bluuebunny/crossref_metadata_2025_split huggingface dataset:
Method Development for Identification and Verification of Protein Metal‐Binding Sites
Authors: Healy Kelly Lynn 
Published: June 2021

Cadmium is a nonessential heavy metal that is found in many common items such as batteries, pigments, wheat, and tobacco, and is known to cause nephrotoxicity, hepatotoxicity, and osteotoxicity. Furthermore, studies have demonstrated that cadmium exposure leads to an accumulation of mutations by disrupting the DNA repair activities of mismatch repair proteins and polynucleotide kinase, as well as by creating reactive oxygen species in the cell. In response to heavy metal exposure, organisms such as bacteria, plants, and animals express metallothionein (MT). MT is rich in cysteine residues, promoting binding to divalent metal cations such as zinc and cadmium. MT has a very slow evolution rate and the cysteine residues are conserved throughout species, indicating that it is essential for life, yet the exact residues where metals bind are unknown. Metal‐binding sites were identified by a three‐dimensional model of rabbit metallothionein‐1A built by predictive protein databases based on homologous structures. Metal‐binding fluorophores were used to quantify the amount of cadmium, zinc, magnesium, and manganese bound to different constructs of MT. Locating binding site mutations in MT is the first step to identifying mutant proteins and correcting function. In addition to identifying the metal‐binding sites in rabbit MT‐1, this project will allow us to use this methodology to locate binding sites in other proteins that are not structurally characterized as well as MT.Support or Funding InformationThis project was funded by St. Mary's Undergraduate Research Fellowship at St. Mary's College of Maryland and by the Cove Point Natural Heritage Trust's Ruth Mathes Scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the Binding Interface between Rad3 and the Cytosolic Iron Sulfur Cluster Assembly Targeting Complex
Authors: Hushka Annelise M , Marquez Melissa , Vo Amanda , Perlstein Deborah 
Published: June 2021

The Cytosolic Iron Sulfur Cluster Assembly (CIA) pathway is a highly conserved pathway that assembles and inserts iron sulfur (FeS) cluster cofactors into a variety of target proteins. These targets are involved in many cellular processes including DNA repair, iron homeostasis, and nucleotide metabolism. For CIA targets to receive the FeS cluster, they must be recruited for FeS cluster insertion by the CIA targeting complex. Although it is known that the targeting complex recognizes and binds to targets, the mechanisms of target recognition are not fully understood. In this work, mutagenesis and affinity co‐purification were utilized to elucidate the binding interface of Met18, a protein in the CIA targeting complex, and Rad3, a DNA helicase that receives an FeS cluster from the CIA pathway. Although this interaction has been investigated by other groups, there has been contradiction as to which portion of Rad3 binds to the targeting complex. Vashist et al. (J. Biol. Chem., 2012, 287, 43351) reported that the N‐terminus of Rad3 is responsible for binding to the targeting complex, while Ito et al. (Mol. Cell., 2010, 39, 632) reported that the C‐terminus of Rad3 is responsible for binding. To help resolve this discrepancy and gain insight into the targeting mechanism of the CIA targeting complex, truncated versions of Rad3 were expressed and isolated, and it was found that the N‐terminal region of the protein is responsible for binding to the targeting complex. Our current work involves investigating a 10 amino acid N‐terminal region of Rad3 identified by Vashist et al. Their group identified this region as participating in binding to Met18. In this work, we split the region into multiple alanine scans to further narrow down the Rad3‐Met18 binding interface. The identification of a region involved in Rad3‐Met18 binding could help define a target recognition motif for the CIA targeting complex.Support or Funding InformationBU Undergraduate Research Opportunities Program; NIH R01 GM121673This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Deletion Mutants Encoding Protein Interactors of the Stress Sensor Mtl1p of Saccharomyces cerevisiae
Authors: Martinez Nelson , Rodriguez‐Medina Jose R. 
Published: June 2021

