bluuebunny/crossref_metadata_2025_split huggingface dataset:
HIV protease inhibitors sensitize human head and neck carcinoma cells to radiation by activating ER stress (762.2)
Authors: Liu Runping , Zhang Xiaoxuan , Wang Xiang , Mikkelsen Ross , Song Shiyu , Zhou Huiping 
Published: June 2021

Head and neck cancers are the eighth most common cancer worldwide. Despite significant advances in the delivery of treatment and surgical reconstruction, the mortality rates for this disease have not improved in the past 4 decades. Our previous study has shown that HIV protease inhibitors (HIV PIs) induce cell apoptosis via activating ER stress. It also has been reported that a few HIV PIs are able to radiosensitize tumor cells. However, the underlying cellular mechanisms remain to be identified. The aim of this study was to examine whether HIV PIs activate the ER stress response and sensitize human head and neck carcinoma cells to radiation. Methods: Human SQ20B and Fadu cells and the most commonly used HIV PIs, lopinavir and ritonavir, were used in this study. The mRNA and protein levels of ER stress‐related genes (CHOP, ATF4, XBP‐1, and GRP78) were detected by real time RT‐PCR and Western blot, respectively. Cell viability and apoptosis were analyzed using Cellometer Vision CBA. After treatment with HIV PIs, cells were irradiated at a dose of 2G or 4G. Colonies were stained and counted 10 days after irradiation. Results: HIV PIs significantly induced activation of ER stress and apoptosis. Treatment of HIV PIs inhibited Akt phosphorylation and increased tumor cell sensitivity to irradiation‐induced cell death. Conclusion: HIV PIs sensitize human head and neck carcinoma cells to radiation by activating ER stress.Grant Funding Source: NIH/NIDDK, China natiional science foundation
Methylthioadenosine phosphorylase is required for prostate cancer growth in vitro and in vivo (766.2)
Authors: Bistulfi Gaia , Morrison Carl , Gillard Bryan , Foster Barbara , Smiraglia Dominic 
Published: June 2021

Methylthioadenosine phosphorylase (MTAP) helps to sustain methionine metabolism, which, either directly or indirectly, plays a significant role in polyamine synthesis, DNA synthesis, and DNA methylation. MTAP is one of the most frequently deleted genes in human cancers, possibly contributing to genetic and epigenetic damage. Because prostate cells have an exceptionally high level of polyamine biosynthesis impinging on methionine metabolism, we investigated MTAP status in human prostate cancer (CaP). In contrast to many other types of cancer, MTAP was never deleted in CaP, as assessed in a sample of 13 cell lines by PCR and 75 CaP patients at different stages of the disease by tissue microarray analysis. In the context of folate deficiency, which stresses methionine metabolism, transcriptional up regulation of MTAP allowed CaP cells to adapt to the metabolic stress in order to provide sufficiency of key metabolites as assessed by HPLC analysis. We therefore hypothesized that MTAP may play an essential role in sustaining CaP growth. We treated a panel of CaP cell lines with a specific MTAP inhibitor. Consistent with MTAP’s importance in prostate cells’ metabolism, we found that the inhibitor was very effective in preventing CaP growth in vitro, with an IC50 between 0.7 and 1.4 µM. Finally, MTAP knock down in LNCaP cells prevented tumor establishment in nude mice. We conclude that MTAP plays a pivotal role in CaP growth and that, because of its importance specific to prostate cells, it could be a novel, ideal target for CaP therapy.Grant Funding Source: AICR American Institute for Cancer Research
Oxidation of DNA by high‐valent metal‐oxo porphyrins Mn‐TMPyP and Fe‐TMPyP (768.12)
Authors: Tandjigora Kaba , Pratviel Genevieve 
Published: June 2021

