scholarly journals A new in vitro assay measuring direct interaction of nonsense suppressors with the eukaryotic protein synthesis machinery

2018 ◽  
Author(s):  
Martin Y. Ng ◽  
Haibo Zhang ◽  
Amy Weil ◽  
Vijay Singh ◽  
Ryan Jamiolkowski ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Single Molecule ◽  
Direct Interaction ◽  
Vitro Assay ◽  
Premature Termination Codons ◽  
Protein Synthesis Machinery ◽  
Nonsense Suppressor ◽  
Nonsense Suppressors ◽  
Selection Of

ABSTRACTNonsense suppressors (NonSups) induce “readthrough”, i.e., the selection of near cognate tRNAs at premature termination codons and insertion of the corresponding amino acid into nascent polypeptide. Prior readthrough measurements utilized contexts in which NonSups can promote readthrough directly, by binding to one or more of the components of the protein synthesis machinery, or indirectly, by several other mechanisms. Here we utilize a new, highly-purified in vitro assay to measure exclusively direct nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 display direct readthrough, with results suggesting that such NonSups act by at least two different mechanisms. In preliminary work we demonstrate the potential of single molecule fluorescence energy transfer measurements to elucidate mechanisms of NonSup-induced direct readthrough, which will aid efforts to identify NonSups having improved clinical efficacy.Table of Contents artwork

Elaboration of Novel TTK1 Inhibitory Leads via QSAR-Guided Selection of Crystallographic Pharmacophores Followed By In vitro Assay

2020 ◽  
Vol 16 ◽  
Author(s):  
Mahmoud A. Al-Sha'er ◽  
Mutasem O. Taha
Keyword(s):  
Standard Error ◽  
Qsar Model ◽  
Qsar Modeling ◽  
Vitro Assay ◽  
Physicochemical Descriptors ◽  
Qsar Models ◽  
Ic50 Values ◽  
Pharmacophore Hypotheses ◽  
Selection Of

Introduction: Tyrosine threonine kinase (TTK1) is a key regulator of chromosome segregation. TTK targeting received recent concern for the enhancement of possible anticancer therapies. Objective: In this regard we employed our well-known method of QSAR-guided selection of best crystallographic pharmacophore(s) to discover considerable binding interactions that anchore inhibitors into TTK1 binding site. Method:Sixtyone TTK1 crystallographic complexes were used to extract 315 pharmacophore hypotheses. QSAR modeling was subsequently used to choose a single crystallographic pharmacophore that when combined with other physicochemical descriptors elucidates bioactivity discrepancy within a list of 55 miscellaneous inhibitors. Results: The best QSAR model was robust and predictive (r2(55) = 0.75, r2LOO = 0.72 , r2press against external testing list of 12 compounds = 0.67), Standard error of estimate (training set) (S)= 0.63 , Standard error of estimate (testing set)(Stest) = 0.62. The resulting pharmacophore and QSAR models were used to scan the National Cancer Institute (NCI) database for new TTK1 inhibitors. Conclusion: Five hits confirmed significant TTK1 inhibitory profiles with IC50 values ranging between 11.7 and 76.6 micM.

Examining P-gp efflux kinetics guided by the BDDCS – Rational selection of in vitro assay designs and mathematical models

2019 ◽  
Vol 132 ◽  
pp. 132-141 ◽  
Author(s):  
Julia Riede ◽  
Ken-Ichi Umehara ◽  
Patrick Schweigler ◽  
Felix Huth ◽  
Hilmar Schiller ◽  
...  
Keyword(s):  
Mathematical Models ◽  
In Vitro Assay ◽  
Rational Selection ◽  
Vitro Assay ◽  
Efflux Kinetics ◽  
Selection Of

scholarly journals A Direct Interaction of Axonin-1 with Ngcam-Related Cell Adhesion Molecule (Nrcam) Results in Guidance, but Not Growth of Commissural Axons

2000 ◽  
Vol 149 (4) ◽  
pp. 951-968 ◽  
Author(s):  
Dora Fitzli ◽  
Esther T. Stoeckli ◽  
Stefan Kunz ◽  
Kingsley Siribour ◽  
Christoph Rader ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Direct Interaction ◽  
Longitudinal Axis ◽  
Vitro Assay ◽  
Commissural Axons ◽  
Related Cell

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti–axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.

scholarly journals In Vitro Assay of Translation Inhibition by Trichothecenes Using a Commercially Available System

Toxins ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 696
Author(s):  
Takahito Toyotome ◽  
Katsuhiko Kamei
Keyword(s):  
Protein Synthesis ◽  
Secondary Metabolites ◽  
Operation Time ◽  
In Vitro Assay ◽  
Ease Of Use ◽  
Vitro Assay ◽  
Vast Number ◽  
Translation Inhibition ◽  
Short Operation Time

Trichothecenes are a family of major secondary metabolites produced by some common filamentous fungi, including plant pathogenic and entomopathogenic fungi. It may be considered difficult to conduct a comparison between the toxicities of trichothecenes with consideration of different conditions and cell lines. In the current study, we developed an in vitro assay based on a commercially available system to estimate the translation inhibition, that is, the main toxicity, of trichothecenes. The assay was applied to estimate the inhibition of protein synthesis by trichothecenes. Initially, we examined the assay using trichothecene dissolved in water followed by an assessment of trichothecene solutions dissolved in acetonitrile. The obtained data showed that the assay tolerated the small amount of acetonitrile. The assay examined in this study has the advantages of a short operation time (one day), ease of use, and data stability, as it is a non-cell-based assay whose components are commercially available. It is expected that this assay will contribute to the evaluation of the toxicity of a vast number of trichothecenes.

