Biochemistry Commons^™

Kynurenine Aminotransferase Iii And Glutamine Transaminase L Are Identical Enzymes That Have Cysteine S-Conjugate Beta-Lyase Activity And Can Transaminate L-Selenomethionine, John T. Pinto, Boris F. Krasnikov, Steven Alcutt, Melanie E. Jones, Thambi Dorai, Arthur J L Cooper

NYMC Faculty Publications

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase …

Ribosomes Left In The Dust: Diverse Strategies For Peptide-Mediated Translation Stalling, Benjamin H. Hudson, Hani S. Zaher

Biology Faculty Research

In two recent papers, Arenz et al. (2014a) and Bischoff et al. (2014) provide structural insights into drug-induced, peptide-mediated stalling of the ribosome.

Cross-Disciplinary Sciences At Gettysburg College: Second Annual Poster Presentation, X-Sig

Student Publications

This booklet includes Biology student presentations by: Taylor Bury, Abigail Dworkin-Brodsky, Mary Pearce, Jasper Leavitt, Morgan Panzer, Ellen Petley, Kalli Qutub, Taylor Randell, Samantha Eck, Lana McDowell, Jenn Soroka, Celina Harris, Natalie Tanke, Alexandra Turano, and Caroline Garliss.

This booklet includes Biochemistry & Molecular Biology student presentations by: Matthew Dunworth, Andrew Sydenstricker, Brianne Tomko, Albert Vill, Warren Campbell, David Van Doren, Kevin Mrugalski, Stacey Heaver, Alecia Achimovich, and Katherine Boas.

This booklet includes Chemistry student presentations by: Kristen Baker, Laura Lee, Kathryn Fodale, Daniel Ruff, Michael Counihan, Ida DiMucci, Joshua Sgroi, Celina Harris, and Natalie Tanke.

This booklet include Health …

Dietary Carbohydrates Influence The Structure And Function Of The Intestinal Alpha-Glucosidases, Mohammad Chegeni

Open Access Dissertations

As the primary products of starch digestion by pancreatic α-amylase, maltooligosaccharides (including maltose) are the main substrates for the α-glucosidases at the intestinal brush border. Here, maltose was shown to induce the formation of a higher molecular weight (HMW) sucrase-isomaltase (SI) species in Caco-2 cells that sorts more quickly to the enterocyte surface to act as a digestive enzyme. As this finding suggested a maltose sensing ability of small intestinal enterocytes, molecular mechanisms associated with the maturation and trafficking of HMW SI were further investigated. A pulse-chase experiment using [ ³⁵S]-methionine revealed a higher rate of early trafficking and …

Effects Of Altered Expression Of The Sumo Conjugating Enzyme, Ubc9 On Mitosis, Meiosis And Conjugation In Tetrahymena Thermophila, Qianyi Yang

Open Access Dissertations

SUMOylation is a critical posttranslational modification in eukaryotic species. Ubc9p is the E2-conjugating enzyme for SUMOylation and consequently it influences multiple cellular pathways. Nuclear proteins are common targets of SUMOylation and regulate nuclear events such as transcription, DNA repair and mitosis. The segregation of the Tetrahymena thermophila genome into two different nuclear compartments provides an unusual context for the analysis of SUMOylation. Each cell contains a transcriptionally silent, diploid germ line micronucleus (MIC) that divides by mitosis and a polyploid transcriptionally active somatic macronucleus (MAC) that divides by an amitotic mechanism. With the long-term goal to exploit these opportunities we …

New Strategies To Reveal Protein Candidates In Protein-Protein Interactome Study, Meng-Chieh Chen

Open Access Theses

Comprehensive protein-protein interaction network analysis can help reveal protein functions in a system-wide manner. A reliable knowledgebase of interaction networks is not only important for selecting the candidates for drug therapies, but also for evaluating the disease risk. In current interaction databases, 322579 interactions comprised of 56460 proteins have been reported (statistical analysis from APID: Agile Protein Interaction DataAnalyzer; http://bioinfow.dep.usal.es/apid/index.htm). The huge datasets are contributed mainly by yeast -two -hybrid (Y2H) screening and affinity-purification followed by mass spectrometry (AP-MS). High false positive rates and failing to cover certain interaction categories are the limitations of these two methods. Here, we developed …

The Non-Canonical Hydroxylase Structure Of Yfcm Reveals A Metal Ion-Coordination Motif Required For Ef-P Hydroxylation, Kan Kobayashi, Assaf Katz, Andrei Rajkovic, Ryohei Ishii, Owen E. Branson, Michael A. Freitas, Ryuichiro Ishitani, Michael Ibba, Osamu Nureki

