bluuebunny/crossref_metadata_2025_split huggingface dataset:
Investigating the role of 53BP1 in DNA repair (554.1)
Authors: Flores Desirey , Grosesser Torsten , Hatherill J. Robert 
Published: June 2021

Hypothesis: 53BP1 foci will return to baseline 24 hrs after radiation exposure, but it will take the BALB/c longer due to their increased radiosensitivity. P53 binding protein No.1 (53BP1) has been shown to accumulate in nuclear areas of a cell that have extensive DNA damage such as double strand breaks (DSB). DSBs are the worse type of DNA damage and cells that have been exposed to damaging agents such as ionizing radiation are susceptible to developing cancer. Micronuclei (MN) were scored as markers of induced DNA damage, which appear as extra nuclear bodies that are formed from un‐ or mis‐rejoined DSBs. In the present studies cells from BALB/c and SPRET/EiJ mice were used to investigate the effects of acute X‐rays and prolonged 32P exposures. 53BP1 foci were imaged after acute exposures and MN were scored after prolonged exposures. BALB/c and SPRET/EiJ cells were challenged with an X‐ray dose of 1 Gy and immunostained to evaluate the number of foci. Both cell lines were also exposed to 32P over a prolonged amount of time to receive a total dose of 0.4 Gy and were then scored for MN. After the acute exposures the number of foci was higher in the cells derived from BALB/c mice compared to SPRET/EiJ cells and after 24 hrs the amount of foci returned to baseline in both cells lines. We conclude that cells from BALB/c mice exposed to radiation sustained more DNA damage than the SPRET/EiJ cell line and 53BP1 foci are localized to areas of DNA damage.Grant Funding Source: NSF ATE DUE 1205059
Posttranslational arginylation is essential for stress response (555.15)
Authors: Kumar Akhilesh , Zhang Fangliang 
Published: June 2021

Arginylation consists of addition of an extra arginine to an existing peptide chain, thus changing the de facto primary sequence as well as the surface change. This process is evolutionarily conserved and mainly mediated by arginyltransferase1 (Ate1). While earlier studies found that the overall arginylation activity often increases during stressing conditions including heat shock, oxidative stress, and injury, little is known about the role of arginylation or Ate1 in stress response. By using multiple types of eukaryotic cells as test models, we found that Ate1 is a pro‐apoptotic factor. Knockout of Ate1 in cells leads to a retardation of stress response and the diminish of apoptosis and cell death during exposure of high doses of various stressing factors including heat shock, oxidative and osmotic stress, and heavy metal. Oppositely, over‐expression of Ate1 leads to cell death. Intriguingly, we also found that the activity of arginylation is essential for the wellbeing of the cells during mitochondria‐dependent metabolism as well as nutrient‐deprivation. Our data suggests that arginylation is a global regulator for metabolism and stress response, and provides an explanation for the observed role of Ate1 in carcinogenesis and injury.Grant Funding Source: NIGMS
Alterations to O‐GlcNAc cycling disrupt mitotic phosphorylation (555.16)
Authors: Whelan Stephen , Tan Ee Phie , Costello Catherine , McComb Mark , Slawson Chad 
Published: June 2021

O‐GlcNAc is a post‐translational modification (PTM) consisting of a single N‐acetylglucosamine moiety attached to serine and threonine residues in nuclear and cytoplasmic proteins. O‐GlcNAc levels can cycle dynamically in response to hormones, nutrients, or stress in order to regulate a multitude of cellular processes. For instance O‐GlcNAc cycling is an important regulator of mitosis. Recently, we demonstrated that cells with a gain of function in either O‐GlcNAc transferase (OGT) or O‐GlcNAcase (OGA) have disrupted mitotic spindles and mitotic exit defects. OGT/OGA gain of function caused the formation of large poorly condensed spindle chromosomes, misaligned kinetochores, and an increase in multipolar spindles. This phenotype was caused partially by a disruption in phosphorylation. In this current study, our objective is to determine how alterations in O‐GlcNAc cycling disrupt mitotic phosphorylation cascades. We employed quantitative proteomics in order to identify changes to mitotic phosphorylation. We were able to quantitate both protein expression changes as well as alterations in PTM’s in OGT or OGA gain of function cells. We identified over 1000 proteins whose expression as well as PTM status was altered by aberrant O‐GlcNAcylation. These results demonstrate the cross‐talk between protein modifications involved in regulating mitotic progression and suggest that diseases that disrupt one PTM will also alter the actions of other PTM’s.Grant Funding Source: NIH‐NHLBI Contracts: HHSN268201000031C ‐ CPC; NIH Grants: P41 RR010888/GM104603 – MSR R21 HL107993 ;
Novel protein engineering of lipase by protein folding memory (567.1)
Authors: Satomura Atsushi , Kuroda Kouichi , Ueda Mitsuyoshi 
Published: June 2021

