Interaction Of Ras Proteins Research Articles

A brief history of RAS and the RAS Initiative.

In this review, I provide a brief history of the discovery of RAS and the GAPs and GEFs that regulate its activity from a personal perspective. Much of this history has been driven by technological breakthroughs that occurred concurrently, such as molecular cloning, cDNA expression to analyze RAS proteins and their structures, and application of PCR to detect mutations. I discuss the RAS superfamily and RAS proteins as therapeutic targets, including recent advances in developing RAS inhibitors. I also describe the role of the RAS Initiative at Frederick National Laboratory for Cancer Research in advancing development of RAS inhibitors and providing new insights into signaling complexes and interaction of RAS proteins with the plasma membrane.

Analysis of RAS protein interactions in living cells reveals a mechanism for pan-RAS depletion by membrane-targeted RAS binders

HRAS, NRAS, and KRAS4A/KRAS4B comprise the RAS family of small GTPases that regulate signaling pathways controlling cell proliferation, differentiation, and survival. RAS pathway abnormalities cause developmental disorders and cancers. We found that KRAS4B colocalizes on the cell membrane with other RAS isoforms and a subset of prenylated small GTPase family members using a live-cell quantitative split luciferase complementation assay. RAS protein coclustering is mainly mediated by membrane association-facilitated interactions (MAFIs). Using the RAS-RBD (CRAF RAS binding domain) interaction as a model system, we showed that MAFI alone is not sufficient to induce RBD-mediated RAS inhibition. Surprisingly, we discovered that high-affinity membrane-targeted RAS binding proteins inhibit RAS activity and deplete RAS proteins through an autophagosome-lysosome-mediated degradation pathway. Our results provide a mechanism for regulating RAS activity and protein levels, a more detailed understanding of which should lead to therapeutic strategies for inhibiting and depleting oncogenic RAS proteins.

Characterizing the Influence of Two Small Molecule Targets Towards the Ras-Related Protein Cdc42

Ras-related Protein-Protein Interactions (PPIs) are critical to events involved in cellular proliferation, inhibition of cell death, and cell transformation. It is therefore importantto characterize molecular features of Ras protein interactions that dictate their function as ‘timing switches’ in signal transduction pathways. This is an important step towards targeting the inhibition of cell transformation as well as controlling abnormal signaling activity of tumor-causing Ras variants. Cell division cycle 42 (Cdc42) is a Ras-related protein, and previousstudies from this laboratory have outlined differences in conformational dynamics in an important region that interacts with various effectors, known as the Switch 1 region. These differences resulted in an increase rate of GTP hydrolysis in the presence of an effector protein that inhibits GTP hydrolysis when bound to wild type Cdc42. These findings suggest that induced conformational changes in key effector-binding regions of Ras-related proteins may serve to alter effector protein interactions. As such, the targeting of small molecules towards this Switch 1 region of Cdc42 may facilitate changes in local conformation as well as dynamics without affecting the overall stability of the protein. We present preliminary data, using biochemical and biophysical approaches to characterize the influence of two small molecule targets on the interaction between Cdc42 and effector proteins that alter normal Cdc42-stimulated activity.

Modeling of RAS complexes supports roles in cancer for less studied partners

BackgroundRAS protein interactions have predominantly been studied in the context of the RAF and PI3kinase oncogenic pathways. Structural modeling and X-ray crystallography have demonstrated that RAS isoforms bind to canonical downstream effector proteins in these pathways using the highly conserved switch I and II regions. Other non-canonical RAS protein interactions have been experimentally identified, however it is not clear whether these proteins also interact with RAS via the switch regions.ResultsTo address this question we constructed a RAS isoform-specific protein-protein interaction network and predicted 3D complexes involving RAS isoforms and interaction partners to identify the most probable interaction interfaces. The resulting models correctly captured the binding interfaces for well-studied effectors, and additionally implicated residues in the allosteric and hyper-variable regions of RAS proteins as the predominant binding site for non-canonical effectors. Several partners binding to this new interface (SRC, LGALS1, RABGEF1, CALM and RARRES3) have been implicated as important regulators of oncogenic RAS signaling. We further used these models to investigate competitive binding and multi-protein complexes compatible with RAS surface occupancy and the putative effects of somatic mutations on RAS protein interactions.ConclusionsWe discuss our findings in the context of RAS localization to the plasma membrane versus within the cytoplasm and provide a list of RAS protein interactions with possible cancer-related consequences, which could help guide future therapeutic strategies to target RAS proteins.

Multivalent Small-Molecule Pan-RAS Inhibitors

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may providechemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found tobind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Biochemical and Biophysical Characterization of a Small Molecule Interaction with the Ras-Related Protein Cdc42

Deleterious Protein-Protein Interactions (PPIs) involving Ras-related proteins have been known to play significant roles in events leading to cellular proliferation, inhibition of cell death, and cell transformation. Therefore, it is important to characterize molecular features of Ras protein interactions that dictate their function as ‘timing switches’ in signal transduction pathways. This is a critical step towards targeting the inhibition of cell transformation as well as controlling abnormal signaling activity of tumor-causing Ras variants.

The RAS-Effector Interaction as a Drug Target.

