In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the “off signal” afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5′NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.

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C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

Biochemical Journal ◽

10.1042/bj3360367 ◽

1998 ◽

Vol 336 (2) ◽

pp. 367-371 ◽

Cited By ~ 30

Author(s):

Leen AMERY ◽

Chantal BREES ◽

Myriam BAES ◽

Chiaki SETOYAMA ◽

Retsu MIURA ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Amino Acid ◽

Fluorescent Protein ◽

Consensus Sequence ◽

C Terminus ◽

Targeting Signal ◽

Fluorescent Pattern ◽

Green Fluorescent ◽

Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

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Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

Journal of Clinical Microbiology ◽

10.1128/jcm.03491-14 ◽

2015 ◽

Vol 53 (6) ◽

pp. 1797-1805 ◽

Cited By ~ 5

Author(s):

Alyssa K. Van Dreumel ◽

Wojtek P. Michalski ◽

Leanne M. McNabb ◽

Brian J. Shiell ◽

Nagendrakumar B. Singanallur ◽

...

Keyword(s):

Amino Acid ◽

Diagnostic Test ◽

Nonstructural Protein ◽

Consensus Sequence ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Amino Acid Consensus Sequence ◽

Fmdv Serotypes ◽

Foot And Mouth

An amino acid consensus sequence for the seven serotypes of foot-and-mouth disease virus (FMDV) nonstructural protein 3B, including all three contiguous repeats, and its use in the development of a pan-serotype diagnostic test for all seven FMDV serotypes are described. The amino acid consensus sequence of the 3B protein was determined from a multiple-sequence alignment of 125 sequences of 3B. The consensus 3B (c3B) protein was expressed as a soluble recombinant fusion protein with maltose-binding protein (MBP) using a bacterial expression system and was affinity purified using amylose resin. The MBP-c3B protein was used as the antigen in the development of a competition enzyme-linked immunosorbent assay (cELISA) for detection of anti-3B antibodies in bovine sera. The comparative diagnostic sensitivity and specificity at 47% inhibition were estimated to be 87.22% and 93.15%, respectively. Reactivity of c3B with bovine sera representing the seven FMDV serotypes demonstrated the pan-serotype diagnostic capability of this bioreagent. The consensus antigen and competition ELISA are described here as candidates for a pan-serotype diagnostic test for FMDV infection.

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Balance between mutually exclusive traits shifted by variants of a yeast transcription factor

10.1101/117911 ◽

2017 ◽

Cited By ~ 1

Author(s):

Michael W. Dorrity ◽

Josh T. Cuperus ◽

Jolie A. Carlisle ◽

Stanley Fields ◽

Christine Queitsch

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Amino Acid ◽

Dna Binding ◽

Single Amino Acid ◽

Environmental Cues ◽

Dna Binding Domain ◽

Independent Manner ◽

Half Site

AbstractIn Saccharomyces cerevisiae, the decision to mate or invade relies on environmental cues that converge on a shared transcription factor, Ste12. Specificity toward invasion occurs via Ste12 binding cooperatively with the co-factor Tec1. Here, we characterize the in vitro binding preferences of Ste12 to identify a defined spacing and orientation of dimeric sites, one that is common in pheromone-regulated genes. We find that single amino acid changes in the DNA-binding domain of Ste12 can shift the preference of yeast toward either mating or invasion. These mutations define two distinct regions of this domain, suggesting alternative modes of DNA binding for each trait. Some exceptional Ste12 mutants promote hyperinvasion in a Tec1-independent manner; these fail to bind cooperative sites with Tec1 and bind to unusual dimeric Ste12 sites that contain one highly degenerate half site. We propose a model for how activation of invasion genes could have evolved with Ste12 alone.

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Selection of the same mutation in the U69 protein kinase gene of human herpesvirus-6 after prolonged exposure to ganciclovir in vitro and in vivo

Journal of General Virology ◽

10.1099/0022-1317-82-11-2767 ◽

2001 ◽

Vol 82 (11) ◽

pp. 2767-2776 ◽

Cited By ~ 59

Author(s):

Chaysavanh Manichanh ◽

Camille Olivier-Aubron ◽

Jean-Pierre Lagarde ◽

Jean-Thierry Aubin ◽

Phillipe Bossi ◽

...

