Biochemistry, Biophysics, and Structural Biology Commons^™

Gap Junctional Intercellular Communication: Role Of Cx43 Phosphorylation By Tyrosine Kinases, Ishika Basu

Theses & Dissertations

Phosphorylation of Cx43 is a process that regulates various functionalities of the gap junction including assembly, stability, internalization, turnover, channel permeability and channel gating. Src has been shown to phosphorylate Cx43 at Y247, Y265, Y313 and regulate gap junctions. Intercellular communication in B and T cells is necessary to mediate adaptive immune response. However, there is little-to-no expression of Src in these lymphocytes. An in vitro kinase screen identified Bruton’s tyrosine kinase (BTK) and Interleukin 2-inducible T-cell kinase (ITK) to phosphorylate Cx43. Mass spectrometry identified Tyr residues to be phosphorylated by BTK and ITK, and these residues are similar to …

Proteomic Approaches To Identify Unique And Shared Substrates Among Kinase Family Members, Charles Lincoln Howarth

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.

Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies …

Characterization Of The Function And Regulation Of The Hmpv Phosphoprotein, Rachel Thompson

Theses and Dissertations--Molecular and Cellular Biochemistry

Human metapneumovirus (HMPV) is a non-segmented, negative strand RNA virus (NNSV) that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. Despite the initial identification of HMPV in 2001, there are currently no FDA approved antivirals or vaccines available. Therefore, understanding the mechanism of HMPV replication is critical for the identification of novel therapeutic targets. A key feature in the replication cycle of HMPV and other NNSVs is the formation of membrane-less, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). Recent work on NNSV IBs suggests they display characteristics of biomolecular condensates formed …

Evaluation Of Current Methods To Detect Cellular Leucine-Rich Repeat Kinase 2 (Lrrk2) Kinase Activity, Belén Fernández, Vinita G. Chittoor-Vinod, Jillian H. Kluss, Kaela Kelly, Nicole Bryant, An Phu Tran Nguyen, Syed A. Bukhari, Nathan J. Smith, Antonio Jesús Lara Ordóñez, Elena Fdez, Marie-Christine Chartier-Harlin, Thomas J. Montine, Mark A. Wilson, Darren J. Moore, Andrew B. West, Mark R. Cookson, R. Jeremy Nichols, Sabine Hilfiker

Department of Biochemistry: Faculty Publications

Background: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson’s disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10.

Objective: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols.

Methods: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated …

Regulation Of The Reaction Between Cytochrome C And Cytochrome C Oxidase In The Mitochondria, Anders Nowell

Biological Sciences Undergraduate Honors Theses

Cytochrome c (Cc) is a multifunction protein that has important life and death functions in the cell. In the electron transport chain (ETC), Cc transfers electrons from cytochrome bc₁ to cytochrome c oxidase (CcO), which helps build the electrochemical gradient that drives ATP synthase. The reaction of Cc with CcO is very important in ETC regulatory processes. Previous research shows phosphorylation sites in Cc that affect the binding with CcO, with measurable effects on k_d, k_f, and K_D. These effects result in changes in mitochondrial membrane potentials, respiration, and reactive oxygen species (ROS) …

Exploring The Functionality Of Putative Bop3 Post-Translational Modifications, Liliya Tkachuk

Honors Scholars Collaborative Projects

All eukaryotic cells require that transcribed mRNAs undergo export form the nucleus to the cytoplasm where they can be translated into proteins. This process requires a host of proteins which are conserved between the unicellular budding yeast, S. cerevisiae, and humans. During this process, Mex67 and other associated proteins facilitate the mRNA to travel across the nuclear pore complex (NPC), doorways embedded in the nuclear envelope. Upon the exit of mRNA, Mex67 is released and recycled back into the nucleus to facilitate the export of more mRNA. This occurs through the action of Dbp5, whose activity is regulated through …

Exploring The Functionality Of Putative Bop3 Post-Translational Modifications, Liliya Tkachuk, Rebecca Adams Phd

Belmont University Research Symposium (BURS)

All eukaryotic cells require that transcribed mRNAs undergo export form the nucleus to the cytoplasm where they can be translated into proteins. This process requires a host of proteins which are conserved between the unicellular budding yeast, S. cerevisiae, and humans. During this process, Mex67 and other associated proteins facilitate the mRNA to travel across the nuclear pore complex (NPC), doorways embedded in the nuclear envelope. Upon the exit of mRNA, Mex67 is released and recycled back into the nucleus to provide the export of more mRNA. This release occurs through the action of Dbp5, whose activity is regulated …