scholarly journals Magnetic Tracking of Protein Synthesis in Microfluidic Environments—Challenges and Perspectives

Nanomaterials ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 585 ◽  
Author(s):  
Wegener ◽  
Ennen ◽  
Walhorn ◽  
Anselmetti ◽  
Hütten ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cell Biology ◽  
Ex Vivo ◽  
Microfluidic Devices ◽  
Cellular Protein ◽  
Fine Tuning ◽  
Superparamagnetic Particles ◽  
Magnetic Tracking ◽  
Selection Of

A novel technique to study protein synthesis is proposed that uses magnetic nanoparticles in combination with microfluidic devices to achieve new insights into translational regulation. Cellular protein synthesis is an energy-demanding process which is tightly controlled and is dependent on environmental and developmental requirements. Processivity and regulation of protein synthesis as part of the posttranslational nano-machinery has now moved back into the focus of cell biology, since it became apparent that multiple mechanisms are in place for fine-tuning of translation and conditional selection of transcripts. Recent methodological developments, such as ribosome foot printing, propel current research. Here we propose a strategy to open up a new field of labelling, separation, and analysis of specific polysomes using superparamagnetic particles following pharmacological arrest of translation during cell lysis and subsequent analysis. Translation occurs in polysomes, which are assemblies of specific transcripts, associated ribosomes, nascent polypeptides, and other factors. This supramolecular structure allows for unique approaches to selection of polysomes by targeting the specific transcript, ribosomes, or nascent polypeptides. Once labeled with functionalized superparamagnetic particles, such assemblies can be separated in microfluidic devices or magnetic ratchets and quantified. Insights into the dynamics of translation is obtained through quantifying large numbers of ribosomes along different locations of the polysome. Thus, an entire new concept for in vitro, ex vivo, and eventually single cell analysis will be realized and will allow for magnetic tracking of protein synthesis.

‘Unmasking’ of stored maternal mRNAs and the activation of protein synthesis at fertilization in sea urchins

Development ◽  
1991 ◽  
Vol 111 (2) ◽  
pp. 623-633 ◽  
Author(s):  
L.C. Kelso-Winemiller ◽  
M.M. Winkler
Keyword(s):  
Protein Synthesis ◽  
Sea Urchins ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Translational Machinery ◽  
Maternal Mrnas ◽  
Additional Support ◽  
And Control

The isolation and in vitro assay of maternal mRNPs has led to differing conclusions as to whether maternal mRNAs in sea urchin eggs are in a repressed or ‘masked’ form. To circumvent the problems involved with in vitro approaches, we have used an in vivo assay to determine if the availability of mRNA and/or components of the translational machinery are limiting protein synthesis in the unfertilized egg. This assay involves the use of a protein synthesis elongation inhibitor to create a situation in the egg in which there is excess translational machinery available to bind mRNA. Eggs were fertilized and the rate of entry into polysomes of individual mRNAs was measured in inhibitor-treated and control embryos using 32P-labeled cDNA probes. The fraction of ribosomes in polysomes and the polysome size were also determined. The results from this in vivo approach provide strong evidence for the coactivation of both mRNAs and components of the translational machinery following fertilization. The average polysome size increases from 7.5 ribosomes per message in 15 min embryos to approximately 10.8 ribosomes in 2 h embryos. This result gives additional support to the idea that translational machinery, as well as mRNA, is activated following fertilization. We also found that individual mRNAs are recruited into polysomes with different kinetics, and that the fraction of an mRNA in polysomes in the unfertilized egg correlates with the rate at which that mRNA is recruited into polysomes following fertilization.

scholarly journals Synthesis and Selection of De Novo Proteins That Bind and Impede Cellular Functions of an Essential Mycobacterial Protein

2006 ◽  
Vol 73 (4) ◽  
pp. 1320-1331 ◽  
Author(s):  
Alka Rao ◽  
Geeta Ram ◽  
Adesh Kumar Saini ◽  
Reena Vohra ◽  
Krishan Kumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Large Scale ◽  
De Novo ◽  
Cell Damage ◽  
Vitro Assay ◽  
De Novo Proteins ◽  
Protein Pool ◽  
Protein Libraries ◽  
Selection Of

ABSTRACT Recent advances in nonrational and part-rational approaches to de novo peptide/protein design have shown increasing potential for development of novel peptides and proteins of therapeutic use. We demonstrated earlier the usefulness of one such approach recently developed by us, called “codon shuffling,” in creating stand-alone de novo protein libraries from which bioactive proteins could be isolated. Here, we report the synthesis and selection of codon-shuffled de novo proteins that bind to a selected Mycobacterium tuberculosis protein target, the histone-like protein HupB, believed to be essential for mycobacterial growth. Using a versatile bacterial two-hybrid system that entailed utilization of HupB and various codon-shuffled protein libraries as bait and prey, respectively, we were able to identify proteins that bound strongly to HupB. The observed interaction was also confirmed using an in vitro assay. One of the protein binders was expressed in Mycobacterium smegmatis and was shown to appreciably affect growth in the exponential phase, a period wherein HupB is selectively expressed. Furthermore, the transcription profile of hupB gene showed a significant reduction in the transcript quantity in mycobacterial strains expressing the protein binder. Electron microscopy of the affected mycobacteria elaborated on the extent of cell damage and hinted towards a cell division malfunction. It is our belief that a closer inspection of the obtained de novo proteins may bring about the generation of small-molecule analogs, peptidomimetics, or indeed the proteins themselves as realistic leads for drug candidates. Furthermore, our strategy is adaptable for large-scale targeting of the essential protein pool of Mycobacterium tuberculosis and other pathogens.

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