Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements

Manoharan, Ganesh babu; Guzmán, Camilo; Najumudeen, Arafath K.; Abankwa, Daniel

doi:10.1016/j.ejcb.2023.151314

Cited by 3 publications

(2 citation statements)

References 48 publications

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“…A CLARIOstar plate reader from BMG Labtech was used for BRET and fluorescence intensity measurement. The BRET protocol was adapted as described by us 73 .…”

Section: Methodsmentioning

confidence: 99%

Identification of an H-Ras nanocluster disrupting peptide

Steffen,

Manoharan,

Pavic

et al. 2024

Commun Biol

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Hyperactive Ras signalling is found in most cancers. Ras proteins are only active in membrane nanoclusters, which are therefore potential drug targets. We previously showed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering via direct interaction with the Ras binding domain (RBD) of Raf. Here, we establish that the B-Raf preference of Gal1 emerges from the divergence of the Raf RBDs at their proposed Gal1-binding interface. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B- and C-Raf-RBDs. Its 23-mer core fragment is sufficient to interfere with H-Ras nanoclustering, modulate Ras-signalling and moderately reduce cell viability. These latter two phenotypic effects may also emerge from the ability of L5UR to broadly engage with several RBD- and RA-domain containing Ras interactors. The L5UR-peptide core fragment is a starting point for the development of more specific reagents against Ras-nanoclustering and -interactors.

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“…A CLARIOstar plate reader from BMG Labtech was used for BRET and fluorescence intensity measurement. The BRET protocol was adapted as described by us 73 .…”

Section: Methodsmentioning

confidence: 99%

Identification of an H-Ras nanocluster disrupting peptide

Steffen,

Manoharan,

Pavic

et al. 2024

Commun Biol

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“…Although dimers are the predominant oligomeric form at the physiological expression level [ 20 ], it has been demonstrated that dimerization is a prerequisite process for the formation of higher-order oligomers (i.e., nanoclusters) comprising approximately four to six RAS monomers [ 25 ]. Advances in fluorescence-based approaches and electron microscopy have provided insights into RAS nanoclustering in cells, yet obtaining the residue-specific structural information about nanoclusters remains challenging [ 26 , 27 , 28 , 29 ]. It should be noted that these methods have been carried out using nonnative KRAS (~21 kDa) samples tagged with relatively large fluorescent proteins (~27 kDa), which may affect the orientation of KRAS with respect to the membrane and KRAS self-association.…”

Section: Introductionmentioning

confidence: 99%

Membrane-Driven Dimerization of the Peripheral Membrane Protein KRAS: Implications for Downstream Signaling

Lee

2024

IJMS

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Transient homo-dimerization of the RAS GTPase at the plasma membrane has been shown to promote the mitogen-activated protein kinase (MAPK) signaling pathway essential for cell proliferation and oncogenesis. To date, numerous crystallographic studies have focused on the well-defined GTPase domains of RAS isoforms, which lack the disordered C-terminal membrane anchor, thus providing limited structural insight into membrane-bound RAS molecules. Recently, lipid-bilayer nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses have revealed several distinct structures of the membrane-anchored homodimers of KRAS, an isoform that is most frequently mutated in human cancers. The KRAS dimerization interface is highly plastic and altered by biologically relevant conditions, including oncogenic mutations, the nucleotide states of the protein, and the lipid composition. Notably, PRE-derived structures of KRAS homodimers on the membrane substantially differ in terms of the relative orientation of the protomers at an “α–α” dimer interface comprising two α4–α5 regions. This interface plasticity along with the altered orientations of KRAS on the membrane impact the accessibility of KRAS to downstream effectors and regulatory proteins. Further, nanodisc platforms used to drive KRAS dimerization can be used to screen potential anticancer drugs that target membrane-bound RAS dimers and probe their structural mechanism of action.

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Identification of an H-Ras nanocluster disrupting peptide

Manoharan,

Steffen,

Pavic

et al. 2023

Preprint

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The Ras-MAPK pathway is critical to regulate cell proliferation and differentiation. Its dysregulation is implicated in the onset and progression of numerous types of cancers. To be active, Ras proteins are membrane anchored and organized into nanoclusters, which realize high-fidelity signal transmission across the plasma membrane. Nanoclusters therefore represent potential drug targets. However, targetable protein components of signalling nanoclusters are poorly established.We previously proposed that the nanocluster scaffold galectin-1 (Gal1) enhances H-Ras nanoclustering by stabilizing stacked dimers of H-Ras and Raf via a direct interaction of dimeric Gal1 with the Ras binding domain (RBD) in particular of B-Raf. Here, we provide further supportive evidence for this model. We establish that the B-Raf preference emerges from divergent regions of the Raf RBDs that were proposed to interact with Gal1. We then identify the L5UR peptide, which disrupts this interaction by binding with low micromolar affinity to the B-Raf-RBD. Its 23-mer core fragment is thus sufficient to interfere with Gal1-enhanced H-Ras nanocluster, reduce MAPK-output and cell viability inHRAS-mutant cancer cell lines.Our data therefore suggest that the interface between Gal1 and the RBD of B-Raf can be targeted to disrupt Gal1-enhanced H-Ras nanoclustering. Collectively, our results support that Raf-proteins are integral components of active Ras nanoclusters.

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Detection of Ras nanoclustering-dependent homo-FRET using fluorescence anisotropy measurements

Cited by 3 publications

References 48 publications

Identification of an H-Ras nanocluster disrupting peptide

Identification of an H-Ras nanocluster disrupting peptide

Membrane-Driven Dimerization of the Peripheral Membrane Protein KRAS: Implications for Downstream Signaling

Identification of an H-Ras nanocluster disrupting peptide

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