Biology subjects

Manoharan, G. b.

Publications and source records attributed to Manoharan, G. b..

3 recordsLinked to original sources

bioRxiv2025

Proteomics- and BRET-screens identify SPRY2 as Ras effector that impacts its membrane organisation

K-Ras functions within nanoscale proteo-lipid domains of the plasma membrane, but few regulators of its membrane organisation are known. We combined TurboID-based proximity proteomics with a secondary BRET screen to identify eight novel K-Ras G-domain interactors. We focused on APLP2 and SPRY2 for further characterisation. APLP2 binds K-Ras indirectly via C-Raf, while SPRY2 exhibits properties of a novel effector. Co-immunoprecipitation and BRET assays revealed that the SPRY2 C-terminal fragment (residues 161-315) binds oncogenic RasG12V more strongly than the full-length protein. Both forms localise to the plasma membrane, but this localisation and binding to K-Ras is disrupted by inhibitors of K-Ras membrane anchorage or activity. Mutations at the predicted interface of K-Ras and SPRY2s C-terminal region affect the interaction. Both full-length SPRY2 and its C-terminal fragment promote differentiation of C2C12 muscle cells, a process requiring MAPK pathway inhibition. Finally, SPRY2 also forms homo- or hetero-oligomers with SPRY4. We propose that active K-Ras recruits SPRY2 dimers to the membrane, where they bind Ras and block effector access.

biochemistry↗

bioRxiv2023

Identification of an H-Ras nanocluster disrupting peptide

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 in HRAS-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.

biochemistry↗

bioRxiv2021

Potential of phenothiazines to synergistically block calmodulin and reactivate PP2A in cancer cells

Phenothiazines (PTZ) are well known as inhibitors of monoamine neurotransmitter receptors, notably dopamine receptors. Because of this activity they are used for decades as antipsychotic drugs. In addition, significant anti-cancer properties have been ascribed to them. Several attempts for their repurposing were made, however, their incompletely understood polypharmacology is challenging. Here we examined the potential of PTZ to synergistically act on two cancer associated targets, calmodulin (CaM) and the tumor suppressor protein phosphatase 2A (PP2A). Both proteins are known to modulate the Ras-MAPK pathway activity. Consistently, combinations of a CaM inhibitor and a PP2A activator synergistically inhibited cancer cells with KRAS or BRAF mutations. We identified the covalently reactive PTZ derivative fluphenazine mustard as an inhibitor of Ras driven proliferation and Ras membrane organization. We confirmed its anti-CaM activity in vitro and through a cellular CaM target engagement bioluminescence resonance energy transfer (BRET) assay. Our results suggest that improved PTZ derivatives retaining their synergistic CaM inhibitory and PP2A activating properties, but without neurological side-effects, may be interesting to pursue further as anti-cancer agents.

biochemistry↗