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

EF-P is a bacterial tRNA-mimic protein, which accelerates the ribosome-catalyzed polymerization of poly-prolines. In Escherichia coli, EF-P is post-translationally modified on a conserved lysine residue. The post-translational modification is performed in a two-step reaction involving the addition of a β-lysine moiety and the subsequent hydroxylation, catalyzed by PoxA and YfcM, respectively. The β-lysine moiety was previously shown to enhance the rate of poly-proline synthesis, but the role of the hydroxylation is poorly understood. We solved the crystal structure of YfcM and performed functional analyses to determine the hydroxylation mechanism. In addition, YfcM appears to be structurally distinct from any …

Eukaryotic Gene Expression Patterns Of Microorganisms In The Amazon River Plume Parallel The Biogeochemistry Of Plume Waters, Brian L. Zielinski

USF Tampa Graduate Theses and Dissertations

Unraveling the microbiological processes that occur as water travels from a river's mouth into the ocean is critical to understanding the role of river plumes in global biogeochemical cycles. Metranscriptomics, the gene expression of a whole community of organisms, was utilized to examine six stations along the Amazon River Plume (ARP) in 2010 to test thehypothesis that there were measurable differences in gene expression for key biogeochemical genes along the ARP. This body of work focuses on methods developed to identify which genes are biogeochemically important for a particular environment along extreme salinity, nutrient and community gradients in the ARP, …

Mistranslation Of The Genetic Code, Adil Moghal, Kyle Mohler, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

During mRNA decoding at the ribosome, deviations from stringent codon identity, or “mistranslation,” are generally deleterious and infrequent. Observations of organisms that decode some codons ambiguously, and the discovery of a compensatory increase in mistranslation frequency to combat environmental stress have changed the way we view “errors” in decoding. Modern tools for the study of the frequency and phenotypic effects of mistranslation can provide quantitative and sensitive measurements of decoding errors that were previously inaccessible. Mistranslation with non‐protein amino acids, in particular, is an enticing prospect for new drug therapies and the study of molecular evolution.

Relaxed Substrate Specificity Leads To Extensive Trna Mischarging By Streptococcus Pneumoniae Class I And Class Ii Aminoacyl-Trna Synthetases, Jennifer Shepherd, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases provide the first step in protein synthesis quality control by discriminating cognate from noncognate amino acid and tRNA substrates. While substrate specificity is enhanced in many instances by cis- and trans-editing pathways, it has been revealed that in organisms such as Streptococcus pneumoniae some aminoacyl-tRNA synthetases display significant tRNA mischarging activity. To investigate the extent of tRNA mischarging in this pathogen, the aminoacylation profiles of class I isoleucyl-tRNA synthetase (IleRS) and class II lysyl-tRNA synthetase (LysRS) were determined. Pneumococcal IleRS mischarged tRNA^Ile with both Val, as demonstrated in other bacteria, and Leu in a tRNA sequence-dependent …

Translation Initiation Rate Determines The Impact Of Ribosome Stalling On Bacterial Protein Synthesis, Steven J. Hersch, Sara Elgamal, Assaf Katz, Michael Ibba, William Wiley Navarre

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Ribosome stalling during translation can be caused by a number of characterized mechanisms. However, the impact of elongation stalls on protein levels is variable, and the reasons for this are often unclear. To investigate this relationship, we examined the bacterial translation elongation factor P (EF-P), which plays a critical role in rescuing ribosomes stalled at specific amino acid sequences including polyproline motifs. In previous proteomic analyses of both Salmonella and Escherichia coli efp mutants, it was evident that not all proteins containing a polyproline motif were dependent on EF-P for efficient expression in vivo . The α- and β-subunits of …

Thiosulfoxide (Sulfane) Sulfur: New Chemistry And New Regulatory Roles In Biology, John Toohey, Arthur J L Cooper

NYMC Faculty Publications

The understanding of sulfur bonding is undergoing change. Old theories on hypervalency of sulfur and the nature of the chalcogen-chalcogen bond are now questioned. At the same time, there is a rapidly expanding literature on the effects of sulfur in regulating biological systems. The two fields are inter-related because the new understanding of the thiosulfoxide bond helps to explain the newfound roles of sulfur in biology. This review examines the nature of thiosulfoxide (sulfane, S0) sulfur, the history of its regulatory role, its generation in biological systems, and its functions in cells. The functions include synthesis of cofactors (molybdenum cofactor, …