Some hydrolytic enzymes including lipases and proteases have propeptides in their N‐ or C‐ terminals. Such propeptides seem to function as an intramolecular chaperone to facilitate the folding of their active domain and are cleaved off after completion of the function as chaperone. By introducing mutations into propeptides of lipases and altering the chaperone functions, we have successfully created altered lipases which showed improved activities and stabilities, in spite of the same primary sequences as the original lipase. We named this phenomenon “protein folding memory”, because mutations in the propeptide imprinted memories into the mature enzyme structures [1]. Our study aimed to alter a substrate recognition of Rhizopus oryzae lipase (ROL) by protein folding memory. Eight hydrophilic amino‐acids were introduced into a part of the propeptide essential for the folding of ROL. This propeptide‐mutated ROL showed a 1.3‐fold higher lipase activity than that of WT ROL. In addition, the propeptide‐mutated ROL remarkably exhibited a peptidase activity, which was absent in the original form [2]. Here, we report the structural and functional differences between WT ROL and propeptide‐altered ROL with the same primary sequence.[1] M. Nagayama, et al., Biochemistry, 51, 3547‐3553 (2012) [2] A. Satomura, et al., submitted
Analysis of co‐chaperone interactions with Hsp90 (567.7)
Authors: Wolmarans Annemarie , LaPointe Paul 
Published: June 2021

Heat shock protein 90 (Hsp90) is a highly abundant, ATP‐dependent molecular chaperone that is present in all eukaryotic cells. It is essential for the maturation and regulation of a specific set of substrate (client) proteins, many of which are key players in oncogenesis. Hsp90, as a homodimer, acts in the context of a complicated ATPase cycle. Co‐chaperone proteins guide and modulate Hsp90 through its functional cycle in order to mature clients, by binding to one or more of Hsp90’s domains in a sequential manner. To address Hsp90’s conformational requirements for cycle progression, we analyze two specific co‐chaperones, Aha1 and Sba1, which bind to Hsp90 in its closed, ATP‐bound state. We have constructed 14 Hsp90 variants harboring mutations in the N terminus of Hsp90 which have the potential to block the binding of Aha1 and Sba1. Several N‐terminal mutants show promising results as they are weakly stimulated by Aha1 or cannot be inhibited by Sba1 from the Aha1‐stimulated rate. Interestingly, one specific mutant harboring the D132K mutation does not interact with Sba1. Analysis of these five mutants in combination with previously characterized Hsp90 mutants will allow for the restriction of either Sba1 or Aha1 to one monomer of Hsp90 or the other. This subunit complementation strategy will bring insight into how Aha1 and Sba1 act asymmetrically on Hsp90.Grant Funding Source: Supported by CIHR
Structure, function and evolution of the mitochondrial replicative DNA helicase (570.1)
Authors: Kaguni Laurie , Stiban Johnny , Farnum Gregory , Hovde Stacy , So Minyoung 
Published: June 2021

Mitochondrial DNA (mtDNA) helicase is an essential part of the mtDNA replisome. The mtDNA helicase shows high amino acid sequence homology with bacteriophage T7 gene 4 protein (T7gp4), which has both DNA primase and DNA helicase activities in its N‐terminal domain (NTD) and C‐terminal domain (CTD), respectively. Although the NTD of mtDNA helicase in metazoa does not have detectable primase activity, retention of extensive amino acid sequence homology throughout most eukaryotic lineages suggests other important roles, such as in protein stability and/ or protein‐protein interactions. Here, we investigate the structural and biochemical properties of the NTD of Drosophila mtDNA helicase by constructing two N‐terminal truncated variants: a full‐length NTD (including both the zinc binding domain (ZBD) and the RNA polymerase domain (RPD)), and a ZBD only construct. We show that NTD and ZBD constructs of Drosophila mtDNA helicase contain an iron‐sulfur cluster, most likely a 2Fe‐2S, as determined by mass spectrometry, and UV‐visible and atomic absorption spectroscopy. Mutagenesis studies show that the four conserved cysteine residues in the ZBD responsible for binding zinc in T7gp4, bind the iron‐sulfur cluster. We extend our biochemical studies in vivo through several approaches to identify partner proteins of the Drosophila mtDNA helicase that may elucidate the functional role of its iron‐sulfur cluster.Grant Funding Source: NIH GM45295
Structure‐function relationships in the mitochondrial single‐stranded DNA‐binding protein (573.2)
Authors: Neitzke Orrin , Kaguni Laurie 
Published: June 2021