The Role Of Charge On Dna Packaging And Integrity Within Reconstituted Peptide-Dna Assemblies, Ehigbai Oikeh

Theses and Dissertations--Chemistry

In nature, DNA exists primarily in a highly compacted form. The compaction of DNA in vivo is mediated by cationic proteins; histone in somatic nuclei and arginine-rich peptides called protamines in sperm chromatin. The packaging in the sperm nucleus is significantly higher than somatic nuclei resulting in a final volume roughly 1/20^th that of a somatic nucleus. This tight packaging results in a near crystalline packaging of the DNA helices. While the dense packaging of DNA in sperm nuclei is considered essential for both efficient genetic delivery as well as DNA protection against damage by mutagens and oxidative species, …

Elucidating The Structural And Dynamical Properties Of The Intrinsically Disordered Protein Nrf2 Using Molecular Dynamics Simulations, Megan Nicole Chang

Electronic Thesis and Dissertation Repository

The nuclear factor erythroid 2-related factor 2 (Nrf2) protein is a critical transcription factor for activating the antioxidant response pathway, a primary defense mechanism against disproportionate levels of oxidants in the cell, via the upregulation of cytoprotective genes. Notably, aberrant activation of Nrf2 in cancer cells increases their resistance to chemotherapy, rendering the treatment ineffective. The focus was to uncover the conformational landscape of Nrf2’s Neh4 and Neh5 domains, which participate in crucial interactions for complete transcriptional activation. Since Nrf2 is an intrinsically disordered protein (IDP), molecular dynamics simulations were employed to capture its dynamic nature and conformational heterogeneity. The …

Hsp70 Phosphorylation: A Case Study Of Serine Residues 385 And 400, Sashrika Saini

Masters Theses

Molecular chaperones play a key role in maintaining a healthy cellular proteome by performing protein quality control. Heat shock protein 70s (Hsp70s) are a diverse class of evolutionarily conserved chaperones that interact with short hydrophobic sequences presented in unfolded proteins, promoting productive folding, and preventing proteins from aggregation. Most of the extensive research on chaperone examines mechanism, substrate promiscuity, and engagement with many co-chaperones. Only recently were chaperones recognized to be frequent targets of post-translational modifications (PTMs). Despite the recent rise in PTMs identified, the impact of these modifications on chaperone function, whether singular or in concert with other modifications, …

Post-Translational Modification And Degradation Mechanisms Of The Aryl Hydrocarbon Receptor, Yujie Yang

University of the Pacific Theses and Dissertations

The aryl hydrocarbon receptor (AHR) is a transcription factor first discovered to be activated by exogenous ligands, such as dioxins, and helps promote downstream gene (e.g. CYP1A1) transcription to metabolize the toxicants. With the reports of various AHR targets genes, the expression levels and activities of AHR have been implicated in many physiological and pathological situations. Understanding how AHR protein level is regulated would provide more information to target AHR. AHR stays in the cytosol in the absence of ligand in a complex with HSP90, p23 and XAP2. After ligand activation, AHR translocates into the nucleus, fulfilling its transactivation function …

Posttranslational Modification And Protein Disorder Regulate Protein-Protein Interactions And Dna Binding Specificity Of P53, Robin Levy

USF Tampa Graduate Theses and Dissertations

p53 is an intrinsically disordered transcription factor that suppresses tumor development by arresting the cell cycle and promoting DNA repair. p53 deletions or mutations can lead to cancer due to the inability of cells to respond to stress. The protein levels and post-translational modification state of p53 changes in response to cellular stress like DNA damage. Previous studies have shown that p53 can undergo coupled folding and binding with the E3 ubiquitin ligase, Mdm2, and the histone deacetylase, p300. In normal cells, p53 is kept at a low level by Mdm2, which marks it with ubiquitin, targeting p53 for proteasome …

Deciphering The Ck2-Dependent Phosphoproteome And Its Integration With Regulatory Ptm Networks, Teresa Nunez De Villavicencio Diaz

Electronic Thesis and Dissertation Repository

Protein functions are regulated by the post-translational addition of covalent modifications on certain amino acids. Depending on their distance within the 3-dimensional structure, addition/removal of individual post translational modifications (PTMs) can be impacted by others. This PTM interplay constitutes an essential regulatory mechanism that interconnects the molecular networks in the cell. Protein CK2, a clinically relevant acidophilic Ser/Thr kinase, may be responsible for 10-20% of the human phosphoproteome. Such estimates agree with the number of known substrates, which continues to expand. Furthermore, the demonstration that CK2 participates in hierarchical phosphorylation and has similar sequence determinants to caspases suggest extensive PTM …

Focal Adhesion Kinase And Src Mediate Microvascular Hyperpermeability Caused By Fibrinogen- Γc- Terminal Fragments, Richard S. Beard Jr.