The goal of this project is to regulate the reactivity of high‐valent metal‐oxo porphyrins by the control of the axial ligand trans to the oxo group. This work will allow a better understanding of the mechanism of metalloporphyrins oxidation. The chosen substrate is a DNA model. The oxidation products are analyzed by HPLC and LC/ESI‐MS. Meso‐tetrakis(4‐N‐methylpyridiniumyl)porphyrinatomanganese(III) (Mn‐TMPyP) and its iron analogue (Fe‐TMPyP) mimic the reaction mechanism of natural heme enzymes namely peroxidases and cytochrome P450. Mn‐TMPyP and Fe‐TMPyP become high‐valent metal‐oxo species when activated by dissymmetric peroxide such as KHSO5 and are able to oxidize DNA. Previous work has shown that Mn‐TMPyP/ KHSO5 was sensitive to pH and produced variable oxidation products of the DNA duplex model 5’‐d(CAGCTG) by electron transfer (ET) and oxygen atom transfer (OAT). At pH 8, the main products observed were those of OAT, whereas at pH 6 the dominant products were obtained from ET. Fe‐TMPyP as Mn‐TMPyP is a powerful oxidizing agent; however, its reactivity towards DNA is unknown. What is known is that it exists as a µ‐oxo species in basic and neutral medium. The µ‐oxo characteristic of Fe‐TMPyP should make it insensitive to pH. We showed that its oxidation products did not vary between pH 6 and pH 8. The present work has shown that the oxidation reactivity of manganese and iron porphyrins can be directed based on their proximal axial ligand (L‐ or X‐type). The proximal ligand can be controlled by simple modulation of the pH of the reaction in the case of manganese porphyrin or by the nature of the metal (iron porphyrins form iron µ‐oxo dimers). The control of metalloporphyrins reactivity may also prove useful in fine chemistry where these compounds are used as oxidation catalysts.Grant Funding Source: Supported by NSF‐CHM 1156907
Probing the determinants of the metal binding specificity in HDAC8 (768.7)
Authors: Kim Byungchul 
Published: June 2021

HDACs catalyze the hydrolysis of ε‐N‐acetyl lysine residues on proteins acetylated by histone acetyltransferases. Upon their discovery, this enzyme was identified as Zn2+‐dependent metalloenzymes. However, current activity and binding affinity studies suggest that Fe2+ could be the native metal cofactor in vivo. This has a number of biologically relevant implications, including 1) metal switching regulatory mechanism, 2) substrate specificity based on the identity of metal status. Therefore I am presenting the determinants of metal selectivity in HDAC8 in vitro to provide insight into the intracellular metal specificity in vivo.In HDAC8, the second shell of amino acid surrounding the metal coordinating side chains is predominantly hydrophobic. This is contrary to many prototypical metalloenzymes, such as carbonic anhydrase II (CAII). The lack of second shell hydrogen bonds may be an important factor contributing to the weaker Zn2+ affinity of HDAC8 compared to CAII, allowing facile switching between Zn2+ and Fe2+ cofactors. To investigate these structural determinants, I have prepared HDAC8 mutants and I am measuring the metal ion affinity and dissociation rate constants of wild‐type and mutants. Surprisingly the dissociation rate constants for Zn2+ and Fe2+ from the HDAC8‐metal complex in mutants are comparable despite the large difference in binding affinity.Previous studies indicate that binding to the 1st K+ site inhibits the activity, while binding to the second K+ site activates catalysis possibly through structural stabilization. This indicates that K+ binding to two different sites may affect the Zn2+ binding site. To study this, I am measuring the dissociation rate constant for zinc and iron binding to HDAC8 at varying concentrations of monovalent cation (K+ and Na+). The Zn2+ dissociation rate constant from metal‐bound HDAC8 decreases as the concentration of monovalent cation (K+/Na+) is increased.Grant Funding Source: Supported by NIH's NIGMS 2RO1GM040602‐22
The minor role of Gln 183 in the NADH reductive half‐reaction of E. coli methylenetetrahydrofolate reductase (769.10)
Authors: Zuo Chong , Jolly Amber , Nikolova Andriana , Cao Sirui , Sanchez Jeremy , Trimmer Elizabeth 
Published: June 2021

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10‐methylenetetrahydrofolate (CH2‐H4folate) to 5‐methyltetrahydrofolate (CH3‐H4folate) using FAD as a redox coenzyme. The reaction contains two different half‐reactions, the NADH reductive half‐reaction and the CH2‐H4folate oxidative half‐reaction. The focus of our work is the active‐site amino acid Glutamine 183 (Gln183). Structural studies have implicated the importance of Gln183 in both substrate binding and catalysis of the two half‐reactions, based on direct hydrogen bonds between Gln183 and the substrates NADH and folate. In order to investigate the function of Gln183 in MTHFR catalysis, two mutants with single amino acid changes, Gln183Ala (Q183A) and Gln183Glu (Q183E) have been constructed. Using the steady‐state NADH‐menadione oxidoreductase assay, we have examined the reductive half‐reactions of each mutant enzyme. For reasons of mutant stability, the temperature of the assay was optimized to 4°C. We expected that the change in hydrogen bonds and polarity of the Q183 mutant enzymes would affect their kinetic parameters kcat and Km. However, the mutant enzymes have similar kinetic parameters to the wild‐type enzyme at 4°C, consistent with our previous results at 25°C. Therefore, we conclude that glutamine 183 plays only a minor role in the reductive half‐reaction of MTHFR.Grant Funding Source: Grinnell College
The mode of substrate entry into the 5‐lipoxygenase active site (769.3)
Authors: Mitra Sunayana , Newcomer Marcia 
Published: June 2021