Mitochondrial single‐stranded DNA‐binding protein (mtSSB) plays a key role at the mitochondrial replication fork. Previous studies examining variant human mtSSBs showed a significant decrease in their ability to stimulate the human mitochondrial replicase, DNA polymerase γ (pol γ) in vitro, suggesting that specific structural features are involved in direct interactions with pol γ (Oliveira, M. O., Kaguni, L. S. (2011) J. Biol. Chem. 286, 40649‐40658). We are currently studying the effects of the corresponding variants of the Drosophila melanogaster mtSSB (Dm mtSSB) in coordinating DNA polymerase γ function and by doing so, we hope to understand the biochemical properties of this highly conserved protein that allow for differential stimulation of the human and fly replicases, which themselves differ in oligomeric structure, and nearly ten fold in the level of stimulation by their cognate mtSSBs. We have generated three variants of Dm mtSSB that introduce changes in amino acid residues located on the protein surface, and that may participate in both physical and functional interactions with pol γ. We have examined their ability to bind ssDNA and to stimulate DNA polymerase activity. We expect to draw a structure‐based biochemical comparison between the interactions of Dm mtSSB and Hs mtSSB with their respective pol γ holoenzymes.Grant Funding Source: NIH GM45295
Modes of mitochondrial DNA replication and its regulation in Drosophila melanogaster (573.3)
Authors: Lillsunde Paivi , Oliveira Marcos , Griffith Jack , Jacobs Howard , Kaguni Laurie S. 
Published: June 2021

Although replication of the mitochondrial DNA genome has been studied extensively in mammalian and yeast models, the mechanisms of mitochondrial DNA (mtDNA) replication in the widely‐used model organism Drosophila melanogaster remain largely unknown. We employ a comparative approach to analyze mtDNA replication intermediates using two‐dimensional neutral agarose gel electrophoresis (2DNAGE), and to visualize their architecture by transmission electron microscopy (TEM). Our studies target the nuclear‐encoded replisome proteins (DNA polymerase γ, mtDNA helicase and single‐stranded DNA‐binding protein), as well as transcription factors (mTTF and mTerf5), and their roles in the regulation of mtDNA replication through overexpression of both wild type proteins and biochemical variants in cultured insect cells and fly strains. Whereas 2DNAGE allows the analysis of minimally manipulated DNA preparations, TEM provides molecular details regarding the length of Okazaki fragments and on the presence of double‐stranded DNA and/ or RNA loops at the replication fork. In addition to the evaluation of the modes of replication in normal and perturbed cells, biotin labeling of the replisome proteins enables the determination of their positions on the replicating mtDNA molecule. Together, these methods can be used to identify novel structures and proteins involved in the progression of the replication process.Grant Funding Source: Supported by Academy of Finland and NIH GM45295
Elucidating the role of cyclin‐dependent kinase 5 in insulin exocytosis: studies toward understanding a possible link between neurodegeneration and type II diabetes (576.4)
Authors: Trye Alice , Aguanno Ann 
Published: June 2021

Cyclin dependent kinase 5 (CDK5) is an atypical member of the cyclin dependent kinase family, as it does not play a role in regulation of the cell cycle. Our lab studies CDK5 in the development of mammalian tissues, and has previously shown that chemical inhibition of CDK5 results in an aberrant neuronal phenotype in nervous system cell models. CDK5’s role in the development and functioning of the nervous system is now well established. Recent reports suggest CDK5 plays a role in non‐neuronal tissues. This prompted us to investigate this enzyme in the pancreas; specifically, whether CDK5 is a positive or negative regulator of insulin exocytosis, since published reports are contradictory. We use the acinar cell line AR42J as a model for the pancreas and insulin‐producing cells and have attempted to verify insulin secretion from transdifferentiated AR42J cells using a novel protein assay. Our method, however, proved problematic. Here, we describe a revised method of detecting secreted insulin and the results of chemical inhibition of CDK5 on insulin secretion. Our findings address the effectiveness of this modified method and begin to uncover the role of CDK5 in insulin exocytosis. These results further suggest that this “neuronal” protein is also important in pancreatic development. We propose, therefore, that CDK5 may be a potential link between two correlated diseases, Alzheimer’s Disease (AD) and Type II diabetes.Grant Funding Source: ASBMB UAN Undergraduate Summer Research Award
In vitro testing of putative Escherichia coli MurA inhibitors (582.1)
Authors: Frost Andrea , Boone Benjamin , Song Vanessa , Muzyka Jennifer , Haile January 
Published: June 2021

