Biochemistry Commons^™

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) …

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, …

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, …

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 …

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 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 …

Towards The Identification Of The Molecular Mechanism Responsible For Rpa:Rad52 Complex Formation, Lucas Struble

Theses & Dissertations

Human cells are routinely exposed to DNA-damaging conditions, from both external sources like ionizing radiation and internal sources like normal oxidative metabolism. Damage in the form of double strand breaks (DSBs) is especially problematic. DSBs occurring outside of replication forks can be repaired through two forms of homologous recombination. The first of these is genetic conversion involving either RPA, BRCA1, PALB2, BRCA2, and RAD51, or RPA, RAD52, RAD51, and other unknown factors. The second is single strand annealing involving RPA and RAD52. Familial breast cancers, among numerous others, are characterized by homozygous pathological mutations in the BRCA2 pathway and must …

Regulation Of Liver Mitochondrial Metabolism During Hibernation By Post-Translational Modification, Katherine E. Mathers

Electronic Thesis and Dissertation Repository

Hibernation, characterized by a seasonal reduction in metabolism and body temperature, allows animals to conserve energy when environmental conditions (e.g. temperature, food availability) are unfavourable. During hibernation, small mammals such as the 13-lined ground squirrel (Ictidomys tridecemlineatus) cycle between two distinct metabolic states: torpor, where metabolic rate is suppressed by >95% and body temperature falls to ~5 °C, and interbout euthermia (IBE), where metabolic rate and body temperature rapidly increase and are maintained at euthermic levels several hours. Suppression of metabolism during entrance into torpor is paralleled by rapid suppression of liver mitochondrial metabolism. In my thesis, I …

Phopsphorylation And Ubiquitin Modification At Dna Damage Sites In Response To Double-Strand Breaks, Atanu Paul

Dissertations & Theses (Open Access)

Genomes of all organisms are continuously damaged by numerous exogenous and endogenous sources leading to different kinds of DNA lesions, which if not repaired efficiently may trigger wide-scale genomic instability, a hallmark of cancer development. To overcome this, cells have evolved a sophisticated sensory network called the DNA damage response (DDR) comprised of a large number of distinct protein complexes categorized as sensor, mediator, transducer and effector proteins that amplify the DNA damage signal and activate cell cycle checkpoint to initiate DNA repair or trigger apoptosis where the defect is beyond repair. This intricate signaling pathway is tightly regulated by …

Exploitation And Regulation Of Apoptotic Caspases, Scott Eron

Doctoral Dissertations

Caspases are the cysteine proteases that govern apoptotic cell death. The regulation of these enzymes is critical in order to restrain their death-inducing capabilities until the appropriate moment. Infidelity of caspase regulation and activation underlies a plethora of human diseases ranging from cancer to neurodegeneration. This establishes a pressing need for comprehensive studies of the apoptotic caspases in order to understand all aspects of their regulation, activation, substrate preferences, structure, and function. A detailed structural view of caspase regulation would have lasting implications for future therapeutic avenues targeting caspase function or apoptosis. This dissertation chronicles caspase regulation by phosphorylation as …

Quantitative Proteomic Characterization Of Cx-4945, A Clinical Stage Inhibitor Of Protein Kinase Ck2, Adam J. Rabalski

Electronic Thesis and Dissertation Repository

Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to …

Quantification Of Cell Signaling Networks Using Kinase Activity Chemosensors, Jon R. Beck, Edward N. Harris, Cliff I. Stains

Department of Biochemistry: Faculty Publications

The ability to directly determine endogenous kinase activity in tissue homogenates provides valuable insights into signaling aberrations that underlie disease phenotypes. When activity data is collected across a panel of kinases, a unique “signaling fingerprint” is generated that allows for discrimination between diseased and normal tissue. Here we describe the use of peptide-based kinase activity sensors to fingerprint the signaling changes associated with disease states. This approach leverages the phosphorylation-sensitive sulfonamido-oxine (Sox) fluorophore to provide a direct readout of kinase enzymatic activity in unfractionated tissue homogenates from animal models or clinical samples. To demonstrate the application of this technology, we …

Defining The Role Of Phosphorylation And Dephosphorylation In The Regulation Of Gap Junction Proteins, Hanjun Li

Theses & Dissertations

Gap junctions are intercellular channels that permit the free passage of ions, small metabolites, and signaling molecules between neighboring cells. In the diseased human heart, altered ventricular gap junction organization and connexin expression (i.e., remodeling) are key contributors to rhythm disturbances and contractile dysfunction. Connexin43 (Cx43) is the dominant gap junction protein isoform in the ventricle which is under tight regulation by serine/tyrosine phosphorylation. Phosphorylation and dephosphorylation regulate many aspects of Cx43 function including trafficking, assembly and disassembly, electrical and metabolic coupling at the plaque, as well as to modulate the interaction with other proteins.

Serine phosphorylation has long been …

The Role Of Phosphorylation In Pam2 Motif-Containing Proteins Mediated Messenger Rna Deadenylation, Kai-Lieh Huang

Dissertations & Theses (Open Access)

Phosphorylation regulates many cellular processes. However, its role in mRNA deadenylation, a process to remove poly adenosines from the mature mRNA 3’ end tail, is unclear. The length of poly(A) tail determines mRNA stability and translation efficiency. Poly(A)-binding protein (PABP), which binds to newly synthesized poly(A) tails homogeneously and is known as a scaffold protein for PAM2 motif-containing proteins, plays a pivotal role in the shortening of poly (A) tails. This study is to examine the role of phosphorylation of PAM2 motif–containing proteins in regulating their interactions with PABP and mRNA deadenylation function.

