Cellular FRET-Biosensors to Detect Membrane Targeting Inhibitors of N-Myristoylated Proteins

Arafath Kaja Najumudeen,Daniel Abankwa,Monika Köhnke,Kirill Alexandrov,Maja Šolman,Matthew Bogyo

doi:10.1371/journal.pone.0066425

Arafath Kaja Najumudeen, Daniel Abankwa + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0066425

Copy DOI

Abstract

Hundreds of eukaryotic signaling proteins require myristoylation to functionally associate with intracellular membranes. N-myristoyl transferases (NMT) responsible for this modification are established drug targets in cancer and infectious diseases. Here we describe NANOMS (NANOclustering and Myristoylation Sensors), biosensors that exploit the FRET resulting from plasma membrane nanoclustering of myristoylated membrane targeting sequences of Gαi2, Yes- or Src-kinases fused to fluorescent proteins. When expressed in mammalian cells, NANOMS report on loss of membrane anchorage due to chemical or genetic inhibition of myristoylation e.g. by blocking NMT and methionine-aminopeptidase (Met-AP). We used Yes-NANOMS to assess inhibitors of NMT and a cherry-picked compound library of putative Met-AP inhibitors. Thus we successfully confirmed the activity of DDD85646 and fumagillin in our cellular assay. The developed assay is unique in its ability to identify modulators of signaling protein nanoclustering, and is amenable to high throughput screening for chemical or genetic inhibitors of functional membrane anchorage of myristoylated proteins in mammalian cells.

Highlights

Covalent protein lipidation is an important protein modification in eukaryotic cells that enables the reversible association of hundreds of proteins with the membrane
Nanoclustering is essential for Ras activity and disruption of clustering leads to a reduction in Ras activity and prevents its robust biological signaling [13]. These experimental data are supported by computational simulations, which suggest that lipidanchors of Ras spontaneously organize into membrane nanocluster in mammalian cells [14,15]
In order to design biosensors that would detect functional membrane anchorage of myristoylated proteins in mammalian cells, we exploited the fact that fluorescently tagged myristoylated membrane anchors can display high FRET due to nanoclustering [16]

Summary

Introduction

Covalent protein lipidation is an important protein modification in eukaryotic cells that enables the reversible association of hundreds of proteins with the membrane. We report the design and application of three FRETbiosensors that can detect membrane anchorage of N-myristoylated proteins in mammalian cells. We demonstrate that these biosensors report on the inhibition of NMTs and Met-APs and can potentially be employed in cell-based high-throughput screening.

Results

Conclusion