scholarly journals The Tribbles 2 (TRB2) pseudokinase binds to ATP and autophosphorylates in a metal-independent manner

2015 ◽  
Vol 467 (1) ◽  
pp. 47-62 ◽  
Author(s):  
Fiona P. Bailey ◽  
Dominic P. Byrne ◽  
Krishnadev Oruganty ◽  
Claire E. Eyers ◽  
Christopher J. Novotny ◽  
...  
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Phosphoryl Transfer ◽  
Divalent Metal Ions ◽  
Cellular Functions ◽  
Independent Manner ◽  
Calcium Calmodulin Dependent ◽  
Related Family ◽  
Cancer Phenotypes

The human Tribbles (TRB)-related pseudokinases are CAMK (calcium/calmodulin-dependent protein kinase)-related family members that have evolved a series of highly unusual motifs in the ‘pseudocatalytic’ domain. In canonical kinases, conserved amino acids bind to divalent metal ions and align ATP prior to efficient phosphoryl-transfer to substrates. However, in pseudokinases, atypical residues give rise to diverse and often unstudied biochemical and structural features that are thought to be central to cellular functions. TRB proteins play a crucial role in multiple signalling networks and overexpression confers cancer phenotypes on human cells, marking TRB pseudokinases out as a novel class of drug target. In the present paper, we report that the human pseudokinase TRB2 retains the ability to both bind and hydrolyse ATP weakly in vitro. Kinase activity is metal-independent and involves a catalytic lysine residue, which is conserved in TRB proteins throughout evolution alongside several unique amino acids in the active site. A similar low level of autophosphorylation is also preserved in the closely related human TRB3. By employing chemical genetics, we establish that the nucleotide-binding site of an ‘analogue-sensitive’ (AS) TRB2 mutant can be targeted with specific bulky ligands of the pyrazolo-pyrimidine (PP) chemotype. Our analysis confirms that TRB2 retains low levels of ATP binding and/or catalysis that is targetable with small molecules. Given the significant clinical successes associated with targeting of cancer-associated kinases with small molecule inhibitors, it is likely that similar approaches will be useful for further evaluating the TRB pseudokinases, with the translation of this information likely to furnish new leads for drug discovery.

scholarly journals Modular structure of chromosomal proteins HMG-14 and HMG-17: definition of a transcriptional enhancement domain distinct from the nucleosomal binding domain.

1995 ◽  
Vol 15 (12) ◽  
pp. 6663-6669 ◽  
Author(s):  
L Trieschmann ◽  
Y V Postnikov ◽  
A Rickers ◽  
M Bustin
Keyword(s):  
Amino Acids ◽  
Structural Features ◽  
Binding Domain ◽  
Full Length ◽  
Modular Structure ◽  
Chromosomal Proteins ◽  
Nucleosome Core ◽  
Terminal Amino

Chromosomal proteins HMG-14 and HMG-17 are the only known nuclear proteins which specifically bind to the nucleosome core particle and are implicated in the generation and/or maintenance of structural features specific to active chromatin. The two proteins facilitate polymerase II and III transcription from in vitro- and in vivo-assembled circular chromatin templates. Here we used deletion mutants and specific peptides to identify the transcriptional enhancement domain and delineate the nucleosomal binding domain of the HMG-14 and -17 proteins. Deletion of the 22 C-terminal amino acids of HMG-17 or 26 C-terminal amino acids of HMG-14 reduces significantly the ability of the proteins to enhance transcription from chromatin templates. In contrast, N-terminal truncation mutants had the same transcriptional enhancement activity as the full-length proteins. We conclude that the negatively charged C-terminal region of the proteins is required for transcriptional enhancement. Chromatin transcription enhancement assays, which involve binding competition between the full-length proteins and peptides derived from their nucleosomal binding regions, indicate that the minimal nucleosomal binding domain of human HMG-17 is 24 amino acids long and spans residues 17 to 40. The results suggest that HMG-14 and -17 proteins have a modular structure and contain distinct functional domains.

scholarly journals Bioactive fractions and compound of Ardisia crispa roots exhibit anti-arthritic properties mediated via angiogenesis inhibition in vitro