We identified nine full‐length proteins that interact physically with the cytosolic domain of the Cell Wall Integrity (CWI) Pathway sensor Mtl1p in Saccharomyces cerevisiae using membrane yeast two hybrid (MYTH) assays. We tested eight viable null mutants for functionality when exposed to 0.5mM H2O2 or to a low‐ glucose environment.ObjectivesThe objectives of our study are to identify protein interactors of Mtl1p that exhibit growth phenotypes in the null mutant and may therefore be useful for designing novel antifungal pharmacotherapies.MethodsTo assess the roles of these protein interactors in stress resistance, cells bearing a deletion of these genes were challenged either by exposure to 0.5mM H2O2 or when grown on 0.05% glucose medium and compared for growth defects with a wild‐type strain. Likewise, double deletion combinations of sensor and interactors were tested in growth assays. The phosphorylation status of the CWI MAPK, Slt2p, was assessed by Western blot (WB) under each condition.ResultsIn low glucose concentration (0.05% w/v) all strains grew slower over a period of 8 hours compared to growth in medium with 2% glucose. We observed a marked decrease in the phosphorylation levels of Slt2p for all strains, including the mtl1Δ strain when cells were put in low glucose for two hours, suggesting a generalized effect which is not dependent on the expression of MTL1. When exposed to 0.5mM H2O2, the single deletion strains egd2Δ and ras2Δ experience enhanced sensitivity over the wild type and their untreated strains.ConclusionsWe successfully identified interactors of the cytosolic domain of Mtl1p whose deletions confer particular susceptibility phenotypes upon exposure to H2O2 and for growth on low glucose suggesting functionality under these conditions. By using sensor‐interactor double mutants we also identified a genetic interaction with the sensor protein Mtl1p.Support or Funding InformationThis project is supported by NIH Grants G12MD007600, U54MD007587, R25GM061838, and by Grant NIGMS INBRE P20GM103475 to the University of Puerto Rico, Medical Sciences Campus.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Basigin‐variant‐2 binding domain in the Ig0 domain of Basigin‐variant‐1 stimulates an immune response in the mouse monocyte RAW 264.7 cell line
Authors: Tompa Abigail Delanie , Ochrietor Judith D 
Published: June 2021

There are two main protein products of the Basigin gene. One protein, known as Basigin‐variant‐1 is expressed in the neural retina, specifically by the photoreceptor neurons. The other protein, known as Basigin‐variant‐2 is expressed throughout the body, including monocytes, as well as Müller glial cells and the retinal pigmented epithelium of the eye. A study by this laboratory indicates that the two Basigin gene products interact via their extracellular domains. A different study by another research group indicates that the Ig0 domain of Basigin‐variant‐1 can elicit an immune response in several cell lines. The purpose of the present study was to determine if the region of the Ig0 domain of Basigin‐variant‐1 thought to interact with Basigin‐variant‐2 is the same region that elicits an immune response. Recombinant versions of the Basigin‐variant‐1 Ig0 domain were incubated with mouse monocytic RAW 264.7 cells. After 24 hours, the cell culture medium was collected and assayed for the expression of the proinflammatory cytokine interleukin‐6 (IL‐6) via an ELISA. Cells treated with a control protein generated from the expression vector used to make the Basigin‐variant‐1 recombinant proteins served as the controls. The data indicate that the region of the Ig0 domain used to interact with Basigin‐variant‐2 does indeed stimulate production of a significantly greater amount of IL‐6 than the control protein. The data suggest that Basigin‐variant‐1 interacts with Basigin‐variant‐2 to stimulate a proinflammatory response in monocytes. This study is important for a better understanding of immune response aspects of diabetic retinopathy and other diseases in which the retinal pigmented epithelium is compromised.Support or Funding InformationUNF Office of Academic AffairsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanisms of Nuclear Export of the RNA‐Binding Protein LARP6
Authors: Zepeda Samantha Kathleen , Lewis Karen A. 
Published: June 2021

