Cyanide is an adequate agonist of the plant hormone ethylene for studying signalling of sensor kinase ETR1 at the molecular level

2012 ◽  
Vol 444 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Melanie M. A. Bisson ◽  
Georg Groth
Keyword(s):  
Plant Hormone ◽  
Specific Binding ◽  
Receptor Protein ◽  
Wild Type ◽  
Intrinsic Signal ◽  
Signal Perception ◽  
Functional Studies ◽  
Ethylene Binding ◽  
Autokinase Activity

The plant hormone ethylene is involved in many developmental processes and responses to environmental stresses in plants. Although the elements of the signalling cascade and the receptors operating the ethylene pathway have been identified, a detailed understanding of the molecular processes related to signal perception and transfer is still lacking. Analysis of these processes using purified proteins in physical, structural and functional studies is complicated by the gaseous character of the plant hormone. In the present study, we show that cyanide, a π-acceptor compound and structural analogue of ethylene, is a suitable substitute for the plant hormone for in vitro studies with purified proteins. Recombinant ethylene receptor protein ETR1 (ethylene-resistant 1) showed high level and selective binding of [14C]cyanide in the presence of copper, a known cofactor in ethylene binding. Replacement of Cys65 in the ethylene-binding domain by serine dramatically reduced binding of radiolabelled cyanide. In contrast with wild-type ETR1, autokinase activity of the receptor is not reduced in the ETR1-C65S mutant upon addition of cyanide. Additionally, protein–protein interaction with the ethylene signalling protein EIN2 (ethylene-insensitive 2) is considerably sustained by cyanide in wild-type ETR1, but is not affected in the mutant. Further evidence for the structural and functional equivalence of ethylene and cyanide is given by the fact that the ethylene-responsive antagonist silver, which is known to allow ligand binding but prevent intrinsic signal transduction, also allows specific binding of cyanide, but shows no effect on autokinase activity and ETR1–EIN2 interaction.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy

2015 ◽  
Vol 112 (43) ◽  
pp. 13237-13242 ◽  
Author(s):  
Lorenzo Sborgi ◽  
Francesco Ravotti ◽  
Venkata P. Dandey ◽  
Mathias S. Dick ◽  
Adam Mazur ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Nmr Spectroscopy ◽  
Specific Binding ◽  
3D Structure ◽  
High Mobility ◽  
Atomic Resolution ◽  
Receptor Protein ◽  
Filament Formation ◽  
Cryo Electron Microscopy

Inflammasomes are multiprotein complexes that control the innate immune response by activating caspase-1, thus promoting the secretion of cytokines in response to invading pathogens and endogenous triggers. Assembly of inflammasomes is induced by activation of a receptor protein. Many inflammasome receptors require the adapter protein ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)], which consists of two domains, the N-terminal pyrin domain (PYD) and the C-terminal CARD. Upon activation, ASC forms large oligomeric filaments, which facilitate procaspase-1 recruitment. Here, we characterize the structure and filament formation of mouse ASC in vitro at atomic resolution. Information from cryo-electron microscopy and solid-state NMR spectroscopy is combined in a single structure calculation to obtain the atomic-resolution structure of the ASC filament. Perturbations of NMR resonances upon filament formation monitor the specific binding interfaces of ASC-PYD association. Importantly, NMR experiments show the rigidity of the PYD forming the core of the filament as well as the high mobility of the CARD relative to this core. The findings are validated by structure-based mutagenesis experiments in cultured macrophages. The 3D structure of the mouse ASC-PYD filament is highly similar to the recently determined human ASC-PYD filament, suggesting evolutionary conservation of ASC-dependent inflammasome mechanisms.

scholarly journals Fusobacterium nucleatum facilitates cetuximab resistance in colorectal cancer via the PI3K/AKT and JAK/STAT3 pathways

2020 ◽  
Author(s):  
Hu Han ◽  
Yan Li ◽  
Wan Qin ◽  
Lu Wang ◽  
Han Yin ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fusobacterium Nucleatum ◽  
Therapy Resistance ◽  
Tumor Burden ◽  
Wild Type ◽  
Poor Clinical Outcome ◽  
Functional Studies ◽  
Cetuximab Therapy

