bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancer regulation by H3K4 methyltransferases MLL3/MLL4
Authors: Ge Kai
Published: June 2021

Enhancers control cell type‐specific gene expression and are marked by H3K4me1. Active enhancers are further marked by H3K27ac. We previously identified CBP/p300 as major H3K27 acetyltransferases and MLL3/4 as major enhancer H3K4me1 methyltransferases (EMBO J 2011; eLife 2013). During differentiation of adipocytes, myocytes and ES cells, MLL3/4 co‐localizes with lineage‐determining TFs (LDTFs) on active enhancers and are required for enhancer activation, cell type‐specific gene expression, and cell differentiation (eLife 2013; PNAS 2016). Further, we show that while enhancer‐priming by MLL3/4 is dispensable for cell identity maintenance, it controls cell fate transition by orchestrating CBP/p300‐mediated enhancer activation. MLL4 protein, rather than MLL4‐mediated H3K4me1, controls p300 recruitment to enhancers, suggesting that MLL4 may regulate enhancer activation independent of its enzymatic activity (PNAS 2016; NAR 2017).

Our recent findings identify Brd4 as an enhancer epigenomic reader that links active enhancers with cell identity gene induction in differentiation. Our data suggest a model of sequential actions of epigenomic regulators on enhancers: 1) pioneer TFs and LDTFs recruit MLL3/4 to prime enhancer regions and label them with H3K4me1; 2) MLL3/4 facilitate the binding of CBP/p300, which activate enhancers and label them with H3K27ac; 3) H3K27ac and acetylated TFs are recognized by Brd4, which recruits Mediator and RNA Pol II to establish enhancerpromoter interactions and activate cell type‐specific gene expression (Nat Comm, 2017).

To investigate the role of MLL3/4‐mediated H3K4me1 in enhancer activation and cell differentiation, we have generated two mouse lines: one expressing H3.3K4M, a lysine‐4‐to‐methionine (K4M) mutation of histone H3.3 that inhibits H3K4 methylation, and the other carrying conditional double knockout of MLL3/4 enzymatic SET domain. Unexpectedly, expression of H3.3K4M, or deletion of the SET domain, in lineage‐specific precursor cells destabilizes MLL3/4, prevents enhancer activation, and impairs cell differentiation and tissue development (NAR 2018). We are using CRISPR to generate enzyme‐dead knockin mutants of MLL3/4 in mice. The phenotypes of these mice will be reported at the meeting.

By immunoprecipitation followed by mass spectrometry analysis, we have found that endogenous MLL3/4 complex interacts with SWI/SNF chromatin remodeling complex in cells. The biological significance of such physical interactions will be reported at the meeting.

Support or Funding Information

Supported by the Intramural Research Program of NIDDK, NIH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isothermal Calorimetric Measurements of Metal Ion Interaction with TAR RNA
Authors: Song Jessica , Grover Neena
Published: June 2021

The trans‐activation response (TAR) element of HIV‐1 is essential for transcriptional elongation in HIV‐1. It consists of a stem‐loop containing a trinucleotide bulge (UCU) and is capped by a hexaloop sequence located in the 5′ UTR in HIV‐1. The trinucleotide bulge interacts with the arginine‐rich basic domain of the Tat protein to increase the rate of transcription by nearly 100‐fold. Thermodynamic studies in our laboratory have previously examined the effects of TAR RNA interactions with divalent metal ions to show that single nucleotide modifications can lead to differences in RNA‐ion interactions. Thermal denaturation studies show an increase in wild type construct stability in 10 mM Ca2+ or Mg2+ as compared to 1 M KCl. In this study, the thermodynamics of metal ion binding to the TAR RNA sequence is investigated by directly measuring the heat released using isothermal titration calorimetry (ITC). TAR RNA derived constructs were titrated with magnesium, calcium, and arginine containing buffers to measure the enthalpic contributions and the binding affinity of the ions to further understand different types of RNA‐metal ion interactions.