The La Related Protein (LARP) superfamily comprises RNA binding proteins that have diverse RNA targets (including mRNA and tRNA) and exert regulatory functions in the cell. One member of the family, LARP6, regulates the synthesis of type I collagen by binding the 5′ untranslated region of the mRNA and facilitating its localization to the ER membrane. Aberrant collagen expression is a key feature of fibroproliferative diseases such as osteogenesis imperfecta. Therefore, LARP6 is a potential target for treatment of these diseases. However, the mechanisms of LARP6 cellular localization remain unclear. Previously published work transfected a GFP‐tagged LARP6 harboring a deletion of a putative nuclear export sequence (NES‐RBD) within the RNA binding domain. The NES deletion mutant was observed to accumulate in the nucleus, suggesting nucleocytoplasmic shuttling. The purpose of the current study is to understand this nuclear export mechanism in concert with recent structural data for the RNA‐binding domain of LARP6. First, we investigated the localization of endogenous LARP6 in immortalized cell lines using immunocytochemistry and leptomycin B treatment to inhibit classical nuclear transport mechanisms. These experiments demonstrated challenges with the selectivity of commercial antibodies for endogenous levels of human LARP6 in mammalian cells. Additionally, we used a recently‐developed algorithm, LocNES, to identify a second candidate NES motif in the C‐terminus of LARP6 (NES‐CTD) and to clarify the boundaries of the NES‐RBD effectively breaking it into two candidates (NES‐RBD1 and NES‐RBD2). Two approaches were used to test these new candidate motifs. First, the predicted NES motifs were cloned as fusions to GST and purified in preparation for pulldown assays to directly assess interaction with the transporter CRM1. Second, bacterial expression constructs of the wildtype and deletion mutants of the two predicted NES were constructed to analyze the effects of the NES deletions on recombinant protein stability. Chemical and thermal denaturation of the recombinant proteins was monitored by fluorescence spectroscopy to measure the free energy of folding. While all of the GST‐NES fusions are readily expressed in bacteria, only the NES‐CTD candidate motif is readily purified. The high hydrophobicity of the two NES‐RBD candidate motifs appears to prevent their purification. Together, the observed differences in the stability of GST‐NES peptides and the LARP6‐NES deletion mutants suggest that manipulation of the hydrophobic‐rich regions complicates the biochemical efforts to elucidate the mechanism of nuclear export of LARP6.Support or Funding InformationNational Institutes of Health – National Institute of General Medical Sciences (GM119096)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutations in the zDHHC9 protein palmitoyltransferase result in X‐Linked Intellectual Disability (XLID) by distinct mechanisms
Authors: Ramadan Ahmed S , Kirouac Lisa S , Sales Conniff Amanda E , Serraneou Karisa , Dzaferi Medina , Schlosser Sydni S , Pendleton Laura S , Deschenes Robert J 
Published: June 2021

The zDHHC9 gene, which encodes a Ras palmitoyltransferase is highly expressed in the brain and the protein is enriched in the hippocampus, a region known to be involved with learning and memory. Loss of function mutations in the zDHHC9 result in lead X‐Linked Intellectual Disabilities (XLID). Analysis of the disease causing mutations has begun to provide insights into the molecular mechanisms underlining the neuronal function of protein palmitoylation. Previously we published that two missense mutations in zDHHC9 (P150S and R148W) result in a decrease in enzyme autopalmitoylation, the first of the two step reaction mechanism (JBC 289:18582 (2014)). Here we show that a nonsense mutation, R298* that truncates the C‐terminal 66‐amino acids of zDHHC9, results in an enzymatically active, but trafficking defective protein. Specifically, wild type zDHHC9 is localized in the Golgi and traffics via a microtubules based mechanism to distal sites of the neuron. In contrast, the truncated R298* protein is restricted to the Golgi. This suggests that subcellular localization is required to engage specific protein substrates involved with neuronal function. Since it is known that zDHHC9 palmitoylates Ras proteins, we investigated whether the loss of zDHHC9 function changes Ras dependent signaling. Wild type HAP1 cells were compared with HAP1 cells in which the zDHHC9 gene has been deleted. In cells lacking zDHHC9 there was a decrease in S6K and AKT1 phosphorylation, consistent with a reduction in Ras signaling. We are in the process of analyzing additional disease mutations of zDHHC9 using cell lines, rat hippocampal neurons and a yeast palmitoylation functional assay. In addition, Acyl‐RAC and ABE methods, coupled with mass spectrometry are being used to identify zDHHC9 substrates in neurons. These studies will provide insight into the role of palmitoylation in the pathophysiology of XLID.Support or Funding InformationThis study was supported by a grant from NIH R21NS090160 to RJD.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How Coordination Chemistry Controls Reactivity in the B12 Trafficking Protein CblC
Authors: Li Zhu , Brunold Thomas , Warncke Kurt , Banerjee Ruma 
Published: June 2021