AbstractInfectious pathogens contribute to about 20% of the total tumor burden. Fusobacterium nucleatum (Fn) has been associated with the initiation, progression, and therapy resistance in colorectal cancer (CRC). The over-abundance of Fn has been observed in patients with right-sided CRC than in those with left-sided CRC. While the KRAS/NRAS/BRAF wild-type status of the CRC conferred better response to cetuximab in patients with left-sided CRC than with right-sided CRC. However, treatment failure remains the leading cause of tumor relapse and poor clinical outcome in patients with CRC. Here, we have studied the association of Fn to cetuximab resistance. Our functional studies indicate that Fn facilitates resistance of CRC to cetuximab in vitro and in vivo. Moreover, Fn was found to target the PI3K/AKT and JAK/STAT3 pathways, which altered the response to cetuximab therapy. Therefore, assessing the levels and targeting Fn and the associated signaling pathways may allow modulating the treatment regimen and improve prognoses of CRC patients.

scholarly journals Functional studies on human Pex7p: subcellular localization and interaction with proteins containing a peroxisome-targeting signal type 2 and other peroxins

2002 ◽  
Vol 365 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Karen GHYS ◽  
Marc FRANSEN ◽  
Guy P. MANNAERTS ◽  
Paul P. Van VELDHOVEN
Keyword(s):  
Fluorescent Protein ◽  
Specific Binding ◽  
Chinese Hamster ◽  
Translation System ◽  
Chondrodysplasia Punctata ◽  
Rhizomelic Chondrodysplasia Punctata ◽  
Functional Studies ◽  
Targeting Signal

Pex7p is a WD40-containing protein involved in peroxisomal import of proteins containing an N-terminal peroxisome-targeting signal (PTS2). The interaction of human recombinant Pex7p expressed in different hosts/systems with its PTS2 ligand and other peroxins was analysed using various experimental approaches. Specific binding of human Pex7p to PTS2 could be demonstrated only when Pex7p was formed in vitro by a coupled transcription/translation system or synthesized in vivo in Chinese hamster ovary K1 cells transfected with a construct coding for a Pex7p-green fluorescent protein (GFP) fusion protein. Apparently, no cofactors are required and only monomeric Pex7p binds to PTS2. The interaction is reduced upon cysteine alkylation and is impaired upon truncation of the N-terminus of Pex7p. Interaction of Pex7p with other peroxins could not be demonstrated in bacterial or yeast two-hybrid screens, or in pull-down binding assays. The GFP fusion proteins, tagged at either the N- or C-terminus, were able to restore PTS2 import in rhizomelic chondrodysplasia punctata fibroblasts, and Pex7p-GFP was located both in the lumen of peroxisomes and in the cytosol.

scholarly journals The m6A demethylase FTO promotes esophageal cancer progression through YTHDF1-dependent posttranscriptional silencing of AKT3

2021 ◽  
Author(s):  
Chunbao Zang ◽  
Fangfang Zhao ◽  
Dabing Huang ◽  
Lingsuo Kong ◽  
Minghua Xie ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Cancer Progression ◽  
Target Genes ◽  
Specific Binding ◽  
Messenger Rnas ◽  
Rna Seq ◽  
Functional Studies ◽  
Rna Immunoprecipitation

Abstract Background : N 6 -methyladenosine (m 6 A) is the most abundant modification in eukaryotic messenger RNAs (mRNAs), and plays important roles in many bioprocesses. However, its functions in esophageal cancer remain elusive. Methods : Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and transcriptomic RNA sequencing (RNA-seq) were used to screen the target genes of FTO. Western blot, quantitative real-time PCR (RT-qPCR) and immunohistochemical (IHC) were used to detect FTO expression in cell lines and patient tissues. The biological functions of FTO were investigated in vitro and in vivo . RNA pull-down and RNA immunoprecipitation assays were conducted to explore the specific binding of target genes. Results : We discovered that the RNA demethylase FTO was significantly up-regulated in esophageal cancer patients. Knockdown of FTO drastically reduced esophageal cancer cells (ESCCs) proliferation, migration, invasion, and apoptosis. On the other hand, overexpression of FTO significantly promoted ESCCs growth and invasion. Moreover, we found that the m 6 A methyltransferase METTL14 negatively correlates with FTO function on esophageal cancer progression. By using transcriptome-wide m 6 A-Seq and RNA-Seq assays, we identified AKT3 is the target of FTO, which acts in concert in esophageal cancer tumorigenesis and metastasis. Moreover, loss and gain functional studies confirm that YTHDF1 mediates m 6 A-increased translation of AKT3 mRNA. Conclusion : Our results uncovered an METTL14/FTO/YTHDF1/AKT3 signaling network that regulates the esophageal cancer progression.