Antibiotic resistance necessitates the continuous development of novel inhibitors targeting a variety of bacterial pathways. The pathway for the synthesis of peptidoglycan, a necessary component of the bacterial cell wall, is an ideal target for antibiotic development for two reasons: 1) the cell wall is a prokaryote‐specific structure, and 2) the cell wall is necessary for bacterial survival. MurA, the target of current study, is the enzyme that catalyzes the first step in peptidoglycan biosynthesis. Our objective is to develop novel inhibitors that competitively bind to the MurA catalytic sight for UDP‐N‐acetylglucosamine. In an effort to study the effects of various putative inhibitors on MurA, the active enzyme was purified in high concentrations. In vitro tests of the known inhibitor fosfomycin and other potential inhibitors were conducted using the purified enzyme. The Malachite green assay as previously described was used to determine the IC50 values for a number of potential inhibitors. Of the compounds assayed, two salts, VS5 and BCB33b, exhibited consistent IC50 values of 160 uM and 300 uM respectively. The IC50 values of the two putative inhibitors are the lowest we have seen out of all the tested compounds; therefore, VS5 and BCB33b will be tested further in vitro, in vivo, and in silico in their salt and acid forms for comparison against the known inhibitor fosfomycin.Grant Funding Source: Faculty Development Committee, Centre College
Cloning of a 1‐pyrroline‐5‐carboxylate dehydrogenase from Taiwanofungus camphorata (583.1)
Authors: Wen Lisa , Lai Chin‐Chun , Huang Jenq‐Kuen , Lin Chi‐Tsai 
Published: June 2021

A cDNA (1890 bp) encoding a putative 1‐pyrroline‐5‐carboxylate dehydrogenase (P5CD) was cloned from Taiwanofungus camphorata. The DNA sequence encodes a protein of 547 amino acid residues with calculated molecular mass of 59.4 kDa. The deduced amino acid sequence is conserved among the reported P5CDs. To characterize the T. camphorata P5CD, the coding region was subcloned into an expression vector pET‐20b(+) and transformed into E. coli Rosetta (DE3). The recombinant His6‐tagged TcP5CD was expressed and purified by Ni2+‐nitrilotriacetic acid Sepharose. The purified enzyme showed a major band of ~59.4 kDa by 12 % SDS‐PAGE. The Michaelis constant (KM) value for 3,5‐dimethoxybenzaldehyde with NAD+ as cofactor was 0.56 mM. The enzyme’s half‐life of deactivation at 45oC was 4.4 min, and its thermal inactivation rate constant kd was 1.23 x 10‐2 min‐1. The enzyme was most active at pH 7.0. The enzyme’s preferred substrate is veratraldehyde. It can also use other aldehyde as substrates including acetaldehyde and propionaldehyde.Grant Funding Source: National Science Council of the Republic of China under grant NSC95‐2313‐B‐019‐007 to C‐T. L.
Hemoglobin binding and catalytic heme extraction by the IsdB receptor of Staphylococcus aureus (583.7)
Authors: Bowden Catherine , Verstraete Meghan , Eltis Lindsay , Murphy Michael 
Published: June 2021

The Isd (Iron‐regulated surface determinant) system is a multi‐protein transporter that enables Staphylococcus aureus to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall‐anchored surface protein that contains 2 NEAT domains and rapidly extracts heme from Hb. Using a series of recombinant IsdB constructs which included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (KD = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB only extracted heme from oxidized metHb although it also bound oxyHb and the Hb‐CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed heme transfer from metHb to IsdA with a Km for metHb of 0.75 ± 0.07 μN and a kcat of 0.22 ± 0.01 s‐1. The latter is consistent with the transfer of heme from metHb to IsdB as being the rate‐limiting step.Grant Funding Source: Supported by CIHR grant MOP‐49597
Investigating structural determinants of maleamate amidohydrolase (NicF) substrate specificity (584.3)
Authors: Kondow Helena , Snider Mark 
Published: June 2021

