Final Report Summary - NANODYGP (Nanoscale Operation and Dynamics of small GTPases - Identification of novel Isoform specifying Determinants)
Ras GTPases control cell growth, proliferation and differentiation. Ras is frequently hyperactivated in cancer and recognized as one of the major oncogenes. However, no therapeutic drugs against Ras itself have been developed so far. The urgent need for anti-Ras drugs is reflected by the NCI (USA) initiative called Ras central, which is devoting 10M USD towards research on specific Ras inhibitors. Ras is also the founder of the Ras superfamily, of which the family of small GTPases contains more than 150 related proteins. Insight into the molecular mechanisms of Ras has critically shaped our understanding of small GTPase functioning and cell signaling in general.
The focus of this proposal has been an underexplored feature of Ras, termed nanoclustering. Ras nanocluster are subresolution signaling complexes of Ras on the plasma membrane, which are indispensable for Ras signaling. Nanocluster concentrate Ras, thus increasing the efficacy of collision-events between active Ras and cytosolic effectors, which constantly sample the plasma membrane. We have previously described another unknown feature of Ras on the membrane. Upon activation, the preferred orientation of its G domain on the membrane changes, which impacts on its signalling activity by an unknown mechanism.
In this project, we aimed at describing 1) the dynamics of GTPase nanocluster, 2) the relevance of nanoclustering for other small GTPases, 3) the functional characteristics of G domain reorientation and 4) the prevalence of this reorientation in other small GTPases.
Aim 1) has been completed and resulted in a publication (Guzman et al 2014), where we explain that G domain orientation-mutants of H-ras exhibit distinct complexation tendencies with the H-ras nanocluster scaffold Galectin-1. Using mathematical modelling and quantitative fluorescence microscopic methods (FRET, FRAP and STED-FCS) on intact cells, we showed that this directly impacts on the dynamics of nanocluster formation. Both the number and lifetime of H-ras nanocluster are affected by the mutations. This suggests that different H-ras conformers (achieved through orientation-mutations) have different activities as a result of specific nanoclustering properties. Our data suggested the exciting possibility that Ras mutations can affect its signalling output by altering its nanoclustering properties. We followed up on this in aim 3). Aim 2) we published in Köhnke et al. 2012 that small GTPases of the Rab family can also nanocluster. This suggests that nanocluster formation is much more common for membrane anchored signalling proteins, as supported by our previous results on heterotrimeric G proteins (Abankwa D & Vogel H 2007, JCS) and more recently on Src-family kinases (Najumudeen AK et al. 2013). Ad Aim 3): We first aimed at reconstituting the Ras nanocluster system in vitro, however, obtaining fully lipid modified Ras has proven to be too challenging and we shifted our attention to work in intact cells, focusing on the functional consequences of G domain orientation. As a continuation of our results from aim 1), we investigated cancer-associated mutations in the orientation-switch III. We demonstrate an unprecedented mechanism of action showing that such mutations in H-, N- and K-ras mutations augment the nanoclustering of Ras and thus promote its hyperactivation, while having no biochemical defects. This provides a mechanistic explanation for cancer mutations in the switch III region, and represents a fundamental mechanistic insight similar to the 20 year-old finding that codon 12, 13 and 61 mutations render Ras GAP-insensitive. A publication for submission to a high impact journal by the end of this year is in preparation. Ad Aim 4): We followed up on our hypothesis derived from our work on Ras (Abankwa D et al. 2010, PNAS), where we proposed that a combination of helix α4 and hypervariable region residues specifies small GTPase specific functions. One postdoc tested an extensive library of 22 Rab chimera, which however did not conclusively confirm our hypothesis. We therefore shifted our attention to employing Rab-nanoclustering associated FRET as readout for its membrane anchorage. In collaboration with chemists from Poland, we characterized compounds with inhibitory activity against Rab geranylgeranyltransferase (Coxon F et al. 2014).
In summary, this project has in aims 1 and 3 validated a pathophysiological relevance of Ras nanoclustering. Therefore, Ras nanocluster represent a novel drug-target to inhibit Ras signaling activity in diseases such as cancer or in RASopathies. In a follow up project to Köhnke et al. that is not connected to directly this proposal, we have actually identified novel compounds, which inhibit specifically nanocluster and signaling of K-ras, the most notorious oncogene. K-ras is highly mutated in aggressive and highly prevalent cancers, such as pancreatic, colorectal and lung cancers. We expect that our mechanistic insight will be of highest bio-medical relevance, and should receive high attention by a number of pharma and biotech companies. Ultimately, novel drugs that build on our insight would significantly improve cancer patient treatment in the clinical setting.
