Research

19 pages, 5659 KiB

Open AccessArticle

Nanoscopic Spatial Association between Ras and Phosphatidylserine on the Cell Membrane Studied with Multicolor Super Resolution Microscopy

by Anna M. Koester, Kai Tao, Malwina Szczepaniak, Matthew J. Rames and Xiaolin Nan

Biomolecules 2022, 12(8), 1033; https://doi.org/10.3390/biom12081033 - 26 Jul 2022

Cited by 5 | Viewed by 2642

Abstract

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their [...] Read more.

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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20 pages, 5740 KiB

Open AccessArticle

Crystal Structure Reveals the Full Ras–Raf Interface and Advances Mechanistic Understanding of Raf Activation

by Trinity Cookis and Carla Mattos

Biomolecules 2021, 11(7), 996; https://doi.org/10.3390/biom11070996 - 07 Jul 2021

Cited by 28 | Viewed by 4491

Abstract

Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas–CRaf-RBD_CRD [...] Read more.

Ras and Raf-kinase interact through the Ras-binding (RBD) and cysteine-rich domains (CRD) of Raf to signal through the mitogen-activated protein kinase pathway, yet the molecular mechanism leading to Raf activation has remained elusive. We present the 2.8 Å crystal structure of the HRas–CRaf-RBD_CRD complex showing the Ras–Raf interface as a continuous surface on Ras, as seen in the KRas–CRaf-RBD_CRD structure. In molecular dynamics simulations of a Ras dimer model formed through the α4–α5 interface, the CRD is dynamic and located between the two Ras protomers, poised for direct or allosteric modulation of functionally relevant regions of Ras and Raf. We propose a molecular model in which Ras binding is involved in the release of Raf autoinhibition while the Ras–Raf complex dimerizes to promote a platform for signal amplification, with Raf-CRD centrally located to impact regulation and function. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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12 pages, 1702 KiB

Open AccessArticle

Elaiophylin Is a Potent Hsp90/ Cdc37 Protein Interface Inhibitor with K-Ras Nanocluster Selectivity

by Farid A. Siddiqui, Vladimir Vukic, Tiina A. Salminen and Daniel Abankwa

Biomolecules 2021, 11(6), 836; https://doi.org/10.3390/biom11060836 - 04 Jun 2021

Cited by 3 | Viewed by 2759

Abstract

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related [...] Read more.

The natural product elaiophylin is a macrodiolide with a broad range of biological activities. However, no direct target of elaiophylin in eukaryotes has been described so far, which hinders a systematic explanation of its astonishing activity range. We recently showed that the related conglobatin A, a protein–protein interface inhibitor of the interaction between the N-terminus of Hsp90 and its cochaperone Cdc37, blocks cancer stem cell properties by selectively inhibiting K-Ras4B but not H-Ras. Here, we elaborated that elaiophylin likewise disrupts the Hsp90/ Cdc37 interaction, without affecting the ATP-pocket of Hsp90. Similarly to conglobatin A, elaiophylin decreased expression levels of the Hsp90 client HIF1α, a transcription factor with various downstream targets, including galectin-3. Galectin-3 is a nanocluster scaffold of K-Ras, which explains the K-Ras selectivity of Hsp90 inhibitors. In agreement with this K-Ras targeting and the potent effect on other Hsp90 clients, we observed with elaiophylin treatment a submicromolar IC₅₀ for MDA-MB-231 and MIA-PaCa-2 3D spheroid formation. Finally, a strong inhibition of MDA-MB-231 cells grown in the chorioallantoic membrane (CAM) microtumor model was determined. These results suggest that several other macrodiolides may have the Hsp90/ Cdc37 interface as a target site. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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Review

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17 pages, 1771 KiB

Open AccessReview

Lipid Profiles of RAS Nanoclusters Regulate RAS Function

by Yong Zhou and John F. Hancock

Biomolecules 2021, 11(10), 1439; https://doi.org/10.3390/biom11101439 - 30 Sep 2021

Cited by 13 | Viewed by 2295

Abstract

The lipid-anchored RAS (Rat sarcoma) small GTPases (guanosine triphosphate hydrolases) are highly prevalent in human cancer. Traditional strategies of targeting the enzymatic activities of RAS have been shown to be difficult. Alternatively, RAS function and pathology are mostly restricted to nanoclusters on the [...] Read more.

