNA and ubiquitin. The mechanism of polymerase choice is not well understood, nor are the interactions between Ub-PCNA and the TLS polymerases. We studied the structure of the complex between the interacting region of polι …

Neurodegenerative Modeling: Tau Protein, Degradative Pathways, And Gene Expression Profiling Of Human Ipsc-Derived Neural Precursors And Differentiated 3-D Neural Sphere Versus 2-D Monolayer Cultures, Kyle H. Anthoney

Cal Poly Humboldt theses and projects

Human induced pluripotent stem cells offer a model for human brain development and disease by differentiation into brain organoids; however, current neural culture systems lack the microenvironment, neuronal circuits and connectivity, vascular circulation, and immune system that exist in vivo. After differentiation and development of neuronal and non-neuronal cell types within two formats of cell cultures, we can visualize and recapitulate in vivo protein accumulation, gene expression, and degradative processes such as autophagy. Using RNA extraction, purification methods and reverse transcription I compared traditional monolayer cultures and novel 3-D neural sphere cultures via gene expression analysis. This analysis indicated …

Regulation Of Glutamate Receptor (Glr-1) Under Endoplasmic Reticulum Stress In Caenorhabditis Elegans, Janie Aguilera

WWU Graduate School Collection

Neurons communicate with other cells to elicit outputs that include memory and movement. Cells, including neurons, create proteins every day for specific functions and in particular, neurons produce proteins that enable their communication. Proteins found in cellular membranes are synthesized at the endoplasmic reticulum (ER). However, up to 30% of new proteins are improperly folded and must be removed from the cell. A build-up of misfolded proteins can trigger the Unfolded Protein Response (UPR) which initiates other pathways of protein quality control and can determine the fate of a cell. ER-associated protein degradation (ERAD) is a ubiquitin-dependent process in eukaryotic …

Uncovering New Mechanisms Of Cdc34 And Cullin-Ring Activity, Spencer Hill

UNLV Theses, Dissertations, Professional Papers, and Capstones

Ubiquitylation is a cellular regulatory system found in all eukaryotic cells, which has managed to find a role in most pathways imaginable. The system works fundamentally through the ligation of a small protein known as ubiquitin onto a substrate. Depending on the context of the ubiquitin ligation, the substrate can be directed towards a number of cellular fates, the best-studied being degradation of the substrate. While originally thought of as a signal for cellular disposal units to degrade aberrant proteins, we now know that ubiquitin plays a highly nuanced role in cellular epistasis, controlling everything from the cell cycle to …

Cul3 Regulates Cyclin E1 Protein Abundance Via A Degron Located Within The N-Terminal Region Of Cyclin E, Brittney Marie Davidge, Katia De Oliveira Rebola, Larry N. Agbor, Curt D. Sigmund, Jeffrey D. Singer

Biology Faculty Publications and Presentations

mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E–Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and Cul3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which Cul3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how Cul3 mediates cyclin E destruction, we identified a degron on cyclin E that Cul3 targets for ubiquitylation. Recognition of …

The Gsk-3Β-Fbxl21 Axis Regulates Tcap Via Ubiquitin-Mediated Proteasomal Pathway In The Cytoplasm, Jiah Yang

Dissertations & Theses (Open Access)

Protein turnover is one of the most essential mechanisms controlling circadian rhythms. F-Box and Leucine Rich Repeat Protein21 (FBXL21) is a circadian E3 ligase which shows oscillatory mRNA transcripts and protein levels. It was previously found to perform subcellular compartment-specific E3 ligase activities targeting the core clock proteins CRYPTOCHROME(CRY)1/2. Here we identified a new sarcomeric target substrate, Telethonin(TCAP), which also shows circadian oscillation in its mRNA transcript and protein expression and, importantly, interaction with FBXL21 in an anti-phasic manner. Via computational and pharmacological tests, we identified Glycogen Synthase Kinase-3β(GSK-3β) as a regulator of FBXL21. Biochemical and molecular characterizations demonstrated that …

Cullin-3 Dependent Deregulation Of Actn1 Represents A New Pathogenic Mechanism In Nemaline Myopathy, Jordan Blondelle, Kavya Tallapaka, Jane T. Seto, Majid Ghassemian, Madison Clark, Jenni M. Laitila, Adam Bournazos, Jeffrey Singer, Stephan Lange

Biology Faculty Publications and Presentations

Nemaline myopathy is a congenital neuromuscular disorder characterized by muscle weakness, fiber atrophy, and presence of nemaline bodies within myofibers. However, understanding of the underlying pathomechanisms is lacking. Recently, mutations in KBTBD13, KLHL40, and KLHL41, three substrate adaptors for the E3 ubiquitin ligase Cullin-3, have been associated with early-onset nemaline myopathies. We hypothesized that deregulation of Cullin-3 and its muscle protein substrates may be responsible for disease development. Using Cullin-3–knockout mice, we identified accumulation of non-muscle α-actinins (ACTN1 and ACTN4) in muscles of these mice, which we also observed in patients with mutations in KBTBD13. Our …

