Selinsky ACS Philadelphia August 2016
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This document discusses the characterization of bacterial cyclooxygenase (COX) proteins identified in the Peroxibase database. The researchers have cloned and expressed COX proteins from Nostoc and Rhodobacter species, but characterization has been difficult due to insolubility. They are also cloning putative COX proteins from Mycobacterium and Nitrosomonas and will present recent advances. Sequence alignments show conserved active site residues between mammalian, algal, and bacterial COX proteins. Structural predictions suggest the Nostoc COX protein adopts a similar fold to sheep COX-1. Future work involves characterizing the enzymatic activity of bacterial COX homologs and determining their crystal structures.
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govern ligand access to peroxisome proliferator activated receptor (PPARγ). Activation of
PPARγ is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9α,11β-
PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti-
inflammatory drugs suggesting that the cancer chemopreventive properties of these agents
may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these
effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and
9α,11β-PGF2. Structure-activity relationships Thirty-five crystal structures of AKR1C3·NADP + ·inhibitor complexes exist in the PDB. Inspection of these structures shows that if the inhibitor contains a carboxylic acid, it can
often form hydrogen bonds with the catalytic tetrad members Tyr55 and His117. Other
portions of the inhibitor can occupy one of several subpockets (SP), e.g. SP1 Ser118,
Asn167, Phe306, Phe311, and Tyr319 (e.g. occupied by the B-ring of N- cphenylaminobenzoates). The SP2 sub-pocket refers to Ser129, W227, and F311 (e.g.
occupied by the side-chain of PGs), and the SP3 sub-pocket which contains Y24, E192,
S217, S221, Q222, Y305, and F306. While the presence of these sub-pockets can be rationalized to determine the binding mode and can be used as the basis of docking studies,
2014 DURF CONFERENCE POSTER
This study investigated a single nucleotide variation (SNV) in the p70 ribosomal S6 kinase 1 (S6K1) gene found in autistic individuals. The researchers introduced the SNV, which changes the amino acid glutamate to glutamine at position 44 (E44Q), into human S6K1 cDNA. When expressed in HEK 293 cells, the mutant S6K1 showed reduced phosphorylation of downstream targets eEF2 and S6 compared to wild-type S6K1 following insulin stimulation. However, no difference was found in basal protein synthesis between wild-type and mutant transfected cells. Further studies are needed to better characterize the impact of this S6K1 mutation on protein synthesis and other
ProteinSci2007
The document describes an experiment where disulfide bonds were engineered into a1-antitrypsin (a1-AT) to lock the movement of helix F (hF) and the connecting loop (thFs3A) during protease inhibition. Disulfide bonds were introduced between thFs3A and strands 3A or 5A of the beta-sheet. The disulfide between thFs3A and strand 5A, but not 3A, eliminated inhibitory activity, suggesting displacement from strand 5A is required for inhibition. The disulfide between thFs3A and strand 3A slowed polymerization without affecting inhibition. This provides direct evidence that hF/thFs3A displacement from strand 5
103BL-F14_PosterSmallQuality_PhageHunters
1) Researchers isolated and characterized a novel bacteriophage (Horchata) that infects Mycobacterium smegmatis from a soil sample.
2) Transmission electron microscopy revealed that Horchata has an icosahedral head and long non-contractile tail, characteristic of the Siphoviridae morphology.
3) Genome sequencing and annotation showed that Horchata has a 67,863 bp genome with 30 genes of putative function and 71 genes of unknown function.
replicación 2010.pdf
This document summarizes the history of research on the archaeal DNA replication machinery, from early biochemical studies in the 1980s to more recent genetic studies. Key developments include the following:
1) Early studies in the 1980s focused on isolating and characterizing thermostable DNA polymerases from archaea.
2) Genome sequencing of the first archaeon in 1996 revealed that archaeal replication proteins are more similar to eukaryotes, spurring further biochemical study of these proteins.
3) Recent applications of genetic tools in archaea have identified new replication proteins and helped elucidate the cellular roles of known proteins like DNA polymerases.