The lipid-anchored RAS (Rat sarcoma) small GTPases (guanosine triphosphate hydrolases) are highly prevalent in human cancer. Traditional strategies of targeting the enzymatic activities of RAS have been shown to be difficult. Alternatively, RAS function and pathology are mostly restricted to nanoclusters on the plasma membrane (PM). Lipids are important structural components of these signaling platforms on the PM. However, how RAS nanoclusters selectively enrich distinct lipids in the PM, how different lipids contribute to RAS signaling and oncogenesis and whether the selective lipid sorting of RAS nanoclusters can be targeted have not been well-understood. Latest advances in quantitative super-resolution imaging and molecular dynamic simulations have allowed detailed characterization RAS/lipid interactions. In this review, we discuss the latest findings on the select lipid composition (with headgroup and acyl chain specificities) within RAS nanoclusters, the specific mechanisms for the select lipid sorting of RAS nanoclusters on the PM and how perturbing lipid compositions within RAS nanoclusters impacts RAS function and pathology. We also describe different strategies of manipulating lipid composition within RAS nanoclusters on the PM. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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20 pages, 3844 KiB

Open AccessEditor’s ChoiceReview

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention

by Que N. Van, Priyanka Prakash, Rebika Shrestha, Trent E. Balius, Thomas J. Turbyville and Andrew G. Stephen

Biomolecules 2021, 11(3), 377; https://doi.org/10.3390/biom11030377 - 03 Mar 2021

Cited by 17 | Viewed by 3841

Abstract

RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, [...] Read more.

RAS proteins are mutated in approximately 20% of all cancers and are generally associated with poor clinical outcomes. RAS proteins are localized to the plasma membrane and function as molecular switches, turned on by partners that receive extracellular mitogenic signals. In the on-state, they activate intracellular signal transduction cascades. Membrane-bound RAS molecules segregate into multimers, known as nanoclusters. These nanoclusters, held together through weak protein–protein and protein–lipid associations, are highly dynamic and respond to cellular input signals and fluctuations in the local lipid environment. Disruption of RAS nanoclusters results in downregulation of RAS-mediated mitogenic signaling. In this review, we discuss the propensity of RAS proteins to display clustering behavior and the interfaces that are associated with these assemblies. Strategies to therapeutically disrupt nanocluster formation or the stabilization of signaling incompetent RAS complexes are discussed. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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28 pages, 10432 KiB

Open AccessReview

The Ins and Outs of RAS Effector Complexes

by Christina Kiel, David Matallanas and Walter Kolch

Biomolecules 2021, 11(2), 236; https://doi.org/10.3390/biom11020236 - 07 Feb 2021

Cited by 27 | Viewed by 5977

Abstract

RAS oncogenes are among the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differentiation, migration and metabolic status. Including aberrations in these pathways, RAS-dependent signaling is altered in more than half [...] Read more.

RAS oncogenes are among the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differentiation, migration and metabolic status. Including aberrations in these pathways, RAS-dependent signaling is altered in more than half of human cancers. Targeting mutant RAS proteins and their downstream oncogenic signaling pathways has been elusive. However, recent results comprising detailed molecular studies, large scale omics studies and computational modeling have painted a new and more comprehensive portrait of RAS signaling that helps us to understand the intricacies of RAS, how its physiological and pathophysiological functions are regulated, and how we can target them. Here, we review these efforts particularly trying to relate the detailed mechanistic studies with global functional studies. We highlight the importance of computational modeling and data integration to derive an actionable understanding of RAS signaling that will allow us to design new mechanism-based therapies for RAS mutated cancers. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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18 pages, 1786 KiB

Open AccessFeature PaperReview

Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again

by Daniel Abankwa and Alemayehu A. Gorfe

Biomolecules 2020, 10(11), 1522; https://doi.org/10.3390/biom10111522 - 06 Nov 2020

Cited by 29 | Viewed by 4105

Abstract

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation [...] Read more.

Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization while the whole Ras/RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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Other

Jump to: Research, Review

4 pages, 705 KiB

Open AccessCommentary

Spotlight on Accessory Proteins: RTK-RAS-MAPK Modulators as New Therapeutic Targets

by Silke Pudewell and Mohammad Reza Ahmadian

Biomolecules 2021, 11(6), 895; https://doi.org/10.3390/biom11060895 - 16 Jun 2021

Cited by 4 | Viewed by 3181

Abstract

The RTK-RAS-MAPK axis is one of the most extensively studied signaling cascades and is related to the development of both cancers and RASopathies. In the last 30 years, many ideas and approaches have emerged for directly targeting constituent members of this cascade, predominantly [...] Read more.

The RTK-RAS-MAPK axis is one of the most extensively studied signaling cascades and is related to the development of both cancers and RASopathies. In the last 30 years, many ideas and approaches have emerged for directly targeting constituent members of this cascade, predominantly in the context of cancer treatment. These approaches are still insufficient due to undesirable drug toxicity, resistance, and low efficacy. Significant advances have been made in understanding the spatiotemporal features of the constituent members of the RTK-RAS-MAPK axis, which are linked and modulated by many accessory proteins. Given that the majority of such modulators are now emerging as attractive therapeutic targets, a very small number of accessory inhibitors have yet to be discovered. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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13 pages, 2502 KiB

Open AccessBrief Report

Allosteric Kinase Inhibitors Reshape MEK1 Kinase Activity Conformations in Cells and In Silico

by Jakob Fleischmann, Andreas Feichtner, Louis DeFalco, Valentina Kugler, Selina Schwaighofer, Roland G Huber and Eduard Stefan

Biomolecules 2021, 11(4), 518; https://doi.org/10.3390/biom11040518 - 30 Mar 2021

Cited by 4 | Viewed by 2988

Abstract

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted [...] Read more.

Mutations at different stages of the mitogen-activated protein kinase (MAPK) signaling pathway lead to aberrant activation of the involved protein kinase entities. These oncogenic modifications alter signal propagation which converge on the gatekeeper kinases MEK1/2, transmitting the input signal to ERK1/2. Thus, targeted MEK inhibition causes qualitative alterations of carcinogenic MAPK signals. Phosphorylation of the MEK1 activation loop at the positions S218 and S222 by RAF kinases triggers the conformational alignment of MEK’s catalytic pocket to enable ATP-binding and substrate phosphorylation. We have extended a kinase conformation (KinCon) biosensor platform to record MEK1 activity dynamics. In addition to MEK phosphorylation by BRAF, the integration of the phosphorylation-mimetic mutations S218D/S222D triggered opening of the kinase. Structural rearrangement may involve the flexibility of the N terminal MEK1 A-helix. Application of the allosterically acting MEK inhibitors (MEKi) trametinib, cobimentinib, refametinib, and selumetinib converted activated MEK1 KinCon reporters back into a more closed inactive conformation. We confirmed MEK1 KinCon activity dynamics upon drug engagement using the patient-derived melanoma cell line A2058, which harbors the V600E hotspot BRAF mutation. In order to confirm biosensor dynamics, we simulated structure dynamics of MEK1 kinase in the presence and absence of mutations and/or MEKi binding. We observed increased dynamics for the S218D/S222D double mutant particularly in the region of the distal A-helix and alpha-C helix. These data underline that MEK1 KinCon biosensors have the potential to be subjected to MEKi efficacy validations in an intact cell setting. Full article

(This article belongs to the Special Issue Recent Insights into the Regulation and Structure of RAS Membrane Signaling Complexes—Emerging Drug Targeting Opportunities)

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