Biomolecular Research Center Publications and Presentations

Objectives

We previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.

Methods and results

Using intravital microscopy of rat mesenteric microvessels, we show increased extravasation …

Practical Applications And Future Directions Of Genetic Code Expansion: Validation Of Novel Akt1 Substrates And The Design Of A Synthetic Auxotroph Strain Of B. Subtilis, Mcshane M. Mckenna

Electronic Thesis and Dissertation Repository

In Chapter 1, site-specifically phosphorylated variants of the oncogene Akt1 were made in Escherichia coli using the orthogonal translation system that enable genetic code expansion with phosphoserine. The differentially phosphorylated variants of Akt1 were used to validate newly predicted Akt1 substrates. The predicted target sites of the peptide substrates were synthesized and subjected to in vitro kinase assays to quantify the activity of each Akt1 phosphorylated variant towards the predicted peptide. A previously uncharacterized kinase-substrate interaction between Akt1 and a peptide derived from RAB11 Family Interacting Protein 2 (RAB11FIP2) was validated in vitro. Chapter 2 describes the preliminary development of …

Factors Influencing Huntingtin Aggregation At Surfaces: Implications For Huntington’S Disease, Sharon E. Groover

Graduate Theses, Dissertations, and Problem Reports

Huntington’s Disease (HD) is a genetic, neurodegenerative disease characterized by an abnormal polyglutamine (polyQ) expansion in the first exon of the huntingtin protein (htt). The polyQ domain facilitates aggregation and initiates the formation of a diverse collection of aggregate species, including fibrils, oligomers and annular aggregates. The first 17 amino acids of htt (Nt17) directly flank the polyQ domain and is a key factor in htt’s association to membranous structures. In addition to Nt17 being an amphipathic αhelix, it also promotes aggregation through self-association and contains numerous posttranslational modifications (PTMs) that can modulate toxicity and subcellular localization. For in depth …

Investigations Into Signaling Mechanisms Of The Dcbld Receptor Family, Anna Schmoker

Graduate College Dissertations and Theses

Cells communicate to drive all biological processes during organismal development, homeostasis, and disease. Communication, or signaling, is carried out through an orchestration of complex sequential molecular interactions. A signal is typically initiated by an extracellular cue binding to a receptor on the cell membrane, which induces an intracellular response, resulting ultimately in cellular phenotypes such as growth, proliferation, migration, apoptosis or survival. Adaptor proteins are critical to signal transduction, as they facilitate the formation of protein complexes that transduce signals. CT10 regulator of kinase (CRK) and CRK-like (CRKL) form a family of adaptors that facilitate complex formation during developmental signaling, …

Defining The Role Of Tyrosine Phosphorylation In The Regulation Of Connexin43 In Cardiac Diseases, Li Zheng

Theses & Dissertations

Connexins are integral membrane proteins that oligomerize to form gap junction channels. Ions and small molecules diffuse intercellularly through these channels, allowing individual cellular events to synchronize into the functional response of an entire organ. Gap junction channels composed of Connexin43 (Cx43) mediate electrical coupling and impulse propagation in the normal working myocardium. In the failing heart, Cx43 remodeling (decreased expression, altered phosphorylation state, loss at intercalated discs, and increased presence at lateral membranes) contributes to rhythm disturbances and contractile dysfunction. While there is considerable information regarding key interactions of Cx43 in the regulation of gap junction channels, unfortunately, the …

A Novel Switch-Like Function Of Delta-Catenin In Dendrite Development, Ryan Baumert

Dissertations & Theses (Open Access)

The formation of neuronal networks in the brain is tightly regulated, and dependent on the morphology of dendrites, the branch-like signal-receiving structures extending from neurons. Disruptions in dendrite development, or dendritogenesis, can lead to the atypical neuronal connectivity associated with multiple neurodevelopmental diseases. My research addresses molecular processes that underlie dendritogenesis via analysis of a pair of novel interactions involving the protein delta-catenin.