Ef-P Dependent Pauses Integrate Proximal And Distal Signals During Translation, Sara Elgamal, Assaf Katz, Steven J. Hersch, David Newsom, Peter White, William Wiley Navarre, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Elongation factor P (EF-P) is required for the efficient synthesis of proteins with stretches of consecutive prolines and other motifs that would otherwise lead to ribosome pausing. However, previous reports also demonstrated that levels of most diprolyl-containing proteins are not altered by the deletion of efp. To define the particular sequences that trigger ribosome stalling at diprolyl (PPX) motifs, we used ribosome profiling to monitor global ribosome occupancy in Escherichia coli strains lacking EF-P. Only 2.8% of PPX motifs caused significant ribosomal pausing in the Δefp strain, with up to a 45-fold increase in ribosome density observed at …

Poly(Arginine) Derived Cancer-Targeting Peptides For The Development Of A Cancer-Targeted Gene Therapy Approach In Hepg2 Liver Cancer Cells, Stesha C. Joseph

Seton Hall University Dissertations and Theses (ETDs)

Cancer is a disease that has eluded medicinal approaches for many years and as a result new and improved therapeutic approaches are in constant demand. Although chemotherapy and radiation treatments have assisted in suppressing the growth of tumors, their poor selectivity and efficacy are major limitations for effective therapy en route towards the development of a cure for the cancer epidemic. With the mission of conquering cancer at heart, researchers have pursued a new form of cancer therapy, aptly named, a cancer targeting approach. This method revolves around the selection of a suitable biomarker, typically a cell surface receptor …

Determining The Binding Between Saga Subunits And Spliceosomal Components, Peyton J. Spreacker, Rachel L. Stegeman, Vikki M. Weake

The Summer Undergraduate Research Fellowship (SURF) Symposium

Proper gene regulation is vital to the health and development of an organism. Determining the relationship between splicing, transcription, and chromatin structure is vital for understanding gene regulation as a whole. There have been previous studies linking these elements pairwise; however, no evidence exists for a direct link between all three. Recent data shows that splicing components of the U2 small nuclear ribonucleic protein (snRNP) co-purify with Spt-Ada-Gcn5-acetyltransferase (SAGA), a highly conserved transcriptional co-activator and chromatin modifier. We hypothesize that SAGA binds with splicing components through a multi-protein binding surface with certain core components based on preliminary yeast two-hybrid data. …

Exploration Into The Cellular Effects Of Aziridinomitosenes, Christopher M. Mallory

Boise State University Theses and Dissertations

Aziridinomitosenes (AZMs) are organic compounds structurally related to the mitomycins, a class of anti-tumor agents and antibiotics. The cytotoxicity of the mitomycins is correlated to their ability to covalently link complimentary strands of DNA, forming DNA interstrand cross-links (ICLs). Currently, there has been limited investigation into the biological activity of AZMs, likely due to difficulties in their synthesis. Our lab has synthesized and evaluated the cellular effects of two AZMs, (1S, 2S)-6-desmethyl(methylaziridino)mitosene (H/H-AZM) and (1S, 2S)-6-methyl(methylaziridino)mitosene (Me/H-AZM). We hypothesize that AZMs exhibit their cytotoxicity and cellular effects following a similar pathway to that of mitomycin C (MC) including …

Biophysical And Computational Investigations Into G-Quadruplex Structural Polymorphism And Interaction With Small Molecules., Huy Tuan Le

Electronic Theses and Dissertations

In the cell, guanine-rich nucleic acids can self-assemble into unique four stranded tertiary structures known as G-quadruplexes. G-quadruplex formation in the telomere leads inhibits telomerase, an enzyme activated in cancer cells to maintain the telomere and allowing for cancer cells to achieve immortality. G-quadruplex formation in the promoters and 5’-untranslated regions regulates the expression of many oncogenes. Furthermore, G-quadruplex formation during cellular replication promotes genomic instability, a characteristic which enables tumor development. Because of their implication in cancer, G-quadruplex structures have emerged as attractive drug targets for anti-tumor therapeutics. In the current dissertation work, we present three experimental approaches to …

Genetic And Mechanistic Analysis Of Rat Mammary Cancer Susceptibility., Jennifer Sanders

Electronic Theses and Dissertations

Breast cancer is a complex disease, which is influenced by genetic, epigenetic and environmental components. Genetic susceptibility to breast cancer is made up of high, moderate and low penetrance alleles. High and moderate penetrance alleles are rare and constitute only a small percentage of the genetic susceptibility. Most variation in genetic susceptibility is controlled by low- penetrance, common polymorphisms. Comparative genetics uses model organisms to study human disease. Rat strains exhibit different susceptibility phenotypes to chemical induced carcinogenesis. The Wistar-Furth (WF) rat strain is susceptible to chemically induced mammary carcinogenesis, while the Wistar-Kyoto (WKy) and Copenhagen (COP) rat strains are …

