Track topics on Twitter Track topics that are important to you
GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 orthologue, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when co-expressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function. This article is protected by copyright. All rights reserved.
This article was published in the following journal.
Name: The FEBS journal
Local pulse wave velocity can be estimated from the waterhammer equation and is an essential component of wave separation analysis. However, previous studies have demonstrated inaccuracies in the esti...
A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type und...
The aggregation-induced emission (AIE) luminogens are now showing strong potential in mimicking the energy donor of fluorescence resonance energy transfer (FRET) system. Herein, one highly efficient F...
FÖrster resonance energy transfer (FRET)-based biosensors have achieved great success for biological applications. However, what is not extensively appreciated is the growing role as versatile FRET b...
Ratiometric fluorescent probes based on FRET mechanism have attracted great attention due to their large pseudo-Stokes shifts and built-in correction for environmental effects. However, most donors fa...
The molecular nature of insulin resistance in human muscle is still incompletely defined. Our data indicate that acetylation of mitochondrial proteins in humans is regulated by muscle cont...
The management of cancers and their therapeutic guidance was until shortly mostly based on histopathological considerations of the tumor. the development of targeted therapies is a turning...
This is a pilot feasibility study assessing the use of a commercial molecular profiling service in the UK's National Health Service (NHS). The impact of knowledge of a patient's molecular ...
Multicenter observational survey of the presence of Human Papilloma Virus (HPV) on trans vaginal ultrasound (TVUS) probes, and of the behavior of professionals during US examination and pr...
Diverting ileostomies are created to protect a rectal anastomosis or in situations with a risk of intestinal perforation. Currently, the application of a rod to hinder slippage of the loop...
The molecular designing of drugs for specific purposes (such as DNA-binding, enzyme inhibition, anti-cancer efficacy, etc.) based on knowledge of molecular properties such as activity of functional groups, molecular geometry, and electronic structure, and also on information cataloged on analogous molecules. Drug design is generally computer-assisted molecular modeling and does not include pharmacokinetics, dosage analysis, or drug administration analysis.
Signaling proteins which function as master molecular switches by activating Rho GTPases through conversion of guanine nucleotides. Rho GTPases in turn control many aspects of cell behavior through the regulation of multiple downstream signal transduction pathways.
MOLECULAR BIOLOGY techniques used in the diagnosis of disease. Included are such techniques as IN SITU HYBRIDIZATION of chromosomes for CYTOGENETIC ANALYSIS; OLIGONUCLEOTIDE ARRAY SEQUENCE ANALYSIS of gene expression patterns in disease states; identification of pathogenic organisms by analysis of species specific DNA sequences; and detection of mutations with POLYMERASE CHAIN REACTION.
A method of chemical analysis based on the detection of characteristic radionuclides following a nuclear bombardment. It is also known as radioactivity analysis. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)
Measurement of cells' substrate utilization and biosynthetic output for modeling of METABOLIC NETWORKS.
Polymerase Chain Reaction (PCR)
PCR (Polymerase Chain Reaction) uses the ability of DNA polymerase (enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA. These enzymes are essential to DNA replication and usually work in pairs to create two ident...
Within medicine, nutrition (the study of food and the effect of its components on the body) has many different roles. Appropriate nutrition can help prevent certain diseases, or treat others. In critically ill patients, artificial feeding by tubes need t...