Leukotrienes are potent immune‐modulatory compounds involved in the inflammatory response. 5‐Lipoxygenase (5‐LOX) is the enzyme that catalyzes the production of Leukotriene A4 (LTA4), which is further metabolized to pro and anti‐inflammatory compounds from the substrate Arachidonic Acid (AA), a polyunsaturated fatty acid.The crystal structure of a stabilized form of 5‐LOX has been recently reported (Gilbert et al, 2011). However, the structure does not reveal the mode of substrate recognition. The first step in the enzymatic reaction involves the removal of a hydrogen from the central carbon of a pentadiene present in the substrate. It is not known how 5‐LOX distinguishes among three such chemically equivalent pentadienes in AA to produce only 5‐ S‐HPETE, which is subsequently transformed to LTA4. The crystal structure suggests a somewhat crescent shaped active site lined with invariant Leucines and Isoleucines, with no clear access port visible for substrate entry.A series of site directed mutants were produced and the activities of these mutants evaluated. Product analyses of these mutants with AA and substrate analogs suggest entry of the substrate by the opening of the so‐called Phenylalanine (F)‐Tyrosine(Y) ‘cork’. The data is also consistent with the orientation of AA as carboxyl innermost, as predicted from the stereochemistry of the product and mechanistic models of lipoxygenase function.Grant Funding Source: Supported by NIH Grant # HL107887
Structural insights into aspartoacylase deficiency in Canavan disease (769.4)
Authors: Wijayasinghe Yasanandana , Pavlovsky Alexander , Viola Ronald 
Published: June 2021

Canavan disease (CD) is a fatal neurodegenerative disorder with impaired N‐acetyl‐L‐aspartic acid (NAA) metabolism. This disease is caused by mutations in the ASPA gene, leading to a deficiency in the aspartoacylase (ASPA) enzyme that hydrolyzes NAA in the brain. The majority of the defects are missense mutations found remote from the active site, resulting in decreased stability and activity. To study the possible structural perturbations triggered by these mutations, ASPA mutants associated with different disease severities have been cloned, purified, and crystallized. E285A, one of the most common mutants shows that loss of the hydrogen bond interaction with Asp 285 disturbs the active site architecture leading to altered substrate binding. The loss of a hydrophobic interaction in the F295S mutant leads to a local collapse of the structure, contributing to low protein stability. But K213E, a mutant associated with a mild disease phenotype, has minimal structural differences compare to the native enzyme. Our improved understanding of the structural changes that occur in ASPA mutants will allow the design of small molecule chaperones that could lead to recovery of the activity of this defective enzyme and therefor a treatment therapy for Canavan disease.Grant Funding Source: Supported by Canavan Research Foundation, and Jacob's Cure Organization
Regulation of endothelial nitric oxide synthase phosphorylated at Thr495 by interaction with calmodulin (769.8)
Authors: Piazza Michael , Guillemette J. Guy , Dieckmann Thorsten 
Published: June 2021

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes and it is important to understand how this enzyme is regulated. Calmodulin (CaM), a small acidic calcium binding protein, is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains, through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide of eNOS that contains a phosphorylated threonine corresponding to Thr495 in order to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α‐helix. The calcium affinity of the CaM‐target peptide complex is reduced due to phosphorylation and this leads to weaker binding under low physiological calcium concentrations. This study provides an explanation for the reduced NO production by eNOS carrying a phosphorylated Thr495 residue.Grant Funding Source: National Science and Engineering Research Council (NSERC)
Thermal denaturation of Mycobacterium tuberculosis protein Rv0045c monitored by intrinsic tryptophan fluorescence (770.1)
Authors: Drake Lindsey , Hoops Geoffrey 
Published: June 2021