CblC is involved in an early step in cobalamin (or B12) processing following entry of the cofactor into the cytoplasm. CblC catalyzes glutathione‐dependent dealkylation of alkylcobalamin and reductive decyanation of cyanocobalamin (CNCbl). Recently, we found that Caenorhabdiitis elegans CblC (ceCblC) exhibits a robust thiol oxidase activity, converting GSH to GSSG accompanied by O2 scrubbing. This futile oxidative cycle is suppressed in wild‐type human CblC (hCblC) but significant in two of the pathogenic mutants, R161G and R161Q. Based on our biochemical and structural analyses, we have proposed GS‐Co(II) is a key intermediate formed in the catalytic cycle of the thiol oxidase activity of ceCblC. In this study, we aimed to capture GS‐Co(II) intermediate using biophysical tools. But instead, both EPR and MCD experiments revealed Cl‐Co(II) intermediate. Further biochemical assays suggest thiol‐oxidase activity of ceCblC is dependent on Cl− or Br− that is present in the reaction buffer. Based on our new findings, we propose that coordination of the cobalt atom in B12 controls the thiol oxidase activity of CblC, and ligand relay (H2O to Cl− to GS−) on Co(II) is a key step in the mechanism.Support or Funding InformationThis work was supported in part by the National Institutes of Health (DK45776)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glutathione transferase catalyzes the addition of glutathione to nitro‐conjugated linoleic acid
Authors: Steglich Martina , Schopfer Francisco , Alvarez Beatriz , Turell Lucia 
Published: June 2021

Nitroalkene fatty acids are formed in vivo and are currently being tested in two Phase 2 clinical trials. They modulate cellular responses through Michael addition reactions with thiol‐containing proteins. Nitro‐conjugated linoleic acid (NO2‐CLA) is formed preferentially both in vivo and in vitro. There are two main positional isomers of NO2‐CLA, 9‐ and 12‐NO2‐CLA. They both react with low molecular weight thiols at two electrophilic sites located on the β‐ and δ‐carbons giving rise to reversible biphasic kinetics. The fast reaction corresponds to the formation of the β‐adduct (the kinetic product), while the slow reaction corresponds to the δ‐adduct (the thermodynamic product) (Turell, Vitturi, J. Biol. Chem., 2017). With the aim of further characterizing the biological chemistry of this bifunctional nitroalkene, we evaluated if its reaction with glutathione (GSH) is catalyzed by glutathione transferases (GST). The reaction was followed by monitoring NO2‐CLA decay at 330 nm using a stopped flow spectrophotometer. A mu‐type glutathione transferase from the parasitic platyhelminth Echinococcus granulosus (EgGST1) was used. Catalysis was observed in both phases when using a mixture of NO2‐CLA isomers. Remarkably, the δ‐addition was accelerated □13 times more than the β‐addition, 4.4 × 106 vs 3.4 × 105 M−1, respectively, at 2 mM GSH and 10 μM NO2‐CLA (25 °C, pH 7.4), suggesting specificity. To evaluate whether there is a preference of the enzyme for the NO2‐CLA isomer, purified 9‐ or 12‐ NO2‐CLA were used. No difference was observed between them. The reaction between GSH and nitro‐oleic acid (NO2‐OA), a nitroalkene fatty acid with only one electrophilic site in β, was also catalyzed by this enzyme. An acceleration of 1 × 105 M−1 was observed (2 mM GSH, 20 μM NO2‐OA), in line with the value obtained for the β‐addition of NO2‐CLA. We are now further characterizing this reaction which is expected to affect nitroalkene fatty acids metabolism and excretion and thus impact their signaling actions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A novel method for insulin detection
Authors: Slachtova Lenka , Zemanova Jitka , Sacha Pavel , Fabre Benjamin , Zakova Lenka , Jiracek Jiri , Konvalinka Jan 
Published: June 2021