Support or Funding Information

Department of Chemistry and Biochemistry

Colorado College

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamics of the Kink‐Turn Motif in RNA
Authors: McCaskill Darryl Cole , Grover Neena
Published: June 2021

The kink‐turn is a helix‐internal loop‐helix with a three‐nucleotide bulge loop containing a canonical stem (Watson‐Crick base pairing) on one side and non‐canonical stem containing two or three G•A sheared base pairs on the other side. The sheared base pairs in the non‐canonical stem give the construct the flexibility to adopt a 120° kink between the two stems. The kink‐turn is expected to form in the presence of metal‐ions to neutralize the charges on phosphates that are in close proximity. RNA constructs were designed to mimic the Haloarcula Marismortui KT7 from the 28S ribosome. Thermodynamic properties of constructs were tested in a 1 M KCl salt buffer and a 10 mM magnesium buffer at either pH 5.5 or 7. The wild‐type construct shows a −2.36 kcal/mol gain in stability in the magnesium buffer compared to the 1 M KCl buffer at pH 7. Direct measurements of the heat released upon ion‐binding to RNA is being measured using ITC to determine the enthalpy and binding constants of RNA‐magnesium interactions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Tissue‐Specific RNA Exosome Cofactors as an Approach to Define Disease Mechanism
Authors: de Amorim Julia L , Leung Sara , van Hoof Ambro , Corbett Anita H
Published: June 2021

The RNA exosome, a 10‐subunit complex that mediates both RNA processing and degradation, plays a critical role in defining cellular expression profiles. This complex is ubiquitously expressed, essential, and critical for fundamental cellular functions, such as ribosomal RNA processing. Recent studies have linked mutations in genes encoding multiple subunits of the complex to tissue‐specific human disease. For example, mutations in the human EXOSC3 gene, coding for a subunit, cause Pontocerebellar Hypoplasia type 1b (PCH1b), a disease characterized by atrophy of the pons and cerebellum. The missense mutations encode single amino acid changes in conserved regions of the EXOSC3 protein. How these amino acid substitutions confer tissue‐specific phenotypes is not known. One possible mechanism underlying the distinct disease phenotypes could be a decrease in the interaction of the RNA exosome complex with cofactors that confer specificity for RNA targets. However, most studies that identify and characterize RNA exosome cofactors have been carried out in budding yeast and thus, do not provide insight into whether tissue‐specific cofactors could exist. Our studies use immunoprecipitation from neuronal cell culture (N2A) and mouse tissues to define RNA exosome cofactors. Biochemical experiments that employ cultured N2A cells identified RNA exosome interacting proteins that are enriched in both the nucleus and cytoplasm. Preliminary results from this analysis identify an association between the RNA exosome and a large complex of tRNA ligase enzymes, which could link defects in tRNA maturation to disease pathology. We are extending these studies to explore the possibility of tissue‐specific cofactors by immunoprecipitating EXOSC3 and analyzing co‐purifying proteins from the cerebellum (affected in disease) and the cortex (unaffected) in mouse brain. Finally, using CRISPR/Cas9 genome editing, we are testing whether the amino acid changes that occur in disease alter interactions with both known RNA exosome cofactors and interactors identified in our co‐immunoprecipitation studies. These studies will provide insight into both the functional consequences of amino acid substitutions in the RNA exosome that cause disease and the role of cofactors in conferring RNA target specificity.

Support or Funding Information

R01GM130147

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of DED1 Suppressors Using Genomic Sequencing
Authors: Piciw Jenny , Winters Aidan , Drewes‐Tartarotti Tatiana , Bowman John , Whitworth Gregg , Hilliker Angie
Published: June 2021

Ded1, an RNA‐dependent ATPase, promotes translation initiation of many mRNAs in yeast. Ded1 is extremely important for the translation of mRNAs predicted to have substantial secondary structure (Sen et al., 2015). Ded1 interacts directly with eIF4A and eIF4G and this complex has increased helicase activity compared to either Ded1 or eIF4A alone (Hilliker et al., 2011; Gao et al., 2016). Consistent with an important role in translation, DDX3, the human homolog of Ded1, has been shown to be misregulated in several cancers and is hijacked by HIV to allow translation of highly structured HIV mRNAs (Valiente‐Echeverria et al., 2015; Xiang et al., 2016). Because it is critical to translation and influences other translation initiation factors, we are interested in how Ded1 itself is regulated and how that regulation impacts translation.