Abstract 555: Functional Characterization Uncovered Novel Loss-of-function Mutations in ABCA1

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Xin Bi ◽  
James McParland ◽  
Jian Wang ◽  
Adam D McIntyre ◽  
Robert A Hegele ◽  
...  
Keyword(s):  
In Silico ◽  
Cholesterol Efflux ◽  
Functional Characterization ◽  
In Silico Analysis ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Wild Type ◽  
Functional Studies ◽  
Lipid Efflux

Objectives: ABCA1 encodes the membrane protein ATP-binding cassette transporter A1 (ABCA1), a pivotal player in nascent HDL formation via its ability to facilitate cholesterol and phospholipid efflux to apolipoprotein A-I (ApoA-I). ABCA1 variants are frequently found in subjects with primary hypoalphalipoproteinemia, however, their pathogenicity and causal link with the clinical phenotype are not always known. Methods: In silico analysis (Mutation Assessor, PANTHER, PolyPhen-2, PROVEAN, SIFT, and VEST) were performed to predict the functional consequences of ABCA1 missense variants found in our cohort of hypoalphalipoproteinemia. A subset of novel ABCA1 variants were generated in vitro through site-directed mutagenesis and their abilities in mediating lipid efflux to apoA-I were determined using standard methods. Results: A total of 32 mutations in ABCA1 were identified, among which 15 were classified as missense, 9 as nonsense or frameshift, 7 as intronic, and 1 as ”no-protein”. We selected 5 variants that were labeled as pathogenic or possibly pathogenic by in silico analysis to conduct functional studies. Two newly identified mutations in ABCA1, a nonsense mutation (p.E1005X) and a missense mutation (p.S2046R), resulted in complete loss of the canonical lipid efflux function of ABCA1 (2.5% and 1.8% of wild type cholesterol efflux level respectively). These results were concordant with the phenotypic characteristics of the carriers. Three additional mutations (p.G750W and p.R1341T and p.I1085F) resulted in only a partial loss of function (66-75% of wild type cholesterol efflux level). These results were somewhat discordant with the phenotype of the heterozygote carriers (HDL-C levels of 16, 14 and 38 mg/dl respectively), suggesting the presence of additional causal factors. Conclusions: These results support E1005X and S2046R as ABCA1 loss-of-function mutations and highlight the need to conduct functional studies on unknown variants to determine their pathogenicity.

scholarly journals Tn5 Transposase with an Altered Specificity for Transposon Ends

2002 ◽  
Vol 184 (1) ◽  
pp. 233-240 ◽  
Author(s):  
Todd A. Naumann ◽  
William S. Reznikoff
Keyword(s):  
Specific Binding ◽  
Random Mutagenesis ◽  
Initial Step ◽  
Wild Type ◽  
Dam Methylase ◽  
End Sequences ◽  
Tn5 Transposase ◽  
Bacterial Transposon

ABSTRACT Tn5 is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn5 contains two different specific end sequences, termed outside end (OE) and inside end (IE). In Escherichia coli, IE is methylated by Dam methylase (IEME). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IEME-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IEME defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.

scholarly journals Transmembrane glycoprotein gp150 is a substrate for receptor tyrosine phosphatase DPTP10D in Drosophila cells.

1997 ◽  
Vol 17 (12) ◽  
pp. 6859-6867 ◽  
Author(s):  
S J Fashena ◽  
K Zinn
Keyword(s):  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Cytoplasmic Domain ◽  
Receptor Protein ◽  
S2 Cells ◽  
Wild Type ◽  
Downstream Signaling ◽  
Transmembrane Glycoprotein