For some of us, insulin is a large polypeptide, for people with diabetes it is the only cure. Diabetes became a global, epidemic problem with increasing rate and health costs every year. Current treatment of type I diabetes is subcutaneously administered insulin substitution. Therefore, there is an urgent need for development of orally available insulin substitution. Here we developed a sensitive method for measurement of potency of insulin mimetics during drug development. DNA‐linked Inhibitor Antibody Assay DIANA is a sensitive, simple and nonradioactive method suitable for determination of binding constants of insulin mimetics. DIANA uses competitive inhibition of a small molecule in solution with a probe containing a ligand derivative linked to DNA. Thus, DIANA is useful method for characterization of proteinreceptor interactions, such as insulin and its receptors, as well as for high throughput screening of potential new therapeutics.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deregulation of Acanthamoeba castellanii steroidogenesis is amoebicidal and protects cultured corneal cells from Ac attack
Authors: Vanderloop Boden H. , Thomas Crista D. , Zhou Wenxu , Gillespie Amelia A. , Singha Ujjal , Chaudhuri Minu , Neelam Sudha , Niederkorn Jerry , Nes William David 
Published: June 2021

Human infections by amoeba represent rare diseases, yet they are an emerging health problem that can develop into rapid death, as in cases involving the spinal cord or amoeba that penetrate the brain. Our recent papers highlighted in J. Lipid Res, 2017 and ASBMB Today magazine, 2018 focus on development of new sterol methyltransferase (SMT) inhibitors to prevent steroidogenesis in the parasitic amoeba Acanthamoeba castellanii (Ac). Now we show, Ac cultured on unexplored sterol C24‐methyltransferase (SMT) inhibitors 25‐azalanosterol and 25‐thialanosterol iodine salt are highly potent anti‐amoeba drugs exhibiting IC50 25 nM and 15 nM, respectively and a minimum amoebicidal concentration of 1 μM. Both analogs inhibited the cloned 24‐AcSMT and 28‐AcSMT yielding Ki values of 13 nM/37nM and 9nM/16nM, respectively and blocked Ac steroidogenesis at the target enzyme. Against human epithelial kidney cell growth these drugs had no effect to 40 μM or effected cholesterol biosynthesis (selective index greater than 103); in mice at 50 mg/kg, 3H‐analog could rapidly cross the blood brain barrier with minimal effect on liver cholesterol biosynthesis. Against human corneal epithelial cultures infected with Ac, the drugs protected the eye at doses of 500 nM. These new amoeba sterol biosynthesis inhibitors are currently under investigation for their therapeutic potential.Support or Funding InformationThis research was funded through a grant from the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering Living Biosensors using the Hemeprotein Transcription Factor CooA
Authors: Murchison Austin Kevon , Esquerra Raymond 
Published: June 2021

Biosensors are devices that detect the presence of a biological compound. Currently biosensors have applications in food safety, medical diagnosis, and environmental monitoring. The lifetime of use is limited in the current models, so we looked to examine archetypes found in nature to improve the field of biosensors. Bacteria utilize gas sensor proteins to detect changes in their environment to modulate their metabolism. Iron containing transcription factors are responsible for sensing/binding O2, CO, and NO to initiate a biological response. CooA is a heme‐containing transcription factor from the photosynthetic bacterium Rhodospirillum Rubrum. CooA is a homo‐dimer and acts as a carbon monoxide (CO) sensor. CooA senses the abundance of CO in the environment and in response activates the transcription of genes involved in CO metabolism. The goal of this project is to engineer cells capable of sensing CO to induce the expression of Red Fluorescent Protein (mRFP). We designed a CO sensing transcription system based on R. Rubrum for use in E. coli using a single expression plasmid encoding CooA and mRFP transformed into the E. coli strain BL21 DE3. The expression of mRFP will be induced by CooA activity in vivo and quantified using fluorescence spectroscopy. The long‐term goal is to use this template to design and optimize CooA into detecting other chemical signals using a hydrid computational and experimental approach. The outcome is to develop a family of biosensors to detect and respond to specific chemical signals. Using living biosensors, we can expand the uses of these devices to serve as environmental watchdogs monitoring pollution in lakes, streams, and other bodies of water.Support or Funding InformationNSF STC CCC Grant: 1548297, Genentech Foundation MS Dissertation ScholarshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.