About a third of all human cancers harbor mutations in one of the K-, N-, or HRAS genes that encode an abnormal RAS protein locked in a constitutively activated state to drive malignant transformation and tumor growth. Despite more than three decades of intensive research aimed at the discovery of RAS-directed therapeutics, there are no FDA-approved drugs that are broadly effective against RAS-driven cancers. Although RAS proteins are often said to be "undruggable," there is mounting evidence suggesting it may be feasible to develop direct inhibitors of RAS proteins. Here, we review this evidence with a focus on compounds capable of inhibiting the interaction of RAS proteins with their effectors that transduce the signals of RAS and that drive and sustain malignant transformation and tumor growth. These reports of direct-acting RAS inhibitors provide valuable insight for further discovery and development of clinical candidates for RAS-driven cancers involving mutations in RAS genes or otherwise activated RAS proteins. Cancer Res; 77(2); 221-6. ©2017 AACR.

Characterization of the Influence of a Small Molecule on a Ras-Related Protein-Protein Interaction

Mutations and/or abnormal expression affecting Ras-related protein interactions may influence their roles in cell-signaling activity. Thusly, it remains important to characterize molecular features of Ras protein interactions that dictate their function as ‘timing switches’ in signal transduction pathways. This is a critical step towards targeting the inhibition of cell transformation as well as controlling abnormal signaling activity of tumor-causing Ras variants. Cell division cycle 42 (Cdc42) is a member of the Ras family of proteins. Previous structural studies on a Cdc42 construct, Cdc42(T35A), from this laboratory have outlined differences in conformational dynamics in an important region that interacts with various effectors, known as the Switch 1 region, in this variant, as compared to wild type Cdc42. These differences correlated to a reduced binding of an effector protein that inhibits GTP hydrolysis. As such, the targeting of a small molecule towards an important effector-binding region of Cdc42 could facilitate changes in local conformational dynamics without affecting the overall stability of the Ras protein. Moreover, this effort could provide valuable contributions toward their use as Ras inhibitors. We are currently using biochemical and biophysical approaches to characterize the influence of a small molecule (ZCL278) has on the interaction between Cdc42 and a GTPase inhibitor Protein. Molecular modeling studies have suggested that this small molecule can bind to the Switch 1 region of Cdc42 and “Mimic” an interaction with an effector protein. Our preliminary data outlines the influence this small molecule has on Cdc42's interaction with an effector protein that inhibits Cdc42-stimulated GTPase activity. We also present results that examine ZCL278's influence on a Cdc42 variant with altered dynamics in the Switch 1 region.

Ras-Mediated Signal Transduction and Virulence in Human Pathogenic Fungi.

Signal transduction pathways regulating growth and stress responses are areas of significant study in the effort to delineate pathogenic mechanisms of fungi. In-depth knowledge of signal transduction events deepens our understanding of how a fungal pathogen is able to sense changes in the environment and respond accordingly by modulation of gene expression and re-organization of cellular activities to optimize fitness. Members of the Ras protein family are important regulators of growth and differentiation in eukaryotic organisms, and have been the focus of numerous studies exploring fungal pathogenesis. Here, the current data regarding Ras signal transduction are reviewed for three major pathogenic fungi: Cryptococcus neoformans, Candida albicans and Aspergillus fumigatus. Particular emphasis is placed on Ras-protein interactions during control of morphogenesis, stress response and virulence.

Individual rate constants for the interaction of Ras proteins with GTPase-activating proteins determined by fluorescence spectroscopy.

Individual rate constants for the interaction of H-, K-, and N-Ras with GAP-334 and NF1-333 were determined using fluorescent derivatives of guanine nucleotides at the active site of the Ras proteins. Stopped-flow experiments with NF1-333 show a fast concentration-dependent initial phase corresponding to the binding reaction followed by a slower phase, which corresponds to the hydrolysis reaction. With Ras bound to the nonhydrolyzable analogue mant-GppNHp, only the concentration-dependent first phase was observed. The Ras x mant-GppNHp x NF1-333 complexes were also used to measure dissociation rate constants of the Ras-GAP complexes. Using GAP-334 as the catalyst, the concentration-dependent first phase was too fast to be measured by the stopped-flow method, but the subsequent chemical cleavage reaction occurred at a similar rate (5-10 s(-1)) to that seen with NF1-333. With both GAP-334 and NF1-333, after rapidly reaching the initial equilibrium, there was no further time-dependent change on mixing GAPs with Ras x mant-GppNHp. The results obtained provide new insights into the individual steps of the GAP-catalyzed GTPase reaction on Ras. They do not require the postulation of a rate-limiting step occurring before GTP hydrolysis.

Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases.

The ras oncogene products require membrane localization for their function, and this is thought to be accomplished by the addition of a palmitoyl group to a cysteine residue near the carboxyl terminus of the nascent chain. A lipidated carboxyl-terminal cysteine residue is also found in sequence-related yeast sex factors, and in at least two cases, the alpha-carboxyl group is also methyl esterified. To determine if ras proteins are themselves modified by a similar type of methylation reaction, we incubated rat embryo fibroblasts transformed with p53 and activated Ha-ras oncogenes with L-[methyl-3H]methionine under conditions in which the isotope was converted to the methyl donor S-adenosyl-L-[methyl-3H]methionine. By using an assay that detects methyl ester linkages, we found that immunoprecipitated ras proteins are in fact esterified and that the stability of these esters is consistent with a carboxyl-terminal localization. This methylation reaction may be important in regulating the interaction of ras proteins with plasma membrane components. The presence of analogous carboxyl-terminal tetrapeptide sequences in other proteins may provide a general recognition sequence for lipidation and methylation modification reactions.