In neurons, delta-catenin localizes to dendrites and synapses, where it functions in their development and maintenance. Structurally, delta-catenin possesses a central Armadillo domain and a C-terminal PDZ-binding motif. This motif associates with PDZ domain-containing proteins, and is …

Characterization Of Endothelial Nitric Oxide Synthase Serine-600 Phosphorylation, Kevin Patel

Master of Science in Chemical Sciences Theses

Endothelial nitric oxide synthase (eNOS) is part of a family of three nitric oxide synthase (NOS) enzymes that catalyze the production of nitric oxide (NO). NO is a gaseous, free-radical signaling molecule that has a variety of cellular and physiological functions that range from maintaining cardiovascular homeostasis to neurotransmission. The function of NO greatly depends on the concentration and is cell type specific. eNOS is the most regulated of the three NOS isoforms and the mechanisms of regulation can be through protein-protein interactions and posttranslational modifications. A connection with eNOS and the cell cycle has begun to form with recent …

Regulation Of Rna Stability By Terminal Nucleotidyltransferases, Christina Z. Chung

Electronic Thesis and Dissertation Repository

The dysregulation of RNAs has global effects on all cellular pathways. The regulation of RNA metabolism is thus tightly controlled. Terminal RNA nucleotidyltransferases (TENTs) regulate RNA stability and activity through the addition of non-templated nucleotides to the 3′-end. TENT-catalyzed adenylation and uridylation have opposing effects; adenylation stabilizes while uridylation silences or degrades RNA. All TENT homologs were initially characterized as adenylyltransferases; the identification of caffeine-induced death suppressor protein 1 (Cid1) in Schizosaccharomyces pombe as an uridylyltransferase led to the reclassification of many TENTs as uridylyltransferases. Cid1 uridylates mRNAs that are subsequently degraded by the exonuclease Dis-like 3′-5′ exonuclease 2 (Dis3L2), …

Differentially Activating The Oncogenic Kinase Akt1, Nileeka Balasuriya

Electronic Thesis and Dissertation Repository

The proto-oncogene Akt/protein kinase B plays a pivotal role in cell growth and survival. Phosphorylation of Akt at Thr308 and Ser473 activates the kinase following growth factor stimulation. Delineating specific role of each activation site in Akt1 on kinase activation, inhibition and substrate selection remain elusive.

We designed a unique set of tools, relying on genetic code expansion with phosphoserine and in vivo enzymatic phosphorylation, to produce differentially phosphorylated Akt1 variants. We found that having both sites phosphorylated increased the apparent catalytic rate of the enzyme by 1500-fold relative to the unphosphorylated enzyme. This increment was mainly due to the …

Understanding How Map Kinases Influence Endothelial Nitric-Oxide Synthase Activity, Xzaviar Solone

Master of Science in Integrative Biology Theses

Mitogen activated protein kinases (MAPK) p38 and ERK have both been reported to bind endothelial nitric oxide synthase (eNOS) with submicromolar affinity via proposed interactions with a pentabasic non-canonical MAPK binding sequence in the autoinhibitory insertion of eNOS. The neuronal isoform, which lacks the pentabasic motif, did not bind either MAPK significantly. In the present study, the pentabasic motif was validated using predictive modeling programming, and eNOS phosphorylation by MAPKs (P38, ERK and JNK) was examined using in vitro kinase assays and immunoblotting. JNK phosphorylation at Ser¹¹⁴ contrasts with ERK, which phosphorylated Ser⁶⁰⁰, and p38, which phosphorylated …

Myxobacteria Versus Sponge-Derived Alkaloids: The Bengamide Family Identified As Potent Immune Modulating Agents By Scrutiny Of Lc-Ms/Elsd Libraries., Tyler A. Johnson, Johann Sohn, Yvette M Vaske, Kimberly N White, Tanya L Cohen, Helene C Vervoort, Karen Tenney, Frederick A Valeriote, Leonard F Bjeldanes, Phillip Crews

Tyler Johnson

A nuclear factor-κB (NF-κB) luciferase assay has been employed to identify the bengamides, previously known for their anti-tumor activity, as a new class of immune modulators. A unique element of this study was that the bengamide analogs were isolated from two disparate sources, Myxococcus virescens (bacterium) and Jaspis coriacea (sponge). Comparative LC-MS/ELSD and NMR analysis facilitated the isolation of M. viriscens derived samples of bengamide E (8) and two congeners, bengamide E' (13) and F' (14) each isolated as an insperable mixture of diastereomers. Additional compounds drawn from the UC, Santa Cruz repository allowed expansion of the structure activity relationship …