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Joan Anak Blin ◽  
Roslida Abdul Hamid ◽  
Huzwah Khaza’ai
Keyword(s):  
Umbilical Vein ◽  
Tube Formation ◽  
Significant Inhibition ◽  
Plant Roots ◽  
Cellular Functions ◽  
Independent Manner ◽  
Angiogenesis Inhibition ◽  
Umbilical Vein Endothelial Cells ◽  
Ardisia Crispa

Abstract Background Ardisia crispa (Thunb.) A.DC (Primulaceae), is a medicinal herb traditionally used by Asian people as remedies to cure inflammatory related diseases, including rheumatism. The plant roots possess various pharmacological activities including antipyretic, anti-inflammation and antitumor. Previous phytochemical studies of the plant roots have identified long chain alkyl-1,4-benzoquinones as major constituents, together with other phytochemicals. Hexane fraction of the plant roots (ACRH), was previously reported with anti-angiogenic and anti-arthritic properties, while its effect on their anti-arthritic in vitro, is yet unrevealed. Considering the significance of angiogenesis inhibition in developing new anti-arthritic agent, thus we investigated the anti-arthritic potential of Ardisia crispa roots by suppressing angiogenesis, in vitro. Methods Ardisia crispa roots hexane extract (ACRH) was prepared from the plant roots using absolute n-hexane. ACRH was fractionated into quinone-rich fraction (QRF) and further isolated to yield benzoquinonoid compound (BQ), respectively. In vitro experiments using VEGF-induced human umbilical vein endothelial cells (HUVECs) and IL-1β-induced human fibroblast-like synoviocytes for rheumatoid arthritis (HFLS-RA) were performed to evaluate the effects of these samples on VEGF-induced HUVECs proliferation and tube formation, and towards IL-1β-induced HFLS-RA proliferation, invasion, and apoptosis, respectively. Therapeutic concentrations (0.05, 0.5, and 5 μg/mL) tested in this study were predetermined based on the IC50 values obtained from the MTT assay. Results ACRH, QRF, and BQ exerted concentration-independent antiproliferative effects on VEGF-induced HUVECs and IL-1β-induced HFLS-RA, with IC50 values at 1.09 ± 0.18, 3.85 ± 0.26, and 1.34 ± 0.16 μg/mL in HUVECs; and 3.60 ± 1.38, 4.47 ± 0.34, and 1.09 ± 0.09 μg/mL in HFLS-RA, respectively. Anti-angiogenic properties of these samples were verified via significant inhibition on VEGF-induced HUVECs tube formation, in a concentration-independent manner. The invasiveness of IL-1β-induced HFLS-RA was also significantly inhibited in a concentration-independent manner by all samples. ACRH and BQ, but not QRF, significantly enhanced the apoptosis of IL-1β-induced HFLS-RA elicited at their highest concentration (5 μg/mL) (P < 0.05). Conclusions These findings highlight the bioactive fractions and compound from Ardisia crispa roots as potential anti-arthritic agents by inhibiting both HUVECs and HFLS-RA’s cellular functions in vitro, possibly mediated via their anti-angiogenic effects.

Oxytocin analogues with inhibitory properties, containing in position 2 a hydrophobic amino acid of D-configuration

1985 ◽  
Vol 50 (1) ◽  
pp. 132-145 ◽  
Author(s):  
Michal Lebl ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Jiřina Slaninová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inhibitory Activity ◽  
Structural Features ◽  
Inhibitory Effect ◽  
Hydrophobic Amino Acid

Ten analogues derived from oxytocin, deamino-oxytocin and deamino-carba-oxytocin were prepared which contained a D-amino acid in the position 2 of the parent system. The following D-amino acids were introduced: tyrosine, phenylalanine, p-methylphenylalanine, p-ethylphenylalanine and O-ethyltyrosine. Combination of two structural features which alone lead to strong inhibitors (a suitable D-amino acid in position 2 and a penicillamine moiety in position 1) did not enhance the inhibitory effect. Compounds containing D-tyrosine are weak agonists in the uterotonic assay; in case of 1-carba-analogues they can be converted into sulfoxides with low inhibitory activity. Analogues with D-phenylalanine substituted in the para-position are the most potent antagonists of the uterotonic effect of oxytocin (pA2 = 8.73) in vitro.