Keyword(s):

Amino Acid ◽

Protein Kinase ◽

Amino Acid Substitution ◽

Consensus Sequence ◽

Human Herpesvirus ◽

Wild Type ◽

Human Herpesvirus 6 ◽

Genotypic Analysis ◽

Herpesvirus 6

After serial passage in the presence of increasing concentrations of ganciclovir (GCV) in vitro, a human herpesvirus-6 (HHV-6) mutant exhibiting a decreased sensitivity to the drug was isolated. Analysis of drug susceptibility showed that the IC50 of this mutant was 24-, 52- and 3-fold higher than that of the wild-type (wt) IC50 in the case of GCV, cidofovir and foscarnet, respectively. Genotypic analysis showed two single nucleotide changes as compared to the wild-type: an A→G substitution of the U69 protein kinase (PK) gene resulted in an M318V amino acid substitution and the other change, located in the C-terminal part of the U38 gene, resulted in an A961V amino acid substitution within the DNA polymerase. The M318V change was located within the consensus sequence DISPMN of the putative catalytic domain VI of the PK. This change was homologous to the M460V and M460I changes that had been reported previously within the consensus sequence DITPMN of the human cytomegalovirus (HCMV) UL97 PK and associated with the resistance of HCMV to GCV. The M318V change was also detected by PCR in HHV-6-infected PBMCs from an AIDS patient who had been treated with GCV for a long period of time and exhibited a clinically GCV-resistant HCMV infection. These findings provide strong circumstantial evidence that the M318V change of the PK gene is associated with resistance to GCV and raise the question of cross resistance to this drug among different betaherpesviruses.

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Mutations acquired during cell culture isolation may affect antigenic characterisation of influenza A(H3N2) clade 3C.2a viruses

Eurosurveillance ◽

10.2807/1560-7917.es.2016.21.3.30112 ◽

2016 ◽

Vol 21 (3) ◽

Cited By ~ 24

Author(s):

Danuta M Skowronski ◽

Suzana Sabaiduc ◽

Catharine Chambers ◽

Alireza Eshaghi ◽

Jonathan B Gubbay ◽

...

Keyword(s):

Cell Culture ◽

Amino Acid ◽

Influenza A ◽

Consensus Sequence ◽

Haemagglutination Inhibition ◽

Genetic Mutations ◽

In Vitro Cell Culture ◽

Antigenic Relatedness ◽

Cell Culture Isolation

As elsewhere, few (< 15%) sentinel influenza A(H3N2) clade 3C.2a viruses that dominated in Canada during the 2014/15 season could be antigenically characterised by haemagglutination inhibition (HI) assay. Clade 3C.2a viruses that could be HI-characterised had acquired genetic mutations during in vitro cell culture isolation that modified the potential glycosylation motif found in original patient specimens and the consensus sequence of circulating viruses at amino acid positions 158–160 of the haemagglutinin protein. Caution is warranted in extrapolating antigenic relatedness based on limited HI findings for clade 3C.2a viruses that continue to circulate globally.

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ywfE in Bacillus subtilis Codes for a Novel Enzyme, l-Amino Acid Ligase

Journal of Bacteriology ◽

10.1128/jb.187.15.5195-5202.2005 ◽

2005 ◽

Vol 187 (15) ◽

pp. 5195-5202 ◽

Cited By ~ 73

Author(s):

Kazuhiko Tabata ◽

Hajime Ikeda ◽

Shin-ichi Hashimoto

Keyword(s):