The focus of this proposal has been an underexplored feature of Ras, termed nanoclustering. Ras nanocluster are subresolution signaling complexes of Ras on the plasma membrane, which are indispensable for Ras signaling. Nanocluster concentrate Ras, thus increasing the efficacy of collision-events between active Ras and cytosolic effectors, which constantly sample the plasma membrane. We have previously described another unknown feature of Ras on the membrane. Upon activation, the preferred orientation of its G domain on the membrane changes, which impacts on its signalling activity by an unknown mechanism.
In this project, we aimed at describing 1) the dynamics of GTPase nanocluster, 2) the relevance of nanoclustering for other small GTPases, 3) the functional characteristics of G domain reorientation and 4) the prevalence of this reorientation in other small GTPases.
Aim 1) has been completed and resulted in a publication (Guzman et al 2014), where we explain that G domain orientation-mutants of H-ras exhibit distinct complexation tendencies with the H-ras nanocluster scaffold Galectin-1. Using mathematical modelling and quantitative fluorescence microscopic methods (FRET, FRAP and STED-FCS) on intact cells, we showed that this directly impacts on the dynamics of nanocluster formation. Both the number and lifetime of H-ras nanocluster are affected by the mutations. This suggests that different H-ras conformers (achieved through orientation-mutations) have different activities as a result of specific nanoclustering properties. Our data suggested the exciting possibility that Ras mutations can affect its signalling output by altering its nanoclustering properties. We followed up on this in aim 3). Aim 2) we published in Köhnke et al. 2012 that small GTPases of the Rab family can also nanocluster. This suggests that nanocluster formation is much more common for membrane anchored signalling proteins, as supported by our previous results on heterotrimeric G proteins (Abankwa D & Vogel H 2007, JCS) and more recently on Src-family kinases (Najumudeen AK et al. 2013). Ad Aim 3): We first aimed at reconstituting the Ras nanocluster system in vitro, however, obtaining fully lipid modified Ras has proven to be too challenging and we shifted our attention to work in intact cells, focusing on the functional consequences of G domain orientation. As a continuation of our results from aim 1), we investigated cancer-associated mutations in the orientation-switch III. We demonstrate an unprecedented mechanism of action showing that such mutations in H-, N- and K-ras mutations augment the nanoclustering of Ras and thus promote its hyperactivation, while having no biochemical defects. This provides a mechanistic explanation for cancer mutations in the switch III region, and represents a fundamental mechanistic insight similar to the 20 year-old finding that codon 12, 13 and 61 mutations render Ras GAP-insensitive. A publication for submission to a high impact journal by the end of this year is in preparation. Ad Aim 4): We followed up on our hypothesis derived from our work on Ras (Abankwa D et al. 2010, PNAS), where we proposed that a combination of helix α4 and hypervariable region residues specifies small GTPase specific functions. One postdoc tested an extensive library of 22 Rab chimera, which however did not conclusively confirm our hypothesis. We therefore shifted our attention to employing Rab-nanoclustering associated FRET as readout for its membrane anchorage. In collaboration with chemists from Poland, we characterized compounds with inhibitory activity against Rab geranylgeranyltransferase (Coxon F et al. 2014).
In summary, this project has in aims 1 and 3 validated a pathophysiological relevance of Ras nanoclustering. Therefore, Ras nanocluster represent a novel drug-target to inhibit Ras signaling activity in diseases such as cancer or in RASopathies. In a follow up project to Köhnke et al. that is not connected to directly this proposal, we have actually identified novel compounds, which inhibit specifically nanocluster and signaling of K-ras, the most notorious oncogene. K-ras is highly mutated in aggressive and highly prevalent cancers, such as pancreatic, colorectal and lung cancers. We expect that our mechanistic insight will be of highest bio-medical relevance, and should receive high attention by a number of pharma and biotech companies. Ultimately, novel drugs that build on our insight would significantly improve cancer patient treatment in the clinical setting.