A Rational Approach For Creating Peptides Mimicking Antibody Binding, Sameer Sachdeva, Hyun Joo, Jerry Tsai, Bhaskara Jasti, Xiaoling Li

School of Pharmacy Faculty Articles

This study reports a novel method to design peptides that mimic antibody binding. Using the Knob-Socket model for protein-protein interaction, the interaction surface between Cetuximab and EGFR was mapped. EGFR binding peptides were designed based on geometry and the probability of the mapped knob-sockets pairs. Designed peptides were synthesized and then characterized for binding specificity, affinity, cytotoxicity of drug-peptide conjugate and inhibition of phosphorylation. In cell culture studies, designed peptides specifically bind and internalize to EGFR overexpressing cells with three to four-fold higher uptake compared to control cells that do not overexpress EGFR. The designed peptide, Pep11, bound to EGFR …

P53 Phosphomimetics Preserve Transient Secondary Structure But Reduce Binding To Mdm2 And Mdmx, Robin Levy, Emily Gregory, Wade Borcherds, Gary W. Daughdrill

Molecular Biosciences Faculty Publications

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient …

Post-Translational Modifications And Functional Studies Of Dksa In Escherichia Coli, Andrew Charles Isidoridy

Legacy Theses & Dissertations (2009 - 2024)

DksA is a bacterial gene regulator that functions synergistically with the stress alarmone ppGpp to mediate the stringent response. DksA also functions independently of ppGpp to regulate transcription of a number of genes. DksA function is dependent on its binding affinity to RNA polymerase and requires specific interactions between RNAP and catalytic amino acids located on the coiled coil tip, D74 and A76. While much of the previous work on DksA has focused on understanding the mechanisms of action and the numerous gene targets for transcriptional regulation, little is known about the mechanisms by which DksA expression and function may …

Intra- And Inter-Molecular Signaling In A Cardiac Connexin: Role Of Cytoplasmic Domain Dimerization And Phosphorylation, Andrew J. Trease

Theses & Dissertations

As critical mediators of cell-to-cell communication, gap junctions (GJs) are comprised of membrane channels that directly link the cytoplasm of adjacent coupled cells thereby allowing for the passage of ions, small metabolites, and secondary messengers. Each channel is formed by the apposition of two connexons from adjacent cells, each composed of six connexin (Cx) proteins. Each GJ channel functions to promote signal propagation and synchronization of cells and tissues in organs. Furthermore, GJs are essential for proper propagation of cardiac action potentials from one cell to the next, leading to the coordinated contraction and relaxation of heart muscle powering circulation. …

A Comprehensive Catalog Of Post-Translational Modifications Of Drosophila Melanogaster Hox Protein, Sex Combs Reduced, Anirban Banerjee

Electronic Thesis and Dissertation Repository

During formation of the anterior-posterior axis, Homeotic selector (HOX) proteins determine the identity of Drosophila body segments. HOX proteins are transcription factors that regulate gene expression during development. Besides a highly conserved DNA-binding homeodomain (HD), HOX proteins also contain functionally important, evolutionarily conserved small motifs. These short motifs found in HOX proteins may be Short Linear Motifs (SLiMs). SLiMs are proposed to be sites of phosphorylation and this may regulate the activity of HOX proteins. The primary aim of this work was to develop a comprehensive catalogue of the sites of phosphorylation and other post-translational modifications (PTMs) for Fushi tarazu …

Structural And Functional Characterization Of Hyper-Phosphorylated Grk5 Protein Expressed From E. Coli, Joseph M. Krampen, John Tesmer, Qiuyan Chen

The Summer Undergraduate Research Fellowship (SURF) Symposium

G protein-coupled receptor (GPCR) kinases (GRKs) are proteins in the cell responsible for regulating GPCRs located on the cell membrane. GRKs regulate active GPCRs by phosphorylating them at certain sites which causes them to stop normal signaling on the membrane. This ultimately affects how the cell responds to its environment. GRK5 is a kinase of particular interest due to its involvement in the pathology of diseases such as cardiac failure, cancers, and diabetes. Understanding the structure and function of GRK5 is essential for discovering ways to manipulate its behavior with these diseases, but not much is known about how GRK5 …