scholarly journals SumSec: Accurate Prediction of Sumoylation Sites Using Predicted Secondary Structure

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3260 ◽  
Author(s):  
Abdollah Dehzangi ◽  
Yosvany López ◽  
Ghazaleh Taherzadeh ◽  
Alok Sharma ◽  
Tatsuhiko Tsunoda
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
High Sensitivity ◽  
Underlying Mechanism ◽  
Structural Features ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Sumoylation Site ◽  
Translation Process ◽  
Wide Range

Post Translational Modification (PTM) is defined as the modification of amino acids along the protein sequences after the translation process. These modifications significantly impact on the functioning of proteins. Therefore, having a comprehensive understanding of the underlying mechanism of PTMs turns out to be critical in studying the biological roles of proteins. Among a wide range of PTMs, sumoylation is one of the most important modifications due to its known cellular functions which include transcriptional regulation, protein stability, and protein subcellular localization. Despite its importance, determining sumoylation sites via experimental methods is time-consuming and costly. This has led to a great demand for the development of fast computational methods able to accurately determine sumoylation sites in proteins. In this study, we present a new machine learning-based method for predicting sumoylation sites called SumSec. To do this, we employed the predicted secondary structure of amino acids to extract two types of structural features from neighboring amino acids along the protein sequence which has never been used for this task. As a result, our proposed method is able to enhance the sumoylation site prediction task, outperforming previously proposed methods in the literature. SumSec demonstrated high sensitivity (0.91), accuracy (0.94) and MCC (0.88). The prediction accuracy achieved in this study is 21% better than those reported in previous studies. The script and extracted features are publicly available at: https://github.com/YosvanyLopez/SumSec.

scholarly journals Active role of free RNA during the early phase of proteostasis stress

2019 ◽  
Author(s):  
Marion Alriquet ◽  
Adrían Martínez-Limón ◽  
Gerd Hanspach ◽  
Martin Hengesbach ◽  
Gian G. Tartaglia ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Active Form ◽  
Active Role ◽  
Structural Features ◽  
Cellular Functions ◽  
Granule Formation

ABSTRACTTransient sequestration of proteins and RNA is an essential principle of cellular reaction to stress. Compared to polypeptides, less is known about the role of RNA released from polysomes during acute proteostasis stress. Using quantitative mass spectrometry, we identified a set of proteins assembled by free RNA in the heat-shocked mammalian cytosol. RNA-associated proteins displayed higher disorder and larger size, which supports the role of multivalent interactions during the initial phase of the RNA granule formation. Structural features of the free RNA interactors defined them as a subset of RNA-binding proteins. The interactome contained preferentially the active form of eIF2α. The interaction between assembled proteins in vivo required RNA. The reconstitution of the association process in vitro indicated to the multimolecular basis for the increased binding to RNA upon heat shock in the cytosol. Our results reveal how free RNA can participate in reorganization of cellular functions during proteostasis stress.

scholarly journals The rfaE Gene from Escherichia coli Encodes a Bifunctional Protein Involved in Biosynthesis of the Lipopolysaccharide Core Precursor ADP-l-glycero-d-manno-Heptose

2000 ◽  
Vol 182 (2) ◽  
pp. 488-497 ◽  
Author(s):  
Miguel A. Valvano ◽  
Cristina L. Marolda ◽  
Mauricio Bittner ◽  
Mike Glaskin-Clay ◽  
Tania L. Simon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Inner Core ◽  
Structural Features ◽  
Predicted Amino Acid Sequence ◽  
E Coli ◽  
K 12 ◽  
Domain I

ABSTRACT The intermediate steps in the biosynthesis of the ADP-l-glycero-d-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement therfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation ofSalmonella enterica SL1102. DNA sequencing of therfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation inR. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains ofrfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis ofd-glycero-d-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-d-glycero-d-manno-heptose.

scholarly journals γ-Tubulin Complexes and Fibrillar Arrays: Two Conserved High Molecular Forms with Many Cellular Functions

