Bioluminescence as a tool in molecular biology

Bioluminescence has been studied for many years by scientists. There are numerous mechanisms of that phenomenon; among them bacterial bioluminescence is the most frequently found in nature. This type of bioluminescence is determined by the appearance of lux operon, which encodes all elements necessary to produce light emission and it does not require any additional substrates supply. Another commonly found example of bioluminescence mechanism is performed by Photinus pyralis. Luciferase of P. pyralis named FLuc requires D-luciferin as a substrate. Bioluminescence is also characteristic for many deep-sea organisms. Most of them are based on oxidation reaction of coelenterazine to coelenteramide mediated by RLuc or GLuc luciferases. Due to the variety of bioluminescence mechanisms in nature, it has become possible to apply them in many sensitive methods that can be used in molecular biology and medicine. The most significant application of bioluminescence is BLI (bioluminescence imaging). This method is cheap and nontoxic which allows both in vitro and in vivo imaging. BLI applications include, e.g. protein-protein interactions, stem cells labeling, tracking of viral, bacterial, fungal and parasitical infections, and carcinogenesis analyses. Bioluminescence has also been used in the creation of modified cell systems capable of light emission in response to certain analytes and thus very sensitive biosensors have been generated. Other important areas of bioluminescence application are immunoassays, ATP assays, and BART analysis (bioluminescent assay in Real-Time) – a very sensitive technique which allows scientists to estimate nucleic acids amplification.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Ways into Understanding HIF Inhibition

Cancers ◽

10.3390/cancers13010159 ◽

2021 ◽

Vol 13 (1) ◽

pp. 159

Author(s):

Tina Schönberger ◽

Joachim Fandrey ◽

Katrin Prost-Fingerle

Keyword(s):

Protein Interactions ◽

Target Genes ◽

Protein Protein Interactions ◽

Low Oxygen ◽

Drug Candidates ◽

Open Questions ◽

Wide Range ◽

Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

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EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

British Journal of Cancer ◽

10.1038/s41416-020-01250-4 ◽

2021 ◽

Author(s):

Liqing Jia ◽

Xiaolu Ge ◽

Chao Du ◽

Linna Chen ◽

Yanhong Zhou ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Protein Interactions ◽

Elongation Factor ◽

Protein Translation ◽

Protein Protein Interactions ◽

Promising Target ◽

Tgfβ Receptor ◽

Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6021-6029.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6021-6029

Author(s):

R Metz ◽

A J Bannister ◽

J A Sutherland ◽

C Hagemeier ◽

E C O'Rourke ◽

...

Keyword(s):

Protein Interactions ◽

Transcriptional Activation ◽

Binding Protein ◽

Tata Box ◽

Binding Motif ◽

Protein Protein Interactions ◽

High Concentrations ◽

Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v2 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

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Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽

10.3390/biom12010106 ◽

2022 ◽

Vol 12 (1) ◽

pp. 106

Author(s):

Pavel V. Ershov ◽

Yuri V. Mezentsev ◽

Alexis S. Ivanov

Keyword(s):

Protein Interactions ◽

Targeted Delivery ◽

Protein Complexes ◽

Protein Protein Interactions ◽

Good Efficiency ◽

Cellular Targets ◽

Proteolytic Stability ◽

Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

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Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

Chemical Communications ◽

10.1039/d1cc04611j ◽

2021 ◽

Author(s):

Babu Sudhamalla ◽

Anirban Roy ◽

Soumen Barman ◽

Jyotirmayee Padhan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Protein Interactions ◽

Site Specific ◽

Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

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Assay Systems for Profiling Deubiquitinating Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21165638 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5638

Author(s):

Jinhong Cho ◽

Jinyoung Park ◽

Eunice EunKyeong Kim ◽

Eun Joo Song

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Live Cells ◽

Deubiquitinating Enzyme ◽

Protein Protein Interactions ◽

Deubiquitinating Enzymes ◽

Cell Lysates ◽

Cellular Processes

Deubiquitinating enzymes regulate various cellular processes, particularly protein degradation, localization, and protein–protein interactions. The dysregulation of deubiquitinating enzyme (DUB) activity has been linked to several diseases; however, the function of many DUBs has not been identified. Therefore, the development of methods to assess DUB activity is important to identify novel DUBs, characterize DUB selectivity, and profile dynamic DUB substrates. Here, we review various methods of evaluating DUB activity using cell lysates or purified DUBs, as well as the types of probes used in these methods. In addition, we introduce some techniques that can deliver DUB probes into the cells and cell-permeable activity-based probes to directly visualize and quantify DUB activity in live cells. This review could contribute to the development of DUB inhibitors by providing important information on the characteristics and applications of various probes used to evaluate and detect DUB activity in vitro and in vivo.

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In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

Applied and Environmental Microbiology ◽

10.1128/aem.01686-09 ◽

2009 ◽

Vol 76 (1) ◽

pp. 264-274 ◽

Cited By ~ 53

Author(s):

M.-L. Foucault ◽

L. Thomas ◽

S. Goussard ◽

B. R. Branchini ◽

C. Grillot-Courvalin

Keyword(s):

Escherichia Coli ◽

Bioluminescence Imaging ◽

Light Emission ◽

Direct Method ◽

Firefly Luciferase ◽

Intestinal Colonization ◽

Bacterial Populations ◽

Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

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Using backbone-cyclized Cys-rich polypeptides as molecular scaffolds to target protein–protein interactions

Biochemical Journal ◽

10.1042/bcj20180792 ◽

2019 ◽

Vol 476 (1) ◽

pp. 67-83 ◽

Cited By ~ 11

Author(s):

Dipankar Chaudhuri ◽

Teshome Aboye ◽

Julio A. Camarero

Keyword(s):

Protein Interactions ◽

Disulfide Bonds ◽

Biological Degradation ◽

Protein Protein Interactions ◽

Molecular Scaffolds ◽

Pharmaceutical Industries ◽

Therapeutic Leads ◽

New Generation

Abstract The use of disulfide-rich backbone-cyclized polypeptides, as molecular scaffolds to design a new generation of bioimaging tools and drugs that are potent and specific, and thus might have fewer side effects than traditional small-molecule drugs, is gaining increasing interest among the scientific and in the pharmaceutical industries. Highly constrained macrocyclic polypeptides are exceptionally more stable to chemical, thermal and biological degradation and show better biological activity when compared with their linear counterparts. Many of these relatively new scaffolds have been also found to be highly tolerant to sequence variability, aside from the conserved residues forming the disulfide bonds, able to cross cellular membranes and modulate intracellular protein–protein interactions both in vitro and in vivo. These properties make them ideal tools for many biotechnological applications. The present study provides an overview of the new developments on the use of several disulfide-rich backbone-cyclized polypeptides, including cyclotides, θ-defensins and sunflower trypsin inhibitor peptides, in the development of novel bioimaging reagents and therapeutic leads.

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