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  • 1
    Publication Date: 2018-10-27
    Description: The caspase recruitment domain–containing protein 9 (CARD9)–B-cell lymphoma/leukemia 10 (Bcl10) signaling axis is activated in myeloid cells during the innate immune response to a variety of diverse pathogens. This signaling pathway requires a critical caspase recruitment domain (CARD)–CARD interaction between CARD9 and Bcl10 that promotes downstream activation of factors, including NF-κB and the mitogen-activated protein kinase (MAPK) p38. Despite these insights, CARD9 remains structurally uncharacterized, and little mechanistic understanding of its regulation exists. We unexpectedly found here that the CARD in CARD9 binds to Zn2+ with picomolar affinity—a concentration comparable with the levels of readily accessible Zn2+ in the cytosol. NMR solution structures of the CARD9–CARD in the apo and Zn2+-bound states revealed that Zn2+ has little effect on the ground-state structure of the CARD; yet the stability of the domain increased considerably upon Zn2+ binding, with a concomitant reduction in conformational flexibility. Moreover, Zn2+ binding inhibited polymerization of the CARD9–CARD into helical assemblies. Here, we also present a 20-Å resolution negative-stain EM (NS-EM) structure of these filamentous assemblies and show that they adopt a similar helical symmetry as reported previously for filaments of the Bcl10 CARD. Using both bulk assays and direct NS-EM visualization, we further show that the CARD9–CARD assemblies can directly template and thereby nucleate Bcl10 polymerization, a capacity considered critical to propagation of the CARD9–Bcl10 signaling cascade. Our findings indicate that CARD9 is a potential target of Zn2+-mediated signaling that affects Bcl10 polymerization in innate immune responses.
    Print ISSN: 0021-9258
    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 2
    Publication Date: 2018-11-03
    Description: In many eukaryotes, translation initiation is regulated by proteins that bind to the mRNA cap–binding protein eukaryotic translation initiation factor 4E (eIF4E). These proteins commonly prevent association of eIF4E with eIF4G or form repressive messenger ribonucleoproteins that exclude the translation machinery. Such gene-regulatory mechanisms in plants, and even the presence of eIF4E-interacting proteins other than eIF4G (and the plant-specific isoform eIFiso4G, which binds eIFiso4E), are unknown. Here, we report the discovery of a plant-specific protein, conserved binding of eIF4E 1 (CBE1). We found that CBE1 has an evolutionarily conserved eIF4E-binding motif in its N-terminal domain and binds eIF4E or eIFiso4E in vitro. CBE1 thereby forms cap-binding complexes and is an eIF4E-dependent constituent of these complexes in vivo. Of note, plant mutants lacking CBE1 exhibited dysregulation of cell cycle–related transcripts and accumulated higher levels of mRNAs encoding proteins involved in mitosis than did WT plants. Our findings indicate that CBE1 is a plant protein that can form mRNA cap–binding complexes having the potential for regulating gene expression. Because mammalian translation factors are known regulators of cell cycle progression, we propose that CBE1 may represent such first translation factor–associated plant-specific cell cycle regulator.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 3
    Publication Date: 2018-12-15
    Description: RNA stem loop structures have been frequently shown to regulate essential cellular processes. The selenocysteine insertion sequence (SECIS) element, found in the 3′ UTRs of all selenoprotein mRNAs, is an example of such a structure, as it is required for the incorporation of the 21st amino acid, selenocysteine (Sec). Selenoprotein synthesis poses a mechanistic challenge because Sec is incorporated during translation in response to a stop codon (UGA). Although it is known that a SECIS-binding protein (SBP2) is required for Sec insertion, the mechanism of action remains elusive. Additional complexity is present in the synthesis of selenoprotein P (SELENOP), which is the only selenoprotein that contains multiple UGA codons and possesses two SECIS elements in its 3′ UTR. Thus, full-length SELENOP synthesis requires processive Sec incorporation. Using zebrafish Selenop, in vitro translation assays, and 75Se labeling in HEK293 cells, we found here that processive Sec incorporation is an intrinsic property of the SECIS elements. Specifically, we identified critical features of SECIS elements that are required for processive Sec incorporation. A screen of the human SECIS elements revealed that most of these elements support processive Sec incorporation in vitro; however, we also found that the processivity of Sec incorporation into Selenop in cells is tightly regulated. We propose a model for processive Sec incorporation that involves differential recruitment of SECIS-binding proteins.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 4
    Publication Date: 2018-09-01