Ubiquitin Regulation: The Histone Modifying Enzyme's Story, Jianlin Wang, Zhaoping Qiu, Yadi Wu

Pharmacology and Nutritional Sciences Faculty Publications

Histone post-translational modifications influence many fundamental cellular events by regulating chromatin structure and gene transcriptional activity. These modifications are highly dynamic and tightly controlled, with many enzymes devoted to the addition and removal of these modifications. Interestingly, these modifying enzymes are themselves fine-tuned and precisely regulated at the level of protein turnover by ubiquitin-proteasomal processing. Here, we focus on recent progress centered on the mechanisms regulating ubiquitination of histone modifying enzymes, including ubiquitin proteasomal degradation and the reverse process of deubiquitination. We will also discuss the potential pathophysiological significance of these processes.

Preventing P-Gp Ubiquitination Lowers Aβ Brain Levels In An Alzheimer's Disease Mouse Model, Anika M. S. Hartz, Yu Zhong, Andrew N. Shen, Erin L. Abner, Björn Bauer

Sanders-Brown Center on Aging Faculty Publications

One characteristic of Alzheimer’s disease (AD) is excessive accumulation of amyloid-β (Aβ) in the brain. Aβ brain accumulation is, in part, due to a reduction in Aβ clearance from the brain across the blood-brain barrier. One key element that contributes to Ab brain clearance is P-glycoprotein (P-gp) that transports Aβ from brain to blood. In AD, P-gp protein expression and transport activity levels are significantly reduced, which impairs Aβ brain clearance. The mechanism responsible for reduced P-gp expression and activity levels is poorly understood. We recently demonstrated that Aβ₄₀ triggers P-gp degradation through the ubiquitin-proteasome pathway. Consistent with these …

Unique E3 Ligase Complexes Regulate Levels Of Cyclin E, Katia De Oliveira Rebola, Jeffrey Dean Simger

Student Research Symposium

Cyclin E is an evolutionarily conserved protein whose essential function is to promote the cell cycle transition from G1 to S phase. Cul3 is the main component of the pathway that controls Cyclin E ubiquitination, playing an important role in the normal cell cycle regulation in vivo. The binding between Cul3 and Cyclin E does not occur directly and requires a BTB domain protein to act as a substrate adaptor. The substrate linker responsible for this binding is known as RhoBTB3. Muf1, a largely uncharacterized protein of unknown function, is able to bind to RhoBTB3 directly indicating that it might …

Development Of Endogenous Tagging Plasmids For Characterization Of Protein Interactions, Localization, And Post-Translational Modifications Of Tetrahymena Thermophila Rad23, Evan Andrew Wilson

MSU Graduate Theses

Rad23 is a protein involved in both nucleotide excision repair (NER) and proteasome-mediated degradation, and has been suggested to facilitate interactions between these two pathways. The model organism Tetrahymena thermophila, which has a transcriptionally silent micronucleus, provides a useful platform for studying the role of Rad23 in global genome NER (GG-NER). However, the ectopic expression systems used thus far in T. thermophila to study Rad23 are repressed by UV light and do not account for the background expression of endogenous RAD23; these phenomena prevent insightful gains to the true dynamics of Rad23. In this thesis, endogenous tagging …

Proteasome Storage Granules Protect Proteasomes From Autophagic Degradation Upon Carbon Starvation, Richard S. Marshall, Richard D. Vierstra

Biology Faculty Publications & Presentations

26S proteasome abundance is tightly regulated at multiple levels, including the elimination of excess or inactive particles by autophagy. In yeast, this proteaphagy occurs upon nitrogen starvation but not carbon starvation, which instead stimulates the rapid sequestration of proteasomes into cytoplasmic puncta termed proteasome storage granules (PSGs). Here, we show that PSGs help protect proteasomes from autophagic degradation. Both the core protease and regulatory particle sub-complexes are sequestered separately into PSGs via pathways dependent on the accessory proteins Blm10 and Spg5, respectively. Modulating PSG formation, either by perturbing cellular energy status or pH, or by genetically eliminating factors required for …

Role Of The Snf2 Homolog, Irc20, In Yeast Genome Maintenance, Deena Mohamed Galal Eldin Ahmed

Theses

In eukaryotes, DNA is wrapped around histone proteins forming a highly compact structure, the chromatin. All DNA-based processes must occur within the complex organization of the chromatin, and this requires modulation of its structure when needed. This is accomplished by covalent histone modifications that alter histone-DNA contacts, as well as through the actions of ATP-dependent chromatin remodelers. These multi-subunit complexes play major roles in transcription regulation, replication and repairing DNA damage. This thesis aims to characterize a poorly studied member of the SWI/SNF family of ATPases/helicases, Irc20, from Saccharomyces cerevisiae. Previously, Irc20 has been shown to be involved in …