Biochemistry Commons^™

Identification Of Covalent Catechin Adducts Of Enoyl-Acp Reductase (Fabi) From E. Coli Using Mass Spectrometry, Francisco G. Atwi, P. M. Joyner, Rustin Ghidari

Seaver College Research And Scholarly Achievement Symposium

Enoyl-ACP reductase (FabI), an enzyme which is inhibited by catechin, is essential for lipid biosynthesis in bacteria (E. coli). Another FabI inhibitor, triclosan, has strong antibacterial effects,1 but overuse can be detrimental to the environment. Flavonoids, a class of dietary compounds, are known to have potent inhibitory effects on FabI and other kinases.2,3,4 Adducts form as a result of the biochemical reaction between flavonoids and cysteine, a modification which can be verified by m/z shifts on the mass spectra.5 Samples of E. coli protein with overexpressed FabI are utilized: the wild type with ordinary primary structure, and mutant type lacking …

Identification Of Serum Exosome Proteins In Systemic Sclerosis With Interstitial Lung Disease By Aptamer Proteomics, Sonsoles Piera-Velazquez, Simon T. Dillon, Xuesong Gu, Towia A. Libermann, Sergio A. Jimenez

Jefferson Institute of Molecular Medicine Papers and Presentations

OBJECTIVE: A major unmet need for Systemic Sclerosis (SSc) clinical management is the absence of well validated biomarkers for early diagnosis of SSc-associated interstitial lung disease (SSc-ILD). The objective of this study was to identify proteins contained within serum exosomes that may serve as potential biomarkers to differentiate patients with Diffuse SSc without SSc-ILD from patients with Diffuse SSc with SSc-ILD employing aptamer-based proteomics.

METHODS: Serum exosomes were isolated from two cohorts of patients. The first cohort included 15 patients with Diffuse SSc without SSc-ILD and 14 patients with Diffuse SSc with SSc-ILD and the second cohort included 12 patients …

Activity-Based Protein Profiling On Nucleophilic Amino Acid Residues With Pyrocarbonates, James Van Horn

Chemistry & Biochemistry Undergraduate Honors Theses

The complexity of the proteome requires strategies that efficiently characterize protein functions. Activity-Based Protein Profiling (ABPP) is a method that identifies enzyme activity by targeting key catalytic residues. This study focused on histidine, a residue involved in enzymatic catalysis, metal coordination, and protein-protein interactions. Despite its importance, histidine-specific chemical probes are limited due to its variable reactivity. This work developed and characterized a histidine-specific probe using a pyrocarbonate-based mechanism. Singleresidue assays confirmed selective reactivity of the probe with histidine over other nucleophilic amino acids. Further testing on enzymatic proteins and lysed cell proteomes showed that the probe modified histidine residues …

P23h Rhodopsin Accumulation Disrupts Synaptic Protein Levels In Rod Photoreceptors, Samantha Thompson

Graduate Theses, Dissertations, and Problem Reports (ETD)

Rod photoreceptors are specialized neurons located in the retina, the neural tissue that lines the back of the eye. Rod photoreceptors are necessary for dim light and peripheral vision through the photoactivation rhodopsin (Rho). Rho is synthesized in the inner segment of rods before being unidirectionally trafficked to the outer segment. In diseases such as retinitis pigmentosa (RP), Rho trafficking can be disrupted, leading to progressive rod cell death. In many cases of RP, Rho is mislocalized to the rod photoreceptor synapse (“spherule”). The impact of Rho mislocalization on spherule structure and protein organization is poorly understood. We hypothesize that …

Quantitative Proteomic Strategies To Determine Substrate Specificities Of Phosphoprotein Phosphatases, Hieu Trung Nguyen

Dartmouth College Ph.D Dissertations

Reversible phosphorylation is a crucial regulatory mechanism of cellular signaling pathways. Being the most prevalent post-translational modification (PTM) in the cells, with over 75% of all proteins detected to be phosphorylated, phosphorylation regulates a significant number of important cellular processes that have implications in various diseases. Phosphorylation is carried out by protein kinases, which have been extensively studied. However, the opposite reaction, carried out by protein phosphatases, has lagged significantly, exposing a gap of knowledge that is required to be investigated to delineate the kinase-substrate-phosphatase relationship. Phosphoprotein phosphatase family (PPPs), containing seven members of phospho-Serine (pS) and phospho-Threonine (pT) phosphatases, …