We use genetic selections to identify regulators of Ded1 function using a well characterized ded1 mutant. A cold sensitive ded1 mutant (ded1‐dam1) lacks two regions important for Ded1's ability to promote translation; one of these regions promotes the interaction between Ded1 and eIF4G. Yeast containing this mutant as the sole copy of ded1 show a growth defect, a dramatic drop in global translation, and an accumulation of P‐bodies at the restrictive temperature (Hilliker et al., 2011). We have isolated 38 putative mutations of the cold‐sensitive growth conferred by ded1‐dam1 assembly mutant. These 38 ded1‐dam1 suppressor mutants were isolated through one of four mutagenesis processes, including extended growth, EMS mutagenesis, UV mutagenesis and spontaneous mutations.

Classical ways of identifying suppressor mutations, including library production or screening, are labor intensive. To accelerate the identification of suppressors, mutations will be identified using high throughput sequencing. Of the 38 putative suppressors of ded1‐dam1, 28 putative suppressors were sequenced using Ilumina HiSeq 2500 technology. Putative suppressors were chosen for sequencing based on results of mitochondrial mutation screens and the strength of suppressor mutation, as determined by frogging of serial dilutions. We are currently developing a pipeline for processing the data and identifying the relevant SNPs within the genomic data. By identifying suppressors of this well‐defined ded1 mutant, we will identify new regulators of Ded1, an RNA helicase essential for translation initiation.

Support or Funding Information

The Thomas F. and Kate Miller Jeffress Memorial Trust Awards Program in Interdisciplinary Research; HHMI Grant to University of Richmond; Dr. Martha Carpenter Fellowship, University of Richmond

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deciphering the Mechanism of 14‐3‐3ζ‐Mediated Inhibition of Phosducin Binding to the G Protein Transducin Complex
Authors: Chen Yuwen , Gao Meng , Huang Yongqi
Published: June 2021

Phosducin modulates phototransduction by sequestering Gtβγ subunit from Gtα subunit of the heterotrimeric G protein transducin complex during light adaptation. However, the interactions between phosducin and the Gtβγ subunit are inhibited through phosphorylation at S54 and S73 within the N‐terminal domain of phosducin. It has been found that phosphorylation at S54 and S73 of phosducin induces its binding to 14‐3‐3 proteins, which blocks interactions between phosducin and Gtβγ. In this work, molecular dynamics simulations and fluorescence resonance energy transfer (FRET) were performed to elucidate the mechanism of 14‐3‐3ζ‐mediated inhibition of phosducin binding to the Gtβγ complex. To simulate the association between 14‐3‐3ζ and phosducin and the dissociation of phosducin from the Gtβγ complex, coarse‐grained representation of proteins and structure‐based Gô‐model were applied. We investigated the influence of phosphorylation on the dynamics and stability of phosducin/Gtβγ complex and association of phosducin with 14‐3‐3ζ. Our simulations showed that phosphorylation of phosducin on S54 and S73 introduced strong electrostatic repulsion between S54 and S73, resulting in opening of a segment between S41 and E66. Consistently, FRET efficiency measurement showed that the distance between Q16 and Q57 increased after phosphorylation. Binding simulations of 14‐3‐3ζ with phosducin/Gtβγ complex in the presence or absence of S54/S73 phosphorylation showed that 14‐3‐3ζ interacted with the phosphorylated S54 motif first and then interacted with the phosphorylated S73 motif when phosducin was dissociated from the phosducin/Gtβγ complex. These results provide insights into the molecular mechanism of phosphorylation‐regulated and 14‐3‐3ζ‐mediated inhibition of Phosducin binding to the G protein transducin complex.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant number: 21603121) and Hubei University of Technology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation States of PEA‐15 Control Binding Specificity and Regulate Cell Proliferation and Apoptosis
Authors: Crespo Sergio , Hassan Sherouk , Cabezas Andres , Wei Yufeng
Published: June 2021