Amino Acids ◽

Bacillus Subtilis ◽

Amino Acid ◽

High Performance ◽

Consensus Sequence ◽

Reaction Products ◽

In Silico Screening ◽

Vitro Assay ◽

Hydrolysis Of

ABSTRACT The ATP-dependent carboxylate-amine/thiol ligase superfamily is known to contain enzymes catalyzing the formation of various types of peptide, such as d-alanyl-d-alanine, polyglutamate, and γ-peptide, but, curiously, no enzyme synthesizing α-dipeptides of l-amino acids is known. We attempted to find such an enzyme. By in silico screening based on the consensus sequence of the superfamily followed by an in vitro assay with purified enzyme to avoid the degradation of the peptide(s) synthesized, ywfE of Bacillus subtilis was found to code for the activity forming l-alanyl-l-glutamine from l-alanine and l-glutamine with hydrolysis of ATP to ADP. No AMP was formed, supporting the idea that the enzyme belongs to the superfamily. Surprisingly, the enzyme accepted a wide variety of l-amino acids. Among 231 combinations of l-amino acids tested, reaction products were obtained for 111 combinations and 44 kinds of α-dipeptides were confirmed by high-performance liquid chromatography analyses, while no tripeptide or longer peptide was detected and the d-amino acids were inert. From these results, we propose that ywfE encodes a new member of the superfamily, l-amino acid ligase.

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Genetic Variation and Susceptibilities to Protease Inhibitors among Subtype B and F Isolates in Brazil

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.2.253 ◽

1999 ◽

Vol 43 (2) ◽

pp. 253-258 ◽

Cited By ~ 14

Author(s):

Amilcar Tanuri ◽

Ana C. P. Vicente ◽

Koko Otsuki ◽

Carlos A. Ramos ◽

Orlando C. Ferreira ◽

...

Keyword(s):

Genetic Variation ◽

Amino Acid ◽

Protease Inhibitors ◽

Consensus Sequence ◽

Antiretroviral Drugs ◽

Protease Gene ◽

Subtype B ◽

Hiv 1 ◽

The U.S

ABSTRACT The genetic variation of the human immunodeficiency virus type 1 (HIV-1) protease gene (prt) permits the classification of HIV-1 strains into five distinct protease subtypes, which follow thegag subtyping patterns. The susceptibilities of non-B-subtype strains to protease inhibitors (PIs) and other antiretroviral drugs remain largely unknown. Subtype F is the main non-B strain contributing to the Brazilian epidemic, accounting for 15 to 20% of these infections. In this work, we report the findings on 81 isolates from PI-naive Brazilian patients collected between 1993 and 1997. In addition, the relevant PI resistance mutations and their phenotypes were determined in vitro for 15 of these patients (B = 9 and F = 6). Among these, the subtype F samples evidenced high sensitivities in vitro to ritonavir and indinavir, with MICs at which 50 and 90% of the isolates are inhibited similar to those of both the Brazilian and the U.S. subtype B isolates. Analysis of the 81 Brazilianprt sequences demonstrated that the subtype F consensus sequence differs from the U.S. and Brazilian subtype B consensus in eight positions (I15V, E35D, M36I, R41K, R57K, Q61N, L63P, and L89M). The frequency of critical PI resistance substitutions (amino acid changes D30N, V82A/F/T, I84V, N88D, and L90M) among Brazilian isolates is very low (mean, 2.5%), and the associated secondary substitutions (amino acid positions 10L, 20K, 36M, 46M, 48G, 54I, 63P, 71A, and 77A) are infrequent. These observations document the relative rarity of resistance to PIs in the treatment of patients infected with HIV-1 subtype F in South America.

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Phosphorylation of Xenopus transcription factor IIIA by an oocyte protein kinase CK2

Biochemical Journal ◽

10.1042/bj3620375 ◽

2002 ◽

Vol 362 (2) ◽

pp. 375-382 ◽

Cited By ~ 5

Author(s):

Cara J. WESTMARK ◽

Romi GHOSE ◽

Paul W. HUBER

Keyword(s):