The PAM2 motif, a region required for …

Modulating Parkin E3 Ubiquitin Ligase Activity Using Phospho-Ubiquitin Variants, Susanna George

Electronic Thesis and Dissertation Repository

Parkin is a Parkinson’s disease-linked E3 ubiquitin (Ub) ligase that promotes mitophagy by ubiquitination of mitochondrial outer membrane proteins. Phosphorylation of Ub at Ser65 by the PTEN-induced putative kinase 1 activates parkin. The role of other Ub phosphorylation sites and the associated kinases remain unknown. We optimized genetic code expansion to produce pure site-specfically phosphorylated Ub (pUb) variants (pUb^S7, pUb^S12, pUb^S20, pUb^S65) and investigated their activity in a key neurodegenerative pathway. Purification of pUb^S7 revealed a +3 frameshifted protein (Ub ∆7) that was successfully purified away from the pUb. Parkin was …

Novel Mechanisms Governing Autoregulation Of The Src Family Kinase Fyn And Its Crosstalk With Protein Kinase A, Marion Weir

Graduate College Dissertations and Theses

ABSTRACT

Phosphorylation is a post-translational modification important for regulating protein activity and protein binding capacity. It is used in many different signaling pathways within the cell. Src Family Kinases and Protein Kinase A (PKA) are two prototyptical non-receptor tyrosine and serine/ threonine kinases, respectively, which are found in canonical signaling pathways. These two kinases are critical for signaling in essentially every cell of a multicellular organism, and are particularly important in development, cell migration and proliferation. Although both proteins have been intensely studied for many decades, an understanding of the molecular mechanisms which govern their regulation and the regulation that …

Functional Roles For Post-Translational Modifications Of T-Snares In Platelets, Jinchao Zhang

Theses and Dissertations--Molecular and Cellular Biochemistry

Platelets affect vascular integrity by secreting a host of molecules that promote hemostasis and its sequela. Given its importance, it is critical to understand how platelet exocytosis is controlled. Post-translational modifications, such as phosphorylation and acylation, have been shown to affect signaling pathways and platelet function. In this dissertation, I focus on how these modifications affect the t-SNARE proteins, SNAP-23 and syntaxin-11, which are both required for platelet secretion. SNAP-23 is regulated by phosphorylation. Using a proteoliposome fusion assay, I demonstrate that purified IκB Kinase (IKK) phosphorylated SNAP-23, which increased the initial rates of SNARE-mediated liposome fusion. SNAP-23 mutants …

The Rational Design And Evaluation Of Ck2alpha Mutants Bearing Inhibitor-Refractory Amino Acid Substitutions, Sam Reid Fess

Electronic Thesis and Dissertation Repository

CK2 is a ubiquitously expressed and constitutively active serine/threonine protein kinase that is implicated in many cellular functions. Previous studies have indicated that the generation of mutants that are less sensitive to inhibition can be advantageous when studying protein kinases. Importantly, studies have demonstrated that mutants of CK2 rendered less sensitive to inhibition are attainable. To extend these observations, mutants of CK2α were designed and evaluated to test their effect on the inhibition of CK2 by CX-4945 using in vitro enzymatic assays followed by the development of inducible cell lines. CX-4945 is a CK2 inhibitor that has demonstrated anti-tumor activity …

The Role Of Protein Kinases Rog1 And Srf6 In The Wak Stress Response Pathway, Jaepil Eric Yoon

Jaepil Eric Yoon

The wall-associated kinases (WAKs) are receptor kinases that bind cell wall pectin to activate expansion and stress response pathways in Arabidopsis thaliana. When bound to pectin polymers, WAKs initiate expansion by inducing the expression of vacuolar invertases that increase cellular turgor pressure, which pushes out against the cell wall. WAKs also bind pathogen or wounding induced pectin fragments called oligogalacturonic acids (OGs). When bound to OGs, WAKs induce a stress response, which leads to the activation of MAP kinases, MPK3 and MPK6. Phosphoproteomics and mass spectrometry identified receptor kinase SRF6, cytoplasmic kinase ROG1, and nuclear DNA binding protein REM2 to …

Beryllium Is A Potent And Unique Gsk-3Β Inhibitor With Potential To Differentially Regulate Glycogen Synthase And Β-Catenin, Ata Ur Rahman Mohammed Abdul

UNLV Theses, Dissertations, Professional Papers, and Capstones

Glycogen Synthase Kinase-3β (GSK-3β) is an important serine/threonine kinase that phosphorylates/regulates diverse and metabolically important proteins. Some of the important substrates of GSK-3β are glycogen synthase, tau, β-catenin, cyclin D1, axin, c-jun, c-myc, Heat Shock Factor–1, BCL-3, CREB, Histone H1.5, mdm2, p21 (CIP1), pyruvate dehydrogenase and many more. De-regulation of GSK-3β has been implicated in diseases like cancer, Alzheimer’s disease, bipolar disorder and type 2 diabetes mellitus. Currently, GSK-3β is one of the most widely studied proteins and there is a great interest in developing potent and efficient GSK-3β-inhibitors for research as well as therapeutic purposes. We demonstrate that beryllium …