4) Reconstituting an in vitro archaeal DNA replication
Biosensors and Bioelectr
This document describes the development of a novel fluorescent protein-based sensor for detecting 2-oxoglutarate (2OG) levels in living cells. The sensor, termed mOGsor, was created by inserting the 2OG-binding domain GAF from the NifA protein into yellow fluorescent protein (YFP). mOGsor exhibits increased fluorescence intensity upon binding to 2OG in a concentration-dependent manner. Testing showed mOGsor has high specificity for 2OG and fast kinetics. Using mOGsor, the authors were able to monitor real-time changes in 2OG levels in E. coli cells under different nutrient conditions. mOGsor represents an improvement over previous FRET-based 2OG sensors by providing a
publication 3
This document discusses the role of the guanine nucleotide exchange factor C3G in neuronal differentiation. It finds that C3G protein levels increase when human neuroblastoma cells are induced to differentiate through serum starvation or treatment with forskolin or nerve growth factor. Overexpression of C3G stimulates neurite growth and increases responsiveness to differentiation signals, in a process dependent on C3G's catalytic domain and the functions of Rap1 and Cdc42. Knockdown of C3G inhibits forskolin- and nerve growth factor-induced differentiation and enhances cell death from serum starvation. C3G phosphorylation and localization to the Golgi are increased by forskolin and nerve growth factor treatment, and C3G
PIIS2211124716313158
1) Loss of the kinase NEK1 in mice leads to abnormal retention of the cohesin component SMC3 on chromosome arms during meiotic prophase I.
2) Mass spectrometry analysis found that WAPL, a protein involved in cohesin removal, has abnormal elevated phosphorylation at a specific residue in NEK1-deficient mice.
3) This suggests NEK1 may regulate WAPL and cohesin removal during meiosis, though not directly as its loss leads to increased rather than decreased WAPL phosphorylation. NEK1 likely acts through another protein to phosphorylate or dephosphorylate WAPL.
PhD Thesis
- The author conducted a review of literature on the physiological significance of multiple hexokinase (HK) isoforms in plants. HK exists as several isoforms across plant organs and tissues, but the roles of each isoform are not well understood.
- The author then characterized ScHK2, an HK from Solanum chacoense, determining its kinetic properties after cloning, sequencing, expressing and purifying the recombinant enzyme. This was the first kinetic characterization of a pure recombinant plant HK.
- Finally, the author manipulated HK activity levels in potato roots via transformation with ScHK2 constructs to study HK's role in glycolytic regulation and flux control. 23 root clones with varying
Choo et al., 2004
This document provides evidence that common machinery is utilized by the early and late RNA localization pathways in Xenopus oocytes. It presents four key findings: 1) Early and late pathway RNAs require the same short sequence motifs for localization. 2) Competition assays show early and late RNAs compete for common localization factors in vivo. 3) A late localization factor, Vg RBP/Vera, binds specifically to localization elements of early pathway RNAs. 4) Confocal imaging reveals early RNAs associate with microtubules, suggesting transport plays a role in both pathways. Together, these findings suggest the early and late pathways share basal localization factors throughout oogenesis.
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and Back
Slides presented at the University of Edinburgh in Jan 2014, based on our paper "A tale of two drug targets: the evolutionary history of BACE1 and BACE2" http://www.ncbi.nlm.nih.gov/pubmed/24381583
The beta amyloid (APP) cleaving enzyme (BACE1) has been a drug target for Alzheimer's Disease (AD) since 1999 with lead inhibitors now entering clinical trials. In 2011, the paralog, BACE2, became a new target for type II diabetes (T2DM) having been identified as a TMEM27 secretase regulating pancreatic β cell function. However, the normal roles of both enzymes are unclear. This study outlines their evolutionary history and new opportunities for functional genomics. We identified 30 homologs (UrBACEs) in basal phyla including Placozoans, Cnidarians, Choanoflagellates, Porifera, Echinoderms, Annelids, Mollusks and Ascidians (but not Ecdysozoans). UrBACEs are predominantly single copy, show 35–45% protein sequence identity with mammalian BACE1, are ~100 residues longer than cathepsin paralogs with an aspartyl protease domain flanked by a signal peptide and a C-terminal transmembrane domain. While multiple paralogs in Trichoplax and Monosiga pre-date the nervous system, duplication of the UrBACE in fish gave rise to BACE1 and BACE2 in the vertebrate lineage. The latter evolved more rapidly as the former maintained the emergent neuronal role. In mammals, Ka/Ks for BACE2 is higher than BACE1 but low ratios for both suggest purifying selection. The 5' exons show higher Ka/Ks than the catalytic section. Model organism genomes show the absence of certain BACE human substrates when the UrBACE is present. Experiments could thus reveal undiscovered substrates and roles. The human protease double-target status means that evolutionary trajectories and functional shifts associated with different substrates will have implications for the development of clinical candidates for both AD and T2DM. A rational basis for inhibition specificity ratios and assessing target-related side effects will be facilitated by a more complete picture of BACE1 and BACE2 functions informed by their evolutionary context.
