Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation

Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.</p

Nanoclustering augmentation : a novel mechanism of Ras activation in cancer

Proteins of the Ras family are central regulators of crucial cellular processes, such as proliferation, differentiation and apoptosis. Their importance is emphasized in cancer, in which the isoforms H-ras, N-ras and K-ras are misregulated by mutations in approximately 20 – 30 % of cases. Thus, they represent major cancer oncogenes and one of the most important targets for cancer drug development. Ras proteins are small GTPases, which cycle between the GTP-bound active and GDP-bound inactive state. Despite the tremendous research conducted in the last three decades, many fundamental properties of Ras proteins remain poorly understood. For instance, although new concepts have recently emerged, the understanding of Ras behavior in its native environment, the membrane, is still largely missing. On the membrane Ras organizes into nanoscale clusters, also called nanoclusters. They differ between isoforms, but also between activation states of Ras. It is considered that nanoclusters represent the basic Ras signaling units. Recently, it was demonstrated that on the membrane Ras adopts distinct conformations, the so-called orientations, which are dependent on the Ras activations state. The membrane-orientation of H-ras is stabilized by the helix α4 and the C-terminal hypervariable region (hvr). The novel switch III region was proposed to be involved in mediating the change between different H-ras orientations. When the regions involved in this mechanism are mutated, H-ras activity is changed by an unknown mechanism. This thesis has explained the connection between the change of Ras orientation on the membrane and Ras activity. We demonstrated that H-ras orientation mutants exhibit altered diffusion properties on the membrane, which reflect the changes in their nanoclustering. The altered nanoclustering consequently rules the activity of the mutants. Moreover, we demonstrated that specific cancer-related mutations, affecting the switch III region of different Ras isoforms, exhibit increased nanoclustering, which consequently leads to stronger Ras signaling and tumorigenicity. Thus, we have discovered nanoclustering increase as a novel mechanism of Ras activity modulation in cancer. The molecular architecture of complexes formed on the membrane upon Ras activation is another poorly understood property of Ras. The following work has provided novel details on the regulation of Ras nanoclustering by a known H-ras-GTP nanoclustering stabilizer galectin-1 (Gal-1). Our study demonstrated that Gal-1 is not able to bind Ras directly, as it was previously proposed. Instead, its effect on H-ras-GTP nanoclustering is indirect, through binding of the effector proteins. Collectively, our findings represent valuable novel insights in the behavior of Ras, which will help the future research to eventually develop new strategies to successfully target Ras in cancer

Design and reporting principle of NANOMS.

<p>FRET-biosensor design of the three different NANOMS. (<b>A</b>) The myristoylated N-terminal membrane-targeting motifs of mouse Gα_i2 (residues 1–35), human Yes (1–17)- and human Src (1–16)-kinases were genetically fused to the N-terminus of fluorescent proteins mCFP or mCit. The sequence of the employed membrane-targeting motifs can be found in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s006" target="_blank">Table S2</a></b>. (<b>B</b>) Intracellular processing involves cleavage of the N-terminal methionine (grey) by methionine amino-peptidase (Met-AP), NMT-mediated myristoylation on glycine 2 (yellow) and depending on the motif cysteine-palmitoylation (red). (<b>C</b>) Lipid modified reporters spontaneously organize into plasma membrane nanocluster. Tight packing of membrane targeted donor (mCFP)- and acceptor (mCit)-fluorophores (blue and yellow squares, respectively) in nanocluster leads to FRET. FRET can decrease due to loss of nanoclustering or cytoplasmic redistribution of the NANOMS after inhibitor treatment. As membrane anchorage is required for the functioning of myristoylated proteins, NANOMS report on functional membrane anchorage.</p

Cherry-picked chemical compound library screen with Yes-NANOMS.

<p>(<b>A</b>) Chemical structures of chemical compounds that were included in the cherry-picked chemical library. (<b>B</b>) BHK21 cells were transfected with Yes-NANOMS and screened with shown chemical compounds at a final concentration of 10 µM/mL. FRET-response of Yes-NANOMS to the chemical compounds is represented with E_max values. Block line indicates the average E_max and the error bars denote the s.e.m (n≥4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

NANOMS reports on RNAi-mediated depletion of NMT.

<p>(<b>A</b>) HEK293 EBNA cells transiently expressing Yes-NANOMS and (<b>B</b>) HEK293 cells transiently expressing Gi2-NANOMS were treated with NMT1 or NMT2 specific siRNAs or control siRNA. Knock-down efficiencies are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066425#pone.0066425.s004" target="_blank">Figure S4</a></b>. The characteristic E_max-value was determined on flow cytometric FRET data. The error bars denote the s.e.m (n = 4). Samples were statistically compared with the untreated control. See Methods for more on statistical analysis.</p

NANOMS report on chemical inhibition of NMT.

<p>(<b>A</b>) FRET-responses of Yes-, Src- and Gi2-NANOMS transfected BHK cells treated with 4 µM of the specific NMT inhibitor DDD85646. The error bars denote the s.e.m (n = 5). Samples were statistically compared with the untreated control. See Methods for more information on statistical analysis. (<b>B</b>) Confocal sensitized acceptor FRET-imaging of Yes-NANOMS expressed in BHK cells. Cells were treated as indicated. Top row shows acceptor channel images, and bottom row FRET images. The look-up table shows the FRET-index FR, color coded with high FRET levels in black and yellow (value 1) indicating no FRET. Scale bar is representative for all images and corresponds to 10 µm. (<b>C</b>) Dose-response curves of the effect of DDD85646 on the E_max values of Yes- and Src-NANOMS expressed in BHK cells (n = 6).</p

Targeting prohibitins at the cell surface prevents Th17‐mediated autoimmunity

T helper (Th)17 cells represent a unique subset of CD4+ T cells and are vital for clearance of extracellular pathogens including bacteria and fungi. However, Th17 cells are also involved in orchestrating autoimmunity. By employing quantitative surface proteomics, we found that the evolutionarily conserved prohibitins (PHB1/2) are highly expressed on the surface of both murine and human Th17 cells. Increased expression of PHBs at the cell surface contributed to enhanced CRAF/MAPK activation in Th17 cells. Targeting surface‐expressed PHBs on Th17 cells with ligands such as Vi polysaccharide (Typhim vaccine) inhibited CRAF‐MAPK pathway, reduced interleukin (IL)‐17 expression and ameliorated disease pathology with an increase in FOXP3+‐expressing Tregs in an animal model for multiple sclerosis (MS). Interestingly, we detected a CD4+ T cell population with high PHB1 surface expression in blood samples from MS patients in comparison with age‐ and sex‐matched healthy subjects. Our observations suggest a pivotal role for the PHB‐CRAF‐MAPK signalling axis in regulating the polarization and pathogenicity of Th17 cells and unveil druggable targets in autoimmune disorders such as MS

Cellular FRET-Biosensors to detect membrane targeting inhibitors of N-Myristoylated Proteins

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