Differentially Disrupted Spinal Cord And Muscle Energy Metabolism In Spinal And Bulbar Muscular Atrophy, Danielle Debartolo, Frederick Arnold, Y Liu, Elana Molotsky, Hsin-Yao Tang, Diane Merry

Department of Biochemistry and Molecular Biology Faculty Papers

Prior studies showed that polyglutamine-expanded androgen receptor (AR) is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of nicotinamide adenine dinucleotide-dependent (NAD+-dependent) sirtuin 1 is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating postsymptomatic transgenic SBMA mice with the NAD+ precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of …

Elucidation Of Molecular Mechanisms Underlying Novel Multi-Targeted Therapy Strategies For, Qianqian Hu

USF Tampa Graduate Theses and Dissertations

EML4-ALK fusion non-small cell lung cancer (NSCLC) represents a distinct subset of lung cancers that possesses unique molecular and clinical features, paving the way for targeted therapeutic interventions. Despite the promise of ALK-directed therapies, challenges such as metastasis at the time of diagnosis and treatment resistance persist, necessitating deeper research into the underlying mechanisms and potential solutions. In the context of these challenges, our study embarked on two specific objectives. The first goal, elucidated in Chapter 2, revolved around brain metastasis, an event critically facilitated by cell migration. Here, we assessed the anti-migratory activities of several clinical ALK inhibitors in …

Pyrroline-5-Carboxylate Reductase-2 Promotes Colorectal Carcinogenesis By Modulating Microtubule-Associated Serine/Threonine Kinase-Like/Wnt/Β-Catenin Signaling, Raju Lama Tamang, Balawant Kumar, Sagar M. Patel, Ishwor Thapa, Alshomrani Ahmad, Vikas Kumar, Rizwan Ahmad, Donald F. Becker, Dundy Kiran Bastola, Punita Dhawan, Amar B. Singh

Department of Biochemistry: Faculty Publications

Background: Despite significant progress in clinical management, colorectal cancer (CRC) remains the third most common cause of cancer-related deaths. A positive association between PYCR2 (pyrroline-5-carboxylate reductase-2), a terminal enzyme of proline metabolism, and CRC aggressiveness was recently reported. However, how PYCR2 promotes colon carcinogenesis remains ill understood. Methods: A comprehensive analysis was performed using publicly available cancer databases and CRC patient cohorts. Proteomics and biochemical evaluations were performed along with genetic manipulations and in vivo tumor growth assays to gain a mechanistic understanding. Results: PYCR2 expression was significantly upregulated in CRC and associated with poor patient survival, specifically among PYCR …

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 …

Physiological And Proteomic Analysis Of Soybean In Response To Drought And Heat Stress, Amandeep Kaur

Theses and Dissertations

Drought and temperature stress are the two most important factors limiting crop productivity. A physiological and proteomic analysis of soybean was performed to investigate the negative impact of these variables. The previous studies reported the beneficial effect of silicon under stress conditions. Silicon supplementation may promote plant growth under drought stress, although the mechanism to alleviate drought stress is uncertain. The proteomic analysis of chloroplast proteins was carried out to study the response of silicon supplementation under drought stress.

Similarly, temperature stress, mainly heat stress, has an adverse effect on soybean plants. The comparative analysis of two soybean cultivars, DS25-1 …

Eluication Of Lipid Metabolic Pathways In Differentiating Giardia Lamblia Using High Resolution Mass Spectrometry, Cameron Ellis

Open Access Theses & Dissertations

Giardia lamblia is an intestinal protozoan found worldwide, including the U.S. This parasite exists in two morphologic stages - a replicative trophozoite and a relatively dormant yet viable cyst. While exposures of cysts to gastric acid during passage through the human stomach induces excystation, factors in the small intestine, where trophozoites colonize trigger encystation or cyst formation. Transformation into cyst stage is essential for Giardia to survive in the environment for months before infecting new hosts. Because of its small genome size (11.7 Mb), metabolic pathways in Giardia are highly reduced. As far as lipid metabolism is concerned, only limited …