A high-resolution protein architecture of the budding yeast genome.pdf
The genome-wide architecture of chromatin-associated proteins that maintains chromosome integrity and gene regulation is not well defined. Here we use chromatin immunoprecipitation, exonuclease digestion and DNA sequencing (ChIP–exo/seq)1,2 to define this architecture in Saccharomyces cerevisiae. We identify 21 meta- assemblages consisting of roughly 400 different proteins that are related to DNA replication, centromeres, subtelomeres, transposons and transcription by RNA polymerase (Pol) I, II and III. Replication proteins engulf a nucleosome, centromeres lack a nucleosome, and repressive proteins encompass three nucleosomes at subtelomeric X-elements. We find that most promoters associated with Pol II evolved to lack a regulatory region, having only a core promoter. These constitutive promoters comprise a short nucleosome-free region (NFR) adjacent to a +1 nucleosome, which together bind the transcription-initiation factor TFIID to form a preinitiation complex. Positioned insulators protect core promoters from upstream events. A small fraction of promoters evolved an architecture for inducibility, whereby sequence-specific transcription factors (ssTFs) create a nucleosome- depleted region (NDR) that is distinct from an NFR. We describe structural interactions among ssTFs, their cognate cofactors and the genome. These interactions include the nucleosomal and transcriptional regulators RPD3-L, SAGA, NuA4, Tup1, Mediator and SWI–SNF. Surprisingly, we do not detect interactions between ssTFs and TFIID, suggesting that such interactions do not stably occur. Our model for gene induction involves ssTFs, cofactors and general factors such as TBP and TFIIB, but not TFIID. By contrast, constitutive transcription involves TFIID but not ssTFs engaged with their cofactors. From this, we define a highly integrated network of gene regulation by ssTFs.
Fall project
1) The study analyzed the effects of Csk knockouts on development of the initial segment of the mouse epididymis. Csk knockout was expected to promote cell proliferation and differentiation through increased ERK pathway activity due to lack of inhibition of SRC kinases.
2) A tissue-specific Csk conditional knockout mouse model was generated using Cre/lox recombination. Genotyping identified one mouse with the desired genotype.
3) Preliminary results found increased vasculogenesis in the initial segment of Csk knockout mice, suggesting effects on differentiation through the ERK pathway. Immunofluorescence found decreased activity of phospho-SRC in knockouts while other markers were similar to controls.
Single Nucleotide Polymorphism - The new generation therapy
Single Nucleotide Polymorphism - The new generation therapySwami Viekananda Institute of Modern Sciences
This presentation aims at giving a vivid knowledge about Nucleoli, a sub organelle of Nucleus, its role in protein formation. control, localization ad how specifically it is involved in Single Nucleotide polymorphism. The slide also discusses about the involvement of SNPs in Alzheimer’s Disease.
Similar to Selinsky ACS Philadelphia August 2016 (20)
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and Back
The Beta Amyloid Cleaving Enzymes: From Drug Discovery to Evolution and Back
A high-resolution protein architecture of the budding yeast genome.pdf
A high-resolution protein architecture of the budding yeast genome.pdf
Single Nucleotide Polymorphism - The new generation therapy
Single Nucleotide Polymorphism - The new generation therapy
Selinsky ACS Philadelphia August 2016
- 1. Cyclooxygenases • Cyclooxygenases (COX; Prostaglandin H2 synthase) exists as two isoforms in most organisms, a constitutive form (COX-1) and an inducible form (COX-2). • Both COX-1 and COX-2 convert arachidonic acid into prostaglandin G2 with its cyclooxygenase activity, and then into prostaglandin H2 via its peroxidase activity. • COX is a dimeric monotopic membrane protein in higher organisms. • A COX protein was recently expressed from an open reading frame found in the red algae Gracilaria vermiculophylla, and was found to possess both cyclooxygenase and peroxidase activities (1). • Putative bacterial COX proteins are predicted to be structurally similar to their mammalian homologs, but are missing structural features involved in protein dimerization and membrane binding (2,3). Progress on the Characterization of Bacterial Prostaglandin H Synthases Identified in Peroxibase Peroxibase is a database generated for the evolutionary analysis of peroxidases (Fawal, N. Li, Q., et al., Nucleic Acids Research, 2013, 41:D441- 