Rv0045c is a novel esterase of Mycobacterium tuberculosis, the causative bacterium of the still prevalent and deadly disease tuberculosis. The six tryptophan residues in this enzyme were of interest to monitor the denaturation process by intrinsic fluorescence. To simplify the signals, the six tryptophans (W78, W98, W122, W246, W248, W263) were mutated to phenylalanine to preserve the relative size of the residue but remove the fluorescence capability; the goal being to create a quadruple mutant with only one pair of tryptophans interacting. Mutated proteins were synthesized through site directed mutagenesis, overexpressed in E.coli hosts, and purified using a Ni2+ affinity resin. Differential scanning fluorimetry and kinetic assays were performed to test the initial stability of the mutants. W78F and W98F were not as stable as the wild type and therefor were not used in further double and triple mutations. Significant changes in the thermal stability of Rv0045c triple and quadruple mutants will be analyzed via intrinsic fluorescence.Grant Funding Source: NSF TUES
Identification and biochemical characterization of a Kunitz‐type serine protease inhibitor from an insect, Manduca sexta (772.1)
Authors: Watkins Jamilah , Takahashi Daisuke , Hiromasa Yasuaki , Kanost Michael 
Published: June 2021

Hemolymph (blood) of insects contains serine proteases and serine protease inhibitors that function in innate immune responses. The objective for this research was to investigate low molecular weight protease inhibitors from hemolymph of a lepidopteran insect, Manduca sexta. We identified serine protease inhibitor activity against trypsin, chymotrypsin, and elastase in plasma from naïve larvae and in plasma from larvae that were previously injected with Micrococcus luteus. We separated hemolymph plasma proteins using size exclusion chromatography, and the fractions with protease inhibitor activity were analyzed by SDS‐PAGE and liquid chromatography‐electrospray ionization tandem mass spectrometry. A serine protease inhibitor belonging to the Kunitz family was identified, and using primers based on the sequence encoding this protein found in the M. sexta genome sequence, we cloned its cDNA from larval fat body RNA. The cDNA encodes a protein with a secretion signal sequence followed by the mature secreted inhibitor protein of 56 amino acid residues. This sequence is highly similar to a Kunitz inhibitor previously purified from M. sexta (Ramesh, N., Sugumaran, M., Mole, J.E. 1988. J Biol Chem. 263:11523‐7). Using a synthesized and codon‐optimized DNA encoding the protease inhibitor, we are currently producing the recombinant inhibitor in E. coli for further biochemical characterization. We will study its inhibitory activity as a potential regulator of proteases in hemolymph that function in the innate immune system of M. sexta. The genome of M. sexta contains nine genes for single‐domain Kunitz proteins closely spaced on a single scaffold. The ultimate goal of this project is to understand the biological functions of these Kunitz protease inhibitors in innate immunity.Grant Funding Source: Supported by NIH grant GM41247.
Modulation of eukaryotic elongation factor 2 kinase activity via phosphorylation on Ser‐500 (773.1)
Authors: Tavares Clint , Kaoud Tamer , Ferguson Scarlett , Wellmann Rebecca , Stancu Bogdan , Giles David , Chitjian Catrina , Dalby Kevin 
Published: June 2021

Eukaryotic elongation factor 2 kinase (eEF‐2K) is an atypical calcium/calmodulin (Ca2+/CaM)‐dependent kinase known to modulate protein synthesis through the inhibitory phosphorylation of elongation factor 2. Despite its pivotal role in translation regulation and potential role in tumorigenesis, the mechanism of regulation of eEF‐2K activity by phosphorylation is still unclear. Our objective was to analyze the activation of eEF‐2K by phosphorylation at Ser‐500 (phosphorylated by protein kinase A or Ca2+/CaM‐induced autophosphorylation). In vitro analysis indicates that autophosphorylation of eEF‐2K induces Ca2+‐independent activity. Western blotting analysis as well as mutagenesis of Ser‐500 to Ala or Asp suggests that Ser‐500 phosphorylation is responsible for this Ca2+‐independent activity. Interestingly, CaM appears to be essential for this Ca2+‐independent activity of eEF‐2K. Mechanistic studies indicate that Ser‐500 phosphorylation increases eEF‐2K affinity for CaM in the presence or absence of Ca2+. Ser‐500 phosphorylation also increases the rate of Thr‐348 phosphorylation (a key site for the allosteric regulation of eEF‐2K activity). We are currently studying the dynamic nature of Ser‐500 phosphorylation in cells. Phosphorylation of eEF‐2K on Ser‐500 could be a significant mechanism for its regulation, especially upon the influx of Ca2+ during cellular signaling events.Grant Funding Source: NIH (GM059802 and CA167505) to KND
MauG catalysis: a tale of ferryl iron, radicals and long distance hopping (774.8)
Authors: Wilmot Carrie , Yukl Erik , Davidson Victor 
Published: June 2021