We have begun to explore the downstream signaling pathways of receptor protein tyrosine phosphatases (RPTPs) that control axon guidance decisions in the Drosophila central nervous system. We have focused our studies on the adhesion molecule-like gp150 protein, which binds directly to and is an in vitro substrate for the RPTP DPTP10D. Here we show that gp150 and DPTP10D form stable complexes in Drosophila Schneider 2 (S2) cells and in wild-type larval tissue. We also demonstrate that the DPTP10D cytoplasmic domain is sufficient to confer binding to gp150. gp150 has a short cytoplasmic domain containing four tyrosines, all found within sequences similar to immunoreceptor family tyrosine-based activation motifs (ITAMs). We demonstrate that gp150 is tyrosine phosphorylated in wild-type larvae. In S2 cells, gp150 becomes tyrosine phosphorylated following incubation with PTP inhibitors or upon coexpression of the Dsrc tyrosine kinase. Phosphorylated Dsrc and an unknown 40-kDa phosphoprotein form stable complexes with gp150, thereby implicating them in a putative gp150 signaling pathway. When coexpressed with gp150, either full-length DPTP10D or its cytoplasmic domain mediates gp150 dephosphorylation whereas a catalytically inactive DPTP10D cytoplasmic domain does not. The neural RPTP DPTP99A can also induce gp150 dephosphorylation but does not coimmunoprecipitate with gp150. Taken together, the results suggest that gp150 transduces signals via phosphorylation of its ITAM-like elements. Phosphotyrosines on gp150 might function as binding sites for downstream signaling molecules, thereby initiating a signaling cascade that could be modulated in vivo by RPTPs such as DPTP10D.

scholarly journals Mutational analysis of the necdin gene in patients with congenital isolated hypogonadotropic hypogonadism

2011 ◽  
Vol 165 (1) ◽  
pp. 145-150 ◽  
Author(s):  
Daiane Beneduzzi ◽  
Anita K Iyer ◽  
Ericka Barbosa Trarbach ◽  
Acacio P Silveira-Neto ◽  
Letícia G Silveira ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Hypogonadotropic Hypogonadism ◽  
Kallmann Syndrome ◽  
Luciferase Reporter ◽  
Causative Role ◽  
Wild Type ◽  
Protein Activity ◽  
Functional Studies ◽  
Significant Impairment

ContextNecdin activates GNRH gene expression and is fundamental for the development, migration, and axonal extension of murine GNRH neurons. In humans, necdin plays a potential role in the hypogonadotropic hypogonadism phenotype in patients with Prader–Willi syndrome.AimTo investigate necdin gene (NDN) variants in patients with isolated hypogonadotropic hypogonadism (IHH).Patients and methodsWe studied 160 Brazilian patients with IHH, which includes 92 with Kallmann syndrome and 68 with normosmic IHH. Genomic DNA was extracted and the single NDN exon was amplified and sequenced. To measure GNRH transcriptional activity, luciferase reporter plasmids containing GNRH regulatory regions were transiently transfected into GT1-7 cells in the presence and absence of overexpressed wild-type or mutant necdin.ResultsA heterozygous variant of necdin, p.V318A, was identified in a 23-year-old male with Kallmann syndrome. The p.V318A was also present in affected aunt and his father and was absent in 100 Brazilian control subjects. Previous FGFR1 gene analysis revealed a missense mutation (p.P366L) in this family. Functional studies revealed a minor difference in the activation of GNRH transcription by mutant protein compared with wild type in that a significant impairment of the necdin protein activity threshold was observed.ConclusionA rare variant of necdin (p.V318A) was described in a family with Kallmann syndrome associated with a FGFR1 mutation. Familial segregation and in vitro analysis suggested that this non-synonymous variant did not have a direct causative role in the hypogonadism phenotype. NDN mutations are not a frequent cause of congenital IHH.

scholarly journals CheZ Has No Effect on Flagellar Motors Activated by CheY13DK106YW

1998 ◽  
Vol 180 (19) ◽  
pp. 5123-5128 ◽  
Author(s):  
Birgit E. Scharf ◽  
Karen A. Fahrner ◽  
Howard C. Berg
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Response Regulator ◽  
Receptor Protein ◽  
Cross Linking ◽  
Wild Type ◽  
Flagellar Motors ◽  
Run And Tumble ◽  
Formation Of Complexes

ABSTRACT The behaviors of both cheZ-deleted and wild-type cells of Escherichia coli were found to be very sensitive to the level of expression of CheZ, a protein known to accelerate the dephosphorylation of the response regulator CheY-phosphate (CheY-P). However, cells induced to run and tumble by the unphosphorylated mutant protein CheY13DK106YW (CheY**) failed to respond to CheZ, even when CheZ was expressed at high levels. Therefore, CheZ neither affects the flagellar motors directly nor sequesters CheY**. In in vitro cross-linking studies, CheY** promoted trimerization of CheZ to the same extent as wild-type CheY but failed to induce the formation of complexes of higher molecular weight observed with CheY-P. Also, CheY** could be cross-linked to FliM, the motor receptor protein, nearly as well as CheY-P. Thus, to CheZ, CheY** looks like CheY, but to FliM, it looks like CheY-P.

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