Characterization Of A Putative Sir2 Like Deacetylase And Its Role In Sabp2 Dependent Salicylic Acid Mediated Pathways In Plant, Md I. Haq

Electronic Theses and Dissertations

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant …

Key Residues Of Human Cytoplasmic Protein Tyrosine Phosphatase-A And -B For Substrate Binding And Specificity, Byunghyun Park

Open Access Theses

Reversible tyrosine phosphorylation plays an important role in signaling pathways that are essential for regulating cellular growth, differentiation and metabolism. Moreover, several human diseases such as diabetes, obesity and cancers are associated with the deregulation of protein tyrosine phosphatases (PTPs). Several studies provide evidence that PTPs not only contribute to cellular differentiation, but over-expression of these molecules also leads to transformation of non-transfomed cells as well. Based on these results, designing specific PTP inhibitors may ultimately function as potential therapeutic agents to treat various diseases including cancer, diabetes, and autoimmune diseases. EphA2 is a receptor tyrosine kinase which is hypo-phosphorylated …

1,25-Dihydroxyvitamin D Alters Lipid Metabolism And Epithelialto- Mesenchymal Transition In Metastatic Epithelial Breast Cancer Cells, Alle Nicole Barnard

Open Access Theses

Evidence suggests that high vitamin D status (marked by serum 25-hydroxyvitamin D, 25(OH)2 D) is associated with a decreased risk of breast cancer. It has been established that 1,25-dihydroxyvitamin D (1,25(OH) 2 D) can alter glycolysis and the Krebs cycle of breast cancer cells (Jiang et al., 2010; Zheng et al., 2013) but little information is available on 1,25(OH) 2 D's alterations of lipid metabolism in breast cancer cells. Thus, the current research investigates if there was an effect of 1,25(OH) 2 D on proteins that regulate lipid metabolism in MCF10A, MCF10A-ras, MCF10CA1h, and MCF10CA1a epithelial breast cancer cells. While …

Developing Crosslinking Constructs Of Protein Kinase R, Prisma E. Lopez

Honors Scholar Theses

Protein Kinase R (PKR) is a key component of the innate immune antiviral response. PKR is activated upon binding to dsRNA. However, recent studies have shown that PKR can also bind to and become activated by duplex RNAs containing complex secondary structure. The mechanism of PKR binding and activation by these RNAs is currently not known. The approach taken here to determine the mechanism of PKR binding by these RNAs is through the development of PKR constructs that are capable of covalently binding to RNAs. Constructs were created by site-specific incorporation of an unnatural, photoactivatable amino acid within PKR. These …

Trnas As Regulators Of Biological Processes, Medha Raina, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Transfer RNAs (tRNA) are best known for their role as adaptors during translation of the genetic code. Beyond their canonical role during protein biosynthesis, tRNAs also perform additional functions in both prokaryotes and eukaryotes for example in regulating gene expression. Aminoacylated tRNAs have also been implicated as substrates for non-ribosomal peptide bond formation, post-translational protein labeling, modification of phospholipids in the cell membrane, and antibiotic biosyntheses. Most recently tRNA fragments, or tRFs, have also been recognized to play regulatory roles. Here, we examine in more detail some of the new functions emerging for tRNA in a variety of cellular processes …

Recruitment Of The Ribosomal 40s Subunit To The 3'Untranslated Region Of A Viral Mrna, Via The Eif4 Complex, Facilitates Cap-Independent Translation, Sohani Das Sharma

Dissertations, Theses, and Capstone Projects

Translation of uncapped plant viral RNAs can be facilitated by either an internal ribosomal entry site (IRES) in the 5' untranslated region (UTR) or a cap-independent translation element (CITE) in the 3' UTR. Barley yellow dwarf virus (BYDV) mRNA, which lacks both cap and poly(A) tail, has a translation element (3'BTE) in its 3' UTR that is essential for efficient translation initiation at the 5'-proximal AUG. This mechanism requires binding of the eukaryotic initiation factor 4G (eIF4G) subunit of the heterodimer eIF4F to the 3'BTE and base pairing between the 3'BTE and the 5' UTR. Here we investigate how this …

