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A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2.
- Source :
-
Scientific reports [Sci Rep] 2020 Jul 30; Vol. 10 (1), pp. 12879. Date of Electronic Publication: 2020 Jul 30. - Publication Year :
- 2020
-
Abstract
- The cyclin M (CNNM) family of Mg <superscript>2+</superscript> transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable K <subscript>d</subscript> . We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.
- Subjects :
- Humans
Protein Domains
Cyclins antagonists & inhibitors
Cyclins chemistry
Cyclins genetics
Enzyme Inhibitors chemistry
Fluorescence Resonance Energy Transfer
Peptides chemistry
Protein Tyrosine Phosphatases antagonists & inhibitors
Protein Tyrosine Phosphatases chemistry
Protein Tyrosine Phosphatases genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 10
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 32733084
- Full Text :
- https://doi.org/10.1038/s41598-020-69818-x