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Jana Chumová ◽  
Hana Kourová ◽  
Lucie Trögelová ◽  
Geoffrey Daniel ◽  
Pavla Binarová
Keyword(s):  
Higher Plants ◽  
Molecular Forms ◽  
Cellular Functions ◽  
Animal Cells ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Independent Manner ◽  
Spontaneous Nucleation ◽  
Plant Microtubules

Higher plants represent a large group of eukaryotes where centrosomes are absent. The functions of γ-tubulin small complexes (γ-TuSCs) and γ-tubulin ring complexes (γ-TuRCs) in metazoans and fungi in microtubule nucleation are well established and the majority of components found in the complexes are present in plants. However, plant microtubules are also nucleated in a γ-tubulin-dependent but γ-TuRC-independent manner. There is growing evidence that γ-tubulin is a microtubule nucleator without being complexed in γ-TuRC. Fibrillar arrays of γ-tubulin were demonstrated in plant and animal cells and the ability of γ-tubulin to assemble into linear oligomers/polymers was confirmed in vitro for both native and recombinant γ-tubulin. The functions of γ-tubulin as a template for microtubule nucleation or in promoting spontaneous nucleation is outlined. Higher plants represent an excellent model for studies on the role of γ-tubulin in nucleation due to their acentrosomal nature and high abundancy and conservation of γ-tubulin including its intrinsic ability to assemble filaments. The defining scaffolding or sequestration functions of plant γ-tubulin in microtubule organization or in nuclear processes will help our understanding of its cellular roles in eukaryotes.

Some Structural Features of Metaphase Chromosomes of Mice and Men

1967 ◽  
Vol 25 ◽  
pp. 190-191
Author(s):  
Godfrey C. Hoskins ◽  
Betty B. Hoskins
Keyword(s):  
Electron Microscopy ◽  
Electron Micrographs ◽  
Structural Features ◽  
Metaphase Chromosomes ◽  
The Future ◽  
Of Mice And Men ◽  
Mouse Cells ◽  
Human And Mouse

Metaphase chromosomes from human and mouse cells in vitro are isolated by micrurgy, fixed, and placed on grids for electron microscopy. Interpretations of electron micrographs by current methods indicate the following structural features.Chromosomal spindle fibrils about 200Å thick form fascicles about 600Å thick, wrapped by dense spiraling fibrils (DSF) less than 100Å thick as they near the kinomere. Such a fascicle joins the future daughter kinomere of each metaphase chromatid with those of adjacent non-homologous chromatids to either side. Thus, four fascicles (SF, 1-4) attach to each metaphase kinomere (K). It is thought that fascicles extend from the kinomere poleward, fray out to let chromosomal fibrils act as traction fibrils against polar fibrils, then regroup to join the adjacent kinomere.

Correlation Between Cellular Functions and Morphological Changes of Human Trophoblast in Vitro

1975 ◽  
Vol 33 ◽  
pp. 600-601
Author(s):  
John C. Garancis ◽  
Robert O. Hussa ◽  
Michael T. Story ◽  
Donald Yorde ◽  
Roland A. Pattillo
Keyword(s):  
Electron Microscopy ◽  
Cellular Proliferation ◽  
Morphological Changes ◽  
Culture Fluid ◽  
Cellular Functions ◽  
Human Trophoblast ◽  
Transmission Electron ◽  
Marked Activity ◽  
Malignant Trophoblast

Human malignant trophoblast cells in continuous culture were incubated for 3 days in medium containing 1 mM N6-O2'-dibutyryl cyclic adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) and 1 mM theophylline. The culture fluid was replenished daily. Stimulated cultures secreted many times more chorionic gonadotropin and estrogens than did control cultures in the absence of increased cellular proliferation. Scanning electron microscopy revealed remarkable surface changes of stimulated cells. Control cells (not stimulated) were smooth or provided with varying numbers of microvilli (Fig. 1). The latter, usually, were short and thin. The surface features of stimulated cells were considerably different. There was marked increase of microvilli which appeared elongated and thick. Many cells were covered with confluent polypoid projections (Fig. 2). Transmission electron microscopy demonstrated marked activity of cytoplasmic organelles. Mitochondria were increased in number and size; some giant forms with numerous cristae were observed.

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