Methylamine dehydrogenase (MADH) enables some methylotrophic/autotrophic bacteria to grow on methylamine as a sole source of carbon and energy. MADH catalysis depends on the cofactor tryptophan tryptophylquinone (TTQ) that is a posttranslational modification of two Trp residues in the MADH β‐subunit. The maturation of MADH depends on four gene products located in the methylamine utilization (mau) gene cluster. One of these, mauG, encodes a c‐type di‐heme enzyme that completes synthesis of the TTQ cofactor. The potent oxidant is an unusual bis‐Fe(IV) MauG species composed of a ferryl heme (Fe(IV)=O) with an oxidizing equivalent stored as Fe(IV) at the second heme, which has an unusual His, Tyr axial ligation. The bis‐Fe(IV) oxidant is formally Fe(V) and equivalent to Compound I. Completion of TTQ to generate active MADH involves long‐range electron transfer and a radical hopping mechanism to effect catalysis over a 40 Å distance. The MauG catalyzed reaction occurs in three discrete 2‐electron events in a H2O2 or O2 (+ reducing equivalents) dependent process. A crystal structure of MauG in complex with its protein substrate, a precursor form of MADH known as preMADH, has been solved. The crystals are catalytically active. The order of the 2‐electron chemistry catalyzed by MauG was determined through a series of structures from crystals harvested after different amounts of time following crystallization. H2O2 to initiate the reaction was generated by the slow breakdown of polyethylene glycol used in crystallization. These in crystallo data are corroborated by mass spectrometry in solution experiments.Grant Funding Source: Supported by NIH grants R37GM41574, R01GM66569, F32GM97779
Recognition of metal ions using ProMOL to improve protein function assignation (777.1)
Authors: Kovuri Venkata Aditya , Craig Paul , Bernstein Herbert 
Published: June 2021

ProMOL is a plugin for PyMOL that utilizes the 3D visualization and measurement capacities of PyMOL to predict enzyme function by aligning query structures with a collection of annotated active sites found in our motif template library. The focus of this initiative is a response to the >3000 structures in the Protein Data Bank that are labeled as having “unknown function”. ProMOL undergoes constant revision and development from the various team members from RIT and Dowling College. The motif capabilities of ProMOL have been expanded to include metal ions and prosthetic groups that are found in enzyme active sites, as nearly 40% of all the PDB structures contain at least one metal ion. This is biologically significant as metal ions such as Mn, Zn, Ca and Fe, play a vital role in both the structure and function of many different proteins. The addition of metal ion recognition to ProMOL/PyMOL promises to lead us to better function assignments to this collection of protein structures. There are current plans to launch a database with specific information pertaining to the metal ion binding sites and 3D visualization of motifs with metal ions. This work has been supported in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1.Grant Funding Source: Supported by NIGMS 2R15GM078077‐02, 3R15GM078077‐02S1
Characterization of CTCF protein around the ‐224 A/G single nucleotide polymorphism in the neuropeptide receptor Y2 (NPY2R) gene in predisposition to hypertension (777.4)
Authors: Albino‐Rodriguez Elinette , Nunez‐Marrero Angel , Porrata‐Doria Tirtsa , Dutil Julie 
Published: June 2021

Hypertension (HTN) is a complex trait resulting from the interactions of multiple genes and environmental factors. Neuropeptide Y receptor 2 (NPY2R) was identified as a candidate gene for HTN in animal models and human populations. Previous work showed that the luciferase activity induced by the NPY2R promoter with the G allele in position ‐224 is reduced by 42% compared to the promoter containing an A in that position. Furthermore, an electrophoretic shift assay (EMSA) showed an allele‐specific binding with the oligonucleotide containing an A nucleotide in ‐224 position. We were also able to detect a super shift using the CTCF antibody. The aim of this study was to characterize the CTCF protein in HEK 293 cells transfected with NPY2R ‐224 A/G variations. Changes in CTCF expression and distribution were analyzed using western blot and immunofluorescence (IF) experiments. Interestingly, the western blot showed higher quantification of CTCF in HEK 293 cells transfected with NPY2R ‐224 A compared to NPY2R ‐224 G (p < 0.001). Analysis of nuclear localization of CTCF by IF revealed that it is highly distributed in HEK 293 cells transfected with NPY2R ‐224 A compare to a lower distribution in NPY2R ‐224 G. CTCF is the only protein identified so far that mediates enhancer‐blocking activity of insulators. These data provides strong evidence of the CTCF role in the transcriptional regulation.Grant Funding Source: Supported by Grant RR003050/MD007579