444). Thirteen bacterial open reading frames (ORFs) are identified in Peroxibase that encode for proteins predicted to structurally resemble mammalian prostaglandin H synthase. Our lab has begun the systematic cloning and expression of these bacterial ORFs, with characterization of the expressed protein products. Proteins from Nostoc and Rhodobacter species have been expressed, but their characterization has been hampered by protein insolubility. Cloning of putative prostaglandin synthases from the Mycobacterium and Nitrososomas genomes is in progress, and recent advances on these systems will be presented. Margaret Butchy, Stephanie Neumann, Caytlin Nichols, Rebecca Skaf, and Barry S Selinsky Purification of the Nostoc protein. The open reading frame was inserted into a pET16 expression vector with a C- terminal (his)6 tag. Lane 1 – Molecular weight ladder Lane 2 - Total cell protein Lane 3 - cleared cell lysate Lane 4 - Nostoc COX- homolog The final product (purified in 0.3% CHAPS) is greater than 95% pure. The open reading frame from Rhodobacter has been purified to similar homogeneity. References 1. Varvas K, Kasvandik S, Hansen K, Jarving I, Morell, Samel N (2013) Biochim. Biophys. Acta 1831, 863-871. 2. Fawal N, Li Q, et al. (2013) Nucleic Acids Res., 41, D441-444. 3. Gupta K, Selinsky BS (2015) Biochim. Biophys. Acta, 1848, 83-94. 4. Thuresson ED, Lakkides KM et al. (2001) J. Biol. Chem. 276, 10347- 10359. 5. Brash AR, Niraula NP, Boeglin WE, Mashhadi Z (2014) J. Biol. Chem. 289, 13101-13111. 6. Kelley, L.A., Sternberg, M.J. (2009) Protein structure prediction on the Web: a case study using the Phyre server, Nature Protocols, 4, 363-371 Goals of this Study • To complete sequence comparisons of mammalian COX-1 (sheep), algal, and bacterial proteins predicted to possess cyclooxygenase activity. • To perform structural predictions on the algal and bacterial COX proteins to help predict structural similarities. • To express and purify bacterial COX proteins, to ultimately determine their enzymatic activities and three dimensional structures. Sequence comparisons – active site Structural Predictions: The predicted structure for the Nostoc protein (left, in red), the calculated structure for ovine COX (center, in green), and an overlay of the two structures (right). The prokaryotic homologs of PGHS were threaded onto the ovine PGHS-1 template structure (PDB ID: 1Q4G) using the PHYRE v2.0 server (6). The resulting models were gradually relaxed by energy minimization in a solvated environment using the programs VMD and NAMD, using CHARMM42 force fields. The overall quality of this minimized model was evaluated using the MOLPROBITY server. While the EGF-like region of ovine COX-1 (left side of overlay) lacks structural homology with the Nostoc protein, as predicted, the remaining structural elements superimpose with only minor variability. Future Plans • Characterize the enzymatic activity of the bacterial COX homologs. • Attempt crystallization of the bacterial proteins for X-ray structural determination. Abstract Active site: Sequence alignments were performed using ClustalW; graphical output generated in GeneDoc. A conserved Y (541; Y385 in ovine COX-1) is absolutely required for catalytic activity; this residue is conserved in the five homologs. A nearby H (544; 388 in ovine COX-1) is involved in heme binding and is also conserved. Other conserved residues are F527, P538, E (or D) at 536, and hydrophobic residues at 523, 530, 533, and 537. W533, L540, and F527 are required for full enzymatic activity (4). Membrane binding region: There is little sequence similarity here. The periodicity of hydrophobic/hydrophilic residues suggests amphipathic helices in this region. There are four amphipathic helices in the ovine protein that anchor the protein to the endoplasmic reticulum membrane. Heme binding: In the ovine protein, H252 (H207 in ovine COX-1) represents the distal heme residue involved in deprotonation/protonation reactions in the peroxidase reaction. The alternate D residue could perform the same function in proteins from the other organisms, or could indicate that these proteins do not possess peroxidase activity. The Nostoc homolog does lack peroxidase activity (5). Sequence comparisons – heme binding Sequence comparisons – membrane binding Chemistry Department, Villanova University Villanova, PA 19085 Protein expression and purification Elution from Ni-NTA resin. Eluted purified protein contains bound heme, suggesting that it has folded correctly. Activity measurements are forthcoming; this protein has been previously characterized as a linoleate dioxygenase (5).