    Description: Phenylalkylamines, such as the plant compounds ephedrine and pseudoephedrine and the animal neurotransmitters dopamine and adrenaline, compose a large class of natural and synthetic molecules with important physiological functions and pharmaceutically valuable bioactivities. The final steps of ephedrine and pseudoephedrine biosynthesis in members of the plant genus Ephedra involve N-methylation of norephedrine and norpseudoephedrine, respectively. Here, using a plant transcriptome screen, we report the isolation and characterization of an N-methyltransferase (NMT) from Ephedra sinica able to catalyze the formation of (pseudo)ephedrine and other naturally occurring phenylalkylamines, including N-methylcathinone and N-methyl(pseudo)ephedrine. Phenylalkylamine N-methyltransferase (PaNMT) shares substantial amino acid sequence identity with enzymes of the NMT family involved in benzylisoquinoline alkaloid (BIA) metabolism in members of the higher plant order Ranunculales, which includes opium poppy (Papaver somniferum). PaNMT accepted a broad range of substrates with phenylalkylamine, tryptamine, β-carboline, tetrahydroisoquinoline, and BIA structural scaffolds, which is in contrast to the specificity for BIA substrates of NMT enzymes within the Ranunculales. PaNMT transcript levels were highest in young shoots of E. sinica, which corresponded to the location of NMT activity yielding (pseudo)ephedrine, N-methylcathinone, and N-methyl(pseudo)ephedrine, and with in planta accumulation of phenylalkylamines. Co-expression of recombinant genes encoding PaNMT and an ω-transaminase (PP2799) from Pseudomonas putida in Escherichia coli enabled the conversion of exogenous (R)-phenylacetylcarbinol (PAC) and (S)-PAC to ephedrine and pseudoephedrine, respectively. Our work further demonstrates the utility of plant biochemical genomics for the isolation of key enzymes that facilitate microbial engineering for the production of medicinally important metabolites.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 5
    Publication Date: 2018-10-27
    Description: Naturally or surgically induced postmenopausal women are widely prescribed estrogen therapies to alleviate symptoms associated with estrogen loss and to lower the subsequent risk of developing metabolic diseases, including diabetes and nonalcoholic fatty liver disease. However, the molecular mechanisms by which estrogens modulate metabolism across tissues remain ill-defined. We have previously reported that 17β-estradiol (E2) exerts antidiabetogenic effects in ovariectomized (OVX) mice by protecting mitochondrial and cellular redox function in skeletal muscle. The liver is another key tissue for glucose homeostasis and a target of E2 therapy. Thus, in the present study we determined the effects of acute loss of ovarian E2 and E2 administration on liver mitochondria. In contrast to skeletal muscle mitochondria, E2 depletion via OVX did not alter liver mitochondrial respiratory function or complex I (CI) specific activities (NADH oxidation, quinone reduction, and H2O2 production). Surprisingly, in vivo E2 replacement therapy and in vitro E2 exposure induced tissue-specific effects on both CI activity and on the rate and topology of CI H2O2 production. Overall, E2 therapy protected and restored the OVX-induced reduction in CI activity in skeletal muscle, whereas in liver mitochondria E2 increased CI H2O2 production and decreased ADP-stimulated respiratory capacity. These results offer novel insights into the tissue-specific effects of E2 on mitochondrial function.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 6
    Publication Date: 2018-01-20
    Description: Parkinson's disease (PD) is associated with the formation of α-synuclein amyloid fibrils. Elucidating the role of these β-sheet-rich fibrils in disease progression is crucial; however, collecting detailed structural information on amyloids is inherently difficult because of their insoluble, non-crystalline, and polymorphic nature. Here, we show that Raman spectroscopy is a facile technique for characterizing structural features of α-synuclein fibrils. Combining Raman spectroscopy with aggregation kinetics and transmission electron microscopy, we examined the effects of pH and ionic strength as well as four PD–related mutations (A30P, E46K, G51D, and A53T) on α-synuclein fibrils. Raman spectral differences were observed in the amide-I, amide-III, and fingerprint regions, indicating that secondary structure and tertiary contacts are influenced by pH and to a lesser extent by NaCl. Faster aggregation times appear to facilitate unique fibril structure as determined by the highly reproducible amide-I band widths, linking aggregation propensity and fibril polymorphism. Importantly, Raman spectroscopy revealed molecular-level perturbations of fibril conformation by the PD–related mutations that are not apparent through transmission electron microscopy or limited proteolysis. The amide-III band was found to be particularly sensitive, with G51D exhibiting the most distinctive features, followed by A53T and E46K. Relating to a cellular environment, our data would suggest that fibril polymorphs can be formed in different cellular compartments and potentially result in distinct phenotypes. Our work sets a foundation toward future cellular Raman studies of amyloids.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 7
    Publication Date: 2018-06-09