NicF catalyzes the hydrolysis of maleamate to maleate in the degradation of nicotinate to fumarate. Kinetic assays, using 1H NMR and UV spectroscopy, were developed to test substrate analogs for reactivity with NicF. Although (Z)‐methyl 4‐amino‐4‐oxobut‐2‐enoate, E‐maleamate, and succinamate showed evidence to be slow substrates, the first was shown to be hydrolyzed to maleamate and the second was potentially isomerized to maleamate. To quantify the thermodynamic basis of succinamate as a slow substrate, temperature dependence of the NicF‐catalyzed reaction with maleamate, and with succinamate, were determined. An observed difference of ΔΔH‡ = 0.6 kcal/mol and TΔΔS‡ = ‐3.1 kcal/mol at 25°C in the reaction of succinamate compared to that of maleamate indicates that the presence of a double bond in the natural substrate appears to play a larger role in productive substrate binding than in potential electronic effects to enhance the electrophilicity of the substrate. Three NicF variants (F34A, F83A, and T146A) were engineered to further test hypotheses concerning the substrate specificity and chemical mechanism of the NicF‐catalyzed reaction.Grant Funding Source: Supported by a HHMI Undergraduate Science Education Program Award at the College of Wooster
Evaluation of new cathepsin K inhibitors (585.4)
Authors: Ellison Amanda , Wen Lisa , Huang Jenq‐Kuen , McConnell Rose 
Published: June 2021

Cathepsin K is a lysosomal cysteine protease that is predominantly expressed in osteoclasts. It has also been found in elevated levels in people with rheumatoid arthritis, prostate cancer, and breast cancer. Due to its strong collagenase activity, cathepsin K has been described as a major enzyme responsible for the turnover of extracellular matrix proteins and plays a fundamental role in bone resorption. Cathepsin K is a novel drug target for osteoporosis, osteoarthritis, and bone metastasis. Here, we report screening and evaluation of five new synthetic inhibitors against recombinant cathepsin K. The recombinant human procathepsin K was overexpressed in E. coli, purified from inclusion bodies, and activated by pepsin treatment. Cathepsin K inhibition assay was performed spectrophotometrically with N‐Carbobenzoxy‐L‐Phenylalanyl‐Arginine‐4‐nitroanilide hydrochloride as the chromogenic substrate. Data analysis of the effectiveness of these inhibitors will be used to foster future development of newer effective inhibitors for the treatment osteoporosis and possibly rheumatoid arthritis and cancer.Grant Funding Source: National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1) and WIU
Structural and biological characterization of NOS inhibitors with antimicrobial properties (585.5)
Authors: Holden Jeffrey , Li Huiying , Richo Jerry , Lim Nathan , Jing Qing , Kang Soosung , Huang He , Silverman Richard , Poulos Thomas 
Published: June 2021

Nitric oxide produced by bacterial nitric oxide synthase (bNOS) protects Gram‐positive pathogens Bacillus anthracis and Staphylococcus aureus against oxidative stress and a wide variety of antibiotics. Recently, we demonstrated that co‐treatment with NOS inhibitors increases the killing efficacy of a common antimicrobial(1). Taking an interdisciplinary approach using microbial in vitro assays, spectroscopic binding analyses, enzyme kinetics and X‐ray crystal structures we have identified a series of lead inhibitors for future structure‐based drug design. Crystal structures of lead bNOS inhibitors have revealed an unprecedented mode of binding to the bacterial NOS that has not been previously observed in mammalian homologs. Continued exploitation of this novel‐binding mode will be imperative for rationally designing a potent and selective bNOS inhibitor.Grant Funding Source: National Institute of Health
Biochemical response to acute temperature change in Atlantic killifish, Fundulus heteroclitus (586.1)
Authors: Biederman Amanda , Williams E. Eugene 
Published: June 2021

It is documented that after temperature acclimation, the killifish Fundulus heteroclitus shows differences in the activities of a number of enzymes including lactate dehydrogenase (LDH), an important catalyst in anaerobic respiration. Less well understood is the temporal pattern of change in activity from one temperature to another. We acclimated fish to 22°C, and shifted the temperature such that an equilibrium was reached at 4°C after 9 hours. During the shift, we collected liver and white muscle samples and assayed homogenates for the specific activity of LDH. After three hours, LDH activity in liver increased from 22.2 µmol·min‐1·mg protein‐1 to 75.1 µmol·min‐1·mg protein‐1 , but then dropped by nine hours to 25.4 µmol·min‐1·mg protein‐1. Conversely, LDH activity in muscle decreased from 154 µmol·min‐1·mg protein‐1 to 61.3 µmol·min‐1·mg protein‐1 and continued to decrease at a slowed rate. These data demonstrate that F. heteroclitus adjusts LDH activity extremely rapidly in response to temperature change. We are currently determining the response to the opposite temperature shift (4 to 22°C), and we plan to assess several enzymes associated with aerobic respiration and other aspects of metabolism. Together, these analyses will help us determine which major metabolic pathways are most critical to this species during rapid temperature change.Grant Funding Source: Supported by the Guerrieri Foundation and the Salisbury University Department of Biological Sciences