Oxidation Of Cellular Amino Acid Pools Leads To Cytotoxic Mistranslation Of The Genetic Code, Tammy J. Bullwinkle, Noah M. Reynolds, Medha Raina, Adil Moghal, Eleftheria Matsa, Andrei Rajkovic, Huseyin Kayadibi, Farbod Fazlollahi, Christopher Ryan, Nathaniel Howitz, Kym F. Faull, Beth A. Lazazzera, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Aminoacyl-tRNA synthetases use a variety of mechanisms to ensure fidelity of the genetic code and ultimately select the correct amino acids to be used in protein synthesis. The physiological necessity of these quality control mechanisms in different environments remains unclear, as the cost vs benefit of accurate protein synthesis is difficult to predict. We show that in Escherichia coli, a non-coded amino acid produced through oxidative damage is a significant threat to the accuracy of protein synthesis and must be cleared by phenylalanine-tRNA synthetase in order to prevent cellular toxicity caused by mis-synthesized proteins. These findings demonstrate how stress …

Reduced Amino Acid Specificity Of Mammalian Tyrosyl-Trna Synthetase Is Associated With Elevated Mistranslation Of Tyr Codons, Medha Raina, Adil Moghal, Amanda Kano, Mathew Jerums, Paul D. Schnier, Shun Luo, Rohini Deshpande, Pavel D. Bondarenko, Henry Lin, Michael Ibba

Biology, Chemistry, and Environmental Sciences Faculty Articles and Research

Quality control operates at different steps in translation to limit errors to approximately one mistranslated codon per 10,000 codons during mRNA-directed protein synthesis. Recent studies have suggested that error rates may actually vary considerably during translation under different growth conditions. Here we examined the misincorporation of Phe at Tyr codons during synthesis of a recombinant antibody produced in tyrosine-limited Chinese hamster ovary (CHO) cells. Tyr to Phe replacements were previously found to occur throughout the antibody at a rate of up to 0.7% irrespective of the identity or context of the Tyr codon translated. Despite this comparatively high mistranslation rate, …

2-Acylamido Analogues Of N-Acetylglucosamine Prime Formation Of Chitin Oligosaccharides By Yeast Chitin Synthase 2, Jacob Gyore, Archana Parameswar, Carleigh Hebbard, Younghoon Oh, Erfei Bi, Alexei Demchenko, Neil Price, Peter Orlean

Chemistry & Biochemistry Faculty Works

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc2) was the major reaction product. …

Mutagenesis Of 8-Oxoguanine Adjacent To An Abasic Site In Escherichia Coli Cells Proficient Or Deficient In Dna Polymerase Iv, Savas T. Tsikis

Honors Scholar Theses

It is well established that clustered DNA damages or multiply damaged sites (MDS) are the result of ionizing radiation and that they are characterized by an enhanced mutagenic potential. As a model MDS, we have evaluated the mutagenic and cytotoxic properties of the ubiquitous oxidative DNA damage 8-oxoguanine (G^8-oxo) adjacent to the abasic site lesion (Z) using a single stranded M13mp7L2 vector. The recombinant DNA was used to transform wild type E. coli strains and strains deficient in the translesion DNA polymerase of the Y-family, DNA polymerase IV, in the presence or absence of SOS induction. The percent …

Defining The Sites Of Interaction Of The Fancd2, Fance, And Fancl Proteins, Joseph Mcclanaghan

Senior Honors Projects

Fanconi anemia (FA) is a rare genetic disease characterized by congenital defects, bone marrow failure and increased cancer susceptibility. FA is caused by mutations in any one of 16 genes. These genes encode for proteins that function in the FA-BRCA pathway to repair damaged DNA. Because of its important r­­­ole in DNA repair, this pathway is considered a major cellular tumor suppressor pathway, i.e. is critical for the prevention of cancer. Underscoring this fact, several of the FA genes - including BRCA2, BRIP1, PALB2, and RAD51C - are bona fide breast and ovarian cancer susceptibility genes.

My …

Molecular Chaperone Tools For Use Against Neurodegenerative Diseases, Matthew Tinkham

Senior Honors Projects

A noted characteristic found in several neurodegenerative disorders, including Alzheimer’s Disease, Parkinson’s Disease, Huntington’s Disease and bovine spongiform encephalopathy, is the accumulation of amyloid plaques in the brain. Amyloid plaques contain deposits of fibrillar aggregates of misfolded proteins that disrupt normal functionality in neurons. Certain variants of these misfolded proteins are self-replicating; these self-replicating amyloids are termed prions (for infectious protein). We are interested in how protein misfolding contributes to amyloid formation and how molecular chaperone proteins can change the formation of amyloid deposits. Chaperone proteins function by catalyzing the proper folding of other proteins, the refolding of misfolded proteins, …