    Description: Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties. Comprehensive studies of these attributes in a single scaffold have not been reported, making design and optimization of Ab fragments challenging. Here, we constructed libraries of 3E8, an Ab specific to tumor-associated glycoprotein 72 (TAG-72), a mucinous glycoprotein overexpressed in 80% of adenocarcinomas. We cloned, expressed, and characterized scFVs, diabodies, and higher-order multimer constructs with varying linker compositions, linker lengths, and domain orientations. These constructs dramatically differed in their oligomeric states and stabilities, not only because of linker and orientation but also related to the purification method. For example, protein L–purified constructs tended to have broader distributions and higher oligomeric states than has been reported previously. From this library, we selected an optimal construct, 3E8.G4S, for biodistribution and pharmacokinetic studies and in vivo xenograft mouse PET imaging. These studies revealed significant tumor targeting of 3E8.G4S with a tumor-to-background ratio of 29:1. These analyses validated 3E8.G4S as a fast, accurate, and specific tumor-imaging agent.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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  • 8
    Publication Date: 2018-06-16
    Description: Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. Methanosarcina acetivorans is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster. The results reported here support a mechanism for FDR similar to that reported for FTR with notable exceptions. Unlike FTR, FDR contains a rubredoxin [1Fe-0S] center postulated to mediate electron transfer from ferredoxin to the active-site [4Fe-4S] cluster. UV-visible, EPR, and Mössbauer spectroscopic data indicated that two-electron reduction of the active-site disulfide in FDR involves a one-electron-reduced [4Fe-4S]1+ intermediate previously hypothesized for FTR. Our results support a role for an active-site tyrosine in FDR that occupies the equivalent position of an essential histidine in the active site of FTR. Of note, one of seven Trxs encoded in the genome (Trx5) and methanoredoxin, a glutaredoxin-like enzyme from M. acetivorans, were reduced by FDR, advancing the physiological understanding of FDR's role in the redox metabolism of methanoarchaea. Finally, bioinformatics analyses show that FDR homologs are widespread in diverse microbes from the domain Bacteria.
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    Topics: Biology , Chemistry and Pharmacology
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  • 9
    Publication Date: 2018-06-23
    Description: 4-1BBL is a member of the tumor necrosis factor (TNF) superfamily and is the ligand for the TNFR superfamily receptor, 4-1BB. 4-1BB plays an immunomodulatory role in T cells and NK cells, and agonists of this receptor have garnered strong attention as potential immunotherapy agents. Broadly speaking, the structural features of TNF superfamily members, their receptors, and ligand-receptor complexes are similar. However, a published crystal structure of human 4-1BBL suggests that it may be unique in this regard, exhibiting a three-bladed propeller-like trimer assembly that is distinctly different from that observed in other family members. This unusual structure also suggests that the human 4-1BB/4-1BBL complex may be structurally unique within the TNF/TNFR superfamily, but to date no structural data have been reported. Here we report the crystal structure of the human 4-1BB/4-1BBL complex at 2.4-Å resolution. In this structure, 4-1BBL does not adopt the unusual trimer assembly previously reported, but instead forms a canonical bell-shaped trimer typical of other TNF superfamily members. The structure of 4-1BB is also largely canonical as is the 4-1BB/4-1BBL complex. Mutational data support the 4-1BBL structure reported here as being biologically relevant, suggesting that the previously reported structure is not. Together, the data presented here offer insight into structure/function relationships in the 4-1BB/4-1BBL system and improve our structural understanding of the TNF/TNFR superfamily more broadly.
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    Topics: Biology , Chemistry and Pharmacology
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  • 10
    Publication Date: 2018-03-31
    Description: The copper-containing superoxide dismutases (SODs) represent a large family of enzymes that participate in the metabolism of reactive oxygen species by disproportionating superoxide anion radical to oxygen and hydrogen peroxide. Catalysis is driven by the redox-active copper ion, and in most cases, SODs also harbor a zinc at the active site that enhances copper catalysis and stabilizes the protein. Such bimetallic Cu,Zn-SODs are widespread, from the periplasm of bacteria to virtually every organelle in the human cell. However, a new class of copper-containing SODs has recently emerged that function without zinc. These copper-only enzymes serve as extracellular SODs in specific bacteria (i.e. Mycobacteria), throughout the fungal kingdom, and in the fungus-like oomycetes. The eukaryotic copper-only SODs are particularly unique in that they lack an electrostatic loop for substrate guidance and have an unusual open-access copper site, yet they can still react with superoxide at rates limited only by diffusion. Copper-only SOD sequences similar to those seen in fungi and oomycetes are also found in the animal kingdom, but rather than single-domain enzymes, they appear as tandem repeats in large polypeptides we refer to as CSRPs (copper-only SOD-repeat proteins). Here, we compare and contrast the Cu,Zn versus copper-only SODs and discuss the evolution of copper-only SOD protein domains in animals and fungi.
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    Electronic ISSN: 1083-351X
    Topics: Biology , Chemistry and Pharmacology
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