Transcription Factor ◽

Amino Acid ◽

Protein Kinase ◽

Protein Kinase Ck2 ◽

Consensus Sequence ◽

Specific Substrate ◽

Recognition Sequence ◽

Transcription Factor Iiia ◽

Kinase Ck2

Transcription factor IIIA (TFIIIA), isolated from the cytoplasmic 7S ribonucleoprotein complex of Xenopus oocytes, is phosphorylated when incubated with [γ-32P]ATP. This modification is due to a trace kinase activity that remains associated with the factor through several steps of purification. The kinase can use either ATP or GTP, and will phosphorylate casein and phosvitin to the exclusion of TFIIIA. The kinase is reactive with a ten-amino-acid peptide that is a specific substrate for protein kinase CK2 (CK2; formerly casein kinase II). In addition, inhibition of phosphorylation by heparin and stimulation by spermidine indicate that the activity can be ascribed to CK2. Phospho amino acid analysis established that serine is the sole phosphoryl acceptor in TFIIIA. There are four consensus sites for CK2 in TFIIIA; all contain serine residues at the putative site of phosphorylation. TFIIIA immunoprecipitated from oocytes, which were incubated with [32P]orthophosphate, is also phosphorylated exclusively on serine residues. Only the cyanogen bromide fragment, which was derived from the N-terminal end of TFIIIA, is labelled in vivo. A recognition sequence for CK2, located at Ser16 in the β-turn of the first zinc-finger domain, is the only protein kinase consensus sequence present in this peptide. Assays in vitro with site-specific mutants of TFIIIA established that Ser16 is the preferred site of phosphorylation, with some secondary modification at Ser314.

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1067. Identifying and Assaying New Potential Molecular Targets in Fungi Following a Novel Strategy Based on Binding Site (Dis)Similarities with Proteins of the Human Pharmacolome

Open Forum Infectious Diseases ◽

10.1093/ofid/ofab466.1261 ◽

2021 ◽

Vol 8 (Supplement_1) ◽

pp. S626-S626

Author(s):

Johann E Bedoya-Cardona ◽

Marcela Rubio-Carrasquilla ◽

Mario S Valdes-Tresanco ◽

Iliana M Ramirez-Velasquez ◽

Ernesto Moreno

Keyword(s):

Amino Acid ◽

Active Sites ◽

Fungal Infections ◽

Drug Repurposing ◽

Binding Pocket ◽

Amino Acid Identity ◽

Fungal Species ◽

Antifungal Drugs ◽

Specific Inhibitors

Abstract Background Invasive fungal infections account for a high burden of morbidity and mortality. This is aggravated because of the toxicity and resistance problems associated to current antifungal drugs, which in whole target only a handful of fungal molecules. In this scenario, new target identification and drug design, together with drug repurposing, represent promising strategies. Methods We aim to identify and test in vitro potential new therapeutic targets in fungi. Our strategy consists in identifying fungal proteins with active sites (meaning the set of residues lining the binding pocket) that are similar, but not identical!, to sites of proteins from the human pharmacolome. A high structural similarity with a human counterpart allows validation of the fungal target using cross-reactive inhibitors of the human protein. On the other hand, a few amino acid differences in the binding pocket produce local topological and chemical changes that create a "design space" for new specific inhibitors of the fungal target. Results Applying our own bioinformatics approach and taking advantage of the >200 available crystal structures of proteins of the human pharmacolome in complex with inhibitors, we have identified ca. 30 proteins in several fungal species of the genera Histoplasma, Candida, Criptococcus, Aspergillus and Fusarium, whose binding sites share at least 70% amino acid identity with their similar binding pockets in human pharmaceutical targets. So far we have assayed in vitro, in seven different fungal species, ca. 60 known inhibitors of around twenty of the orthologous human proteins. Some of the tested inhibitors have been previously assayed in different species in drug repurposing screenings, while others, to our knowledge, have not been yet tested. Over a dozen of these compounds, targeting eight different protein targets, showed IC50 values in the micromolar order in one or across several species. In general, yeasts were more significantly affected than molds. Conclusion Our results point to new potential fungal targets that can be exploited for the design of new antifungal agents. Ongoing work by our group aims to identify, by virtual screening, specific inhibitors for several of these potential targets. Disclosures All Authors: No reported disclosures

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