Phosphoprotein enriched in astrocytes, 15 KDa (PEA‐15), is a small, cytoplasmic, death‐effector domain (DED) containing protein, found pervasively in mammals. It is ubiquitously expressed in different tissues, and has been known to be involved in several cellular pathways, including apoptosis and cell proliferation. This protein appeals to us and many other scientists because it is present in the cells in two different forms, phosphorylated PEA‐15 (at serine 104 and serine 116) and unphosphorylated PEA‐15, each interacting with different proteins at distinct biological pathways. PEA‐15 in its unphosphorylated state, interacts with ERK1/2 and prevents cell proliferation, while phosphorylated form interacts with Fas‐Associated Death Domain (FADD) protein and inhibits death receptor‐initiated apoptosis. Our initial NMR spectroscopic study showed that PEA‐15 undergoes conformational changes when it becomes phosphorylated at both serine residues, mimicked by serine to aspartic acid mutations (PEA‐15DD). The modulation of conformation switches the binding specificity of PEA‐15 from ERK1/2 to FADD, and changes its function from promoting cell death to cell survival. Based on our most recent results, we hypothesize that the DED conformation is allosterically controlled by the phosphorylation states of the C‐terminal tail, which in turn determines the binding specificity of the protein. Our ongoing research aims to broaden our understanding of the FADD/PEA‐15DD complex in vivo using NMR. In our phosphostasis model, we speculate that the balance between phosphorylated and unphosphorylated PEA‐15 is strictly regulated in different cell types and tissues to maintain the optimal cellular condition, and disruptions of the delicate balance could be detrimental to the cells, resulting in unregulated proliferation or elevated cell death.

Support or Funding Information

U.S. Education Department Minority Science & Engineering Improvement Program Grant # P120A160084 NIH AREA Grant #CA179410

USED Title III Part F, HSI‐STEM & Articulation Grant #P03C160155

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanism of Competitive Binding Between HIF‐1α and CITED2 with TAZ1
Authors: Gao Meng , Huang Yongqi
Published: June 2021

The cellular response to hypoxia is mediated by interactions between the α‐subunit of transcription factor HIF‐1 (HIF‐1α) and the TAZ1 domain of the transcriptional coactivators CBP and p300. CITED2 negatively regulates the transcriptional activity of HIF‐1. Structure studies have revealed that HIF‐1α and CITED2 bind to a partially overlapping surface of TAZ1 with similar affinity. Although the affinity of HIF‐1α and CITED2 for binding to TAZ1 is comparable, fluorescence and NMR experiments indicated that CITED2 efficiently displaced HIF‐1α from the HIF‐1α/TAZ1 complex. In this work, the binding mechanisms for HIF‐1α/TAZ1 and CITED2/TAZ1 interactions were studied by coarse‐grained molecular dynamics simulations. We found that the structure of HIF‐1α/TAZ1 complex was more dynamic than that of the CITED2/TAZ1 complex. Free energy analysis revealed that binding free energy barrier of the CITED2/TAZ1 complex was higher than that of HIF‐1α/TAZ1 complex, suggesting that the cooperativity of CITED2/TAZ1 interaction was stronger than that of HIF‐1α/TAZ1 interaction. Detailed analysis on the binding mechanism showed that CITED2 and HIF‐1α encountered different surfaces of TAZ1 to initiate the binding process. We further investigated the competitive binding between CITED2 and HIF‐1α with TAZ1 by carried out simulations using free CITED2 with HIF‐1α/TAZ1 complex, free HIF‐1α with CITED2/TAZ1 complex, and free CITED2, HIF‐1α and TAZ1. Consistent with previous NMR experiments, we observed formation of a ternary intermediate complex. Our results indicated that the dynamic interactions between HIF‐1α and TAZ1 allowed CITED2 efficiently displaced HIF‐1α from the HIF‐1α/TAZ1 complex.

Support or Funding Information

This work was supported by National Natural Science Foundation of China (Grant 21603121 to Y.H.) and Hubei University of Technology (M. G. and Y.H.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

This 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 Information

BU Undergraduate Research Opportunities Program; NIH R01 GM121673

This 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.

Objectives

The 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.

Methods

To 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.

Results

In 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.