Deciphering Phosphoprotein Phosphatase Signaling Networks Using Proteomics Approaches, Brooke Brauer

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a highly regulated mechanism of cell signaling control and its deregulation is implicated in disease. The kinases that catalyze the addition of phosphate groups onto their substrate proteins have been well studied, their signaling pathways mapped, and their effects on cell and organismal health observed. Knowledge of the phosphatases that reverse the reaction only recently began to come into focus. Phosphoprotein phosphatases (PPPs), long thought to be housekeeping enzymes, are now known to be exquisitely specific towards their substrates, but the exact nature of phosphatase regulation—both upstream and downstream of the phosphatase—is unclear.

PPPs recognize substrates through …

Extending Plasma Proteome Coverage To Enable Biomarker Discovery, Jordan Bruce Burton

Wayne State University Dissertations

Two features make plasma a valued source of health information: First, plasma contains secreted proteins from all tissues in the body and can, therefore, provide information about the health status of all tissues. Second, plasma is available in large quantity using a minimally invasive procedure that is well tolerated. The status of plasma as a valued source of health information is matched by the difficulty in extracting proteome information from the samples. Using state of the art instrumentation a standard analysis of plasma yields quantitative information on approximately 300 proteins. Extreme fractionation strategies coupled with increased mass spectrometry time extends …

Development Of High-Throughput Methods For Screening Venom Peptides, Tanya Napolitano

Dissertations, Theses, and Capstone Projects

Bioactive venom peptides are increasingly being explored as drug leads in pharmaceutical research because of their immense therapeutic potential, however there is a need to more efficiently screen and characterize these peptides to discover medicinal leads more quickly.1,2 The Holford lab has leveraged recent technological achievements in genomics, transcriptomics, and proteomics to produce a leap in the discovery of venom peptides from previously hard to characterize small and neglected venomous animals such as Terebridae, an understudied lineage of the Conoidea Superfamily.4, 5–7 With the rise of -omic technologies the floodgates have opened and venom researchers are awash with large datasets …

Sd2 Ptov1 Interactomics, Joshua Andersen

ScholarsArchive Data

PTOV1 interactome data

Sd1 14-3-3 Interactome For Ptov1 Manuscript, Joshua Andersen

ScholarsArchive Data

14-3-3 interactome

Sd1 14-3-3 Interactome For Ptov1 Manuscript, Joshua Andersen

ScholarsArchive Data

Excel data sheet of MS data from 14-3-3 interactomics

Validation Of A Deployable Proteomic Assay For The Serological Screening Of Sexual Assault Samples, Catherine O'Sullivan Brown

Electronic Theses and Dissertations

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing …

Intrinsic Buffer Hydroxyl Radical Dosimetry For Hydroxyl Radical Protein Footprinting, Addison Roush

Honors Theses

Hydroxyl radical protein footprinting (HRPF) coupled to mass spectrometry is a powerful technique for the analysis of protein topography as it generates covalent mass labels that can survive downstream sample handling, and it is sensitive to the solvent accessibility of amino acid sidechains. Of the multiple platforms for HRPF, fast photochemical oxidation of proteins (FPOP) utilizes a pulsed 248 nm KrF excimer laser to label proteins by photolyzing hydrogen peroxide. FPOP is the most widely used HRPF platform because it labels proteins faster than unfolding can occur. Variations in FPOP sample conditions make it difficult to compare results between experiments …

Dietary Fish Oil Supplement Induces Age-Specific Contractile And Proteomic Responses In Muscles Of Male Rats, David W. Russ, Kalina Dimova, Emily Morris, Marguerite Pacheco, Sean M. Garvey, Stylianos P. Scordilis

Biological Sciences: Faculty Publications

Background: Dietary fish oil (DFO) has been identified as a micronutrient supplement with the potential to improve musculoskeletal health in old age. Few data are available for effects of DFO on muscle contractility, despite the significant negative impact of muscle weakness on age-related health outcomes. Accordingly, the effects of a DFO intervention on the contractile function and proteomic profile of adult and aged in an animal model of aging were investigated.

