Molecular and Functional Profiling of Gαi as an Intracellular pH Sensor.

Heterotrimeric G proteins (Gα, Gß and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. Previously, human Gαi and its yeast homolog Gpa1 have been reported to function as intracellular pH sensors, yet the pH sensing capabilities of Gαi and the underlying mechanism remain to be established. Herein, we identify a pH sensing network within Gαi, and evaluate the consequences of pH modulation on the structure and stability of the G-protein. We find that changes over the physiological pH range significantly alter the structure and stability of Gαi-GDP, with the protein undergoing a disorder-to-order transition as the pH is raised from 6.8 to 7.5. Further, we find that modulation of intracellular pH in HEK293 cells regulates Gαi-Gßγ release. Identification of key residues in the pH-sensing network allowed the generation of low pH mimetics that attenuate Gαi-Gßγ release. Our findings, taken together, indicate that pH-dependent structural changes in Gαi alter the agonist-mediated Gßγ dissociation necessary for proper signaling.

Understanding the perspectives and needs of multiple stakeholders: Identifying key elements of a digital health intervention to protect against environmental hazards.

AirRater is a free environmental health smartphone app developed and available in Australia that collects individual health data and disseminates environmental hazard information to populations. Following previous evaluations with app users, the aim of this study was to better understand how clinicians, government agency and non-government advocacy group representatives perceive an app designed to reduce the impacts of environmental hazards on individual and public health. Nine government agency and non-government advocacy group representatives, along with 11 clinicians based in Australia participated in a semi-structured interview or focus group to explore perspectives on AirRater. Interview and focus group data were transcribed and analysed using the qualitative data analysis software NVivo. Results indicate that for clinicians, apps like AirRater can add value as an