Conclusions

We 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 Information

This 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 Information

UNF Office of Academic Affairs

This 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 Information

National 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 Information

This 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 Information

This 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 Information

This 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 Information

NSF STC CCC Grant: 1548297, Genentech Foundation MS Dissertation 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.
A novel use of water‐soluble CdSe Quantum Dots capped with GSH for Minute Virus of Mice prototype genome labeling
Authors: Rivera Joel , Arroyo Shirley Ann , Diffoot‐Carlo Nanette
Published: June 2021

Viruses can infect the host's cell through different mechanisms. Nevertheless, studies have shown that the viral entry pathway more common of viruses that affect humans is the interaction between glycoproteins of the envelope with receptors in the host's cell membrane. For many years, studies have tried to use fluorescent proteins to understand the mechanisms of a single viral particle, but it has not been effective because in order to emit fluorescence, strong enough to be detected, the viral particle must have many of the fluorescent proteins attached and these can alter the invasion of the host cell. Additionally, some do not have an envelope, and therefore for these types of viruses, the fluorescent proteins are not effective. To solve this problem, we are substituting fluorescent proteins for quantum dots (QDs). These QDs are made from different inorganic compounds that have shown to produce more fluorescence than the protein without requiring the use of many of them. This means that these QDs can help study the mechanisms of infection of a single viral particle. These particles are unstable in aqueous solution, and it can cause the production of toxic molecules for the cell. This obstacle has led new research in how one can modify the synthesis or the compound to make a more stable and effective QDs. In this research, we used a proposed protocol for the synthesis of QDs in a direct aqueous solution to obtain a more stable particle. Additionally, to make the QDs more stable for biological applications, we conjugated glutathione (GSH) to these particles. The GSH allowed the QDs to enter the cell and interact with the genome of the virus used for this study, the minute virus of mice (MVMp). Furthermore, we used the cell line MDA‐MB‐231 as a model to test our experimental approach. This study will help better understand the mechanisms of infection of a single MVMp particle. Additionally, it will open doors to study how this particle can be conjugated with other molecules such as DNA and encapsulated for gene therapy of different diseases.

Support or Funding Information

Financial support from the NIH RISE‐E‐BASE program grant number 1R25GM127191‐01.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activity‐dependent ATP release from neurons and astrocytes
Authors: Tantama Mathew
Published: June 2021

Adenosine triphosphate (ATP) is released as an autocrine and paracrine extracellular signal that can activate a diverse set of purinergic receptors, including P2X ion channels and P2Y G‐protein coupled receptors. For example, during synaptic transmission both neurons and astrocytes can release ATP in an activity‐dependent manner, but it remains a challenge to directly visualize these extracellular ATP dynamics. We are therefore developing genetically‐encoded fluorescent sensors to quantify changes in the levels of extracellular ATP, and we have now engineered a second‐generation sensor with higher affinity for ATP. Using co‐cultures of primary mouse neurons and astrocytes, we demonstrate that our new sensor can be used to detect neuron‐activity dependent release of ATP. We also use our new sensor to detect ATP release during in vitro injury models including hypoosmotic shock and chemical ischemia. Importantly, we also demonstrate that dual‐sensor imaging experiments can be used to correlate changes in extracellular ATP with changes in intracellular signaling, such as via calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) dynamics. Our results show that our sensor will be an effective tool for studying purinergic signaling in real‐time.

Support or Funding Information

NIH/NINDS R21 NS092010

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electrochemical Biosensor Targeting the Cancer Biomarker Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2)
Authors: Bonham Andrew J , Fetter Lisa
Published: June 2021

Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2) is a cell‐surface metalocatalyst which is expressed solely on malignancies of all major human cancer types. ENOX2, along with its ubiquitous parent protein ENOX1, serve as terminal oxidases in plasma membrane electron transport chains, suggesting that such proteins are essential for cell growth. ENOX2 is frequently shed from malignancies into the sera and demonstrates site‐specific transcript variants, and thus presents an attractive target for both diagnostic blood testing and anti‐cancer therapeutic treatments. To better facilitate detection and targeting of ENOX2 in patient sera, we utilized SELEX to generate aptamers against recombinantly expressed ENOX2. These aptamers were characterized via gel mobility shift assays to determine the candidate aptamer with highest affinity for human ENOX2. The candidate with highest affinity was incorporated into an electrochemical DNA‐based biosensor, where changes in DNA confirmation upon target binding result in changed dynamics of an appended redox reporter molecule, ultimately generating a rapid and quantitative change in the current of the electrochemical response. This novel ENOX2‐targeting electrochemical biosensor allows rapid, dose‐responsive readout for the presence of ENOX2 in both buffer and blood serum.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.