Methods: This preliminary study evaluated 14 adult (8 months) and 12 aged (22 months) male, Sprague-Dawley rats consuming a DFO-supplemented diet or a control diet for 8 weeks (7 …

A Proteomic Analysis Of Corydoras Sterbai Secretions And Tissues, Erik Powell Wictor

University of the Pacific Theses and Dissertations

Defensive mechanisms vary widely in the animal kingdom ranging from physical defenses like spines to chemical defenses such as toxins. Toxins in these secretions and tissues can fluctuate from enzymes to lipids to uncharacterized chemicals. Next generation -omics technology and mass spectrometry are extremely important in analyzing these samples because of their ability to distinguish minute amounts of toxic substance within a complicated sample. The goal of this experiment was to look at secretions and tissues from Corydoras sterbai. All samples in this study were proteolyzed using a mixture of Trypsin and Lys-C, fractionated, and run through nanoLC-MS/MS analysis using …

An Exploration Of Protein And Dna Components In Fingerprint Residue, Ashley Borrego

Student Theses

The main focus of this project was to investigate the protein and DNA components in both sebaceous and eccrine fingerprints. This study investigated the relative content of DNA and proteins in eccrine fingerprints to sebaceous fingerprints. All volunteers were instructed to wash and dry their hands prior to depositing parallel thumbprints. Twenty volunteers were instructed to touch their face to produce sebaceous prints, and 5 volunteers were instructed to wear gloves over a heat source to produce sweaty or eccrine prints. Microscopy was used to score the cellular debris of the right fingerprint on a scale of 1-4 based on …

Composition Of The Survival Motor Neuron (Smn) Complex In Drosophila Melanogaster, A. Gregory Matera, Amanda C. Raimer, Casey A. Schmidt, Jo A. Kelly, Gaith N. Droby, David Baillat, Sara Ten Have, Angus I. Lamond, Eric J. Wagner, Kelsey M. Gray

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Spinal Muscular Atrophy (SMA) is caused by homozygous mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. SMN is part of an oligomeric complex with core binding partners, collectively called Gemins. Biochemical and cell biological studies demonstrate that certain Gemins are required for proper snRNP assembly and transport. However, the precise functions of most Gemins are unknown. To gain a deeper understanding of the SMN complex in the context of metazoan evolution, we investigated its composition in Drosophila …

Egfr Signaling From The Early Endosome., Julie A. Gosney

Electronic Theses and Dissertations

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is an integral component of proliferative signaling. When activated by a ligand at the plasma membrane, EGFR dimerizes with another ErbB family receptor, leading to kinase domain activation and transphosphorylation of C-terminus tyrosine residues. These phosphotyrosines act as crucial regulators of EGFR signaling as effector proteins dock to the receptor at these sites. The receptor undergoes clathrin-mediated endocytosis into early endosomes, where it can then be trafficked to a lysosome for degradation. However, the kinase domain of EGFR retains its activity during trafficking, suggesting that EGFR can continue …

Influence Of Dietary Ractopamine And Supranutritional Supplementation Of Vitamin E On Proteome Profile Of Postmortem Beef Longissimus Lumborum Muscle, Hyun Mok Kim

Theses and Dissertations--Animal and Food Sciences

The effects of dietary ingredients on the proteome profile of postmortem beef longissimus lumborum (LL) muscle were evaluated. In the first experiment, the influence of dietary ractopamine on the whole-muscle proteome of beef LL was examined. Five proteins were differentially abundant between ractopamine-fed (RAC) and non-ractopamine fed (CON) groups. The differentially abundant proteins were over-abundant in RAC and were related to muscle structure development (F-actin-capping protein subunit beta-2 and PDZ and LIM domain protein-3), chaperone (heat shock protein beta-1), oxygen transportation (myoglobin), and glycolysis (L-lactate dehydrogenase A chain). These findings indicated that ractopamine influences the abundance of proteins associated with …

Comparative Proteomic Analyses Of The Parietal Lobe From Rhesus Monkeys Fed A High-Fat/Sugar Diet With And Without Resveratrol Supplementation, Relative To A Healthy Diet: Insights Into The Roles Of Unhealthy Diets And Resveratrol On Function, Aaron M. Swomley, Judy C. Triplett, Jeriel T. Keeney, Govind Warrier, Kevin J. Pearson, Julie A. Mattison, Rafael De Cabo, Jian Cai, Jon B. Klein, D. Allan Butterfield

Chemistry Faculty Publications

A diet consisting of a high intake of saturated fat and refined sugars is characteristic of a Western-diet and has been shown to have a substantial negative effect on human health. Expression proteomics were used to investigate changes to the parietal lobe proteome of rhesus monkeys consuming either a high fat and sugar (HFS) diet, a HFS diet supplemented with resveratrol (HFS+RSV), or a healthy control diet for 2 years. Here we discuss the modifications in the levels of 12 specific proteins involved in various cellular systems including metabolism, neurotransmission, structural integrity, and general cellular signaling following a nutritional intervention. …

Metagomics: A Web-Based Tool For Peptide-Centric Functional And Taxonomic Analysis Of Metaproteomics Data, Michael Riffle, Damon H. May, Emma Timmins-Schiffman, Molly P. Mikan, Daniel Jaschob, William S. Noble, Brook L. Nunn

OES Faculty Publications

Metaproteomics is the characterization of all proteins being expressed by a community of organisms in a complex biological sample at a single point in time. Applications of metaproteomics range from the comparative analysis of environmental samples (such as ocean water and soil) to microbiome data from multicellular organisms (such as the human gut). Metaproteomics research is often focused on the quantitative functional makeup of the metaproteome and which organisms are making those proteins. That is: What are the functions of the currently expressed proteins? How much of the metaproteome is associated with those functions? And, which microorganisms are expressing the …

Quantitative Proteomic Investigation Of Disease Models Of Type 2 Diabetes, Mark Gabriel Athanason

USF Tampa Graduate Theses and Dissertations

PANcreatic DERived factor (PANDER, FAM3B) is a member of a superfamily of FAM3 proteins that are uniquely structured and strongly expressed from the endocrine pancreas and co-secreted with insulin. Unique animal models available to our lab have indicated that PANDER can induce a selective hepatic insulin resistant (SHIR) phenotype whereby insulin signaling is blunted yet lipogenesis is increased. The complexity of the biological networks involved with this process warranted the logical approach of employing quantitative mass spectrometry based proteomic analysis using stable isotope labeling of amino acids in cell culture (SILAC) to identify the global proteome differences between the PANDER …

Novel Interactome Of Saccharomyces Cerevisiae Myosin Type Ii Identified By A Modified Integrated Membrane Yeast Two-Hybrid (Imyth) Screen, Ednalise Santiago, Pearl Akamine, Jamie Snider, Victoria Wong, Matthew Jessulat, Viktor Deineko, Alla Gagarinova, Hiroyuki Aoki, Zoran Minic, Sadhna Phanse, Andrea San Antonio, Luis A Cubano, Brian C. Rymond, Mohan Babu, Igor Stagljar, Jose R. Rodriguez-Medina

Biology Faculty Publications

Nonmuscle myosin type II (Myo1p) is required for cytokinesis in the budding yeast Saccharomyces cerevisiae. Loss of Myo1p activity has been associated with growth abnormalities and enhanced sensitivity to osmotic stress, making it an appealing antifungal therapeutic target. The Myo1p tail-only domain was previously reported to have functional activity equivalent to the full-length Myo1p whereas the head-only domain did not. Since Myo1p tail-only constructs are biologically active, the tail domain must have additional functions beyond its previously described role in myosin dimerization or trimerization. The identification of new Myo1p-interacting proteins may shed light on the other …

Investigation Of Respiratory Syncytial Virus Structural Determinants And Exploitation Of The Host Ubiquitin System, Jillian Nicole Whelan

USF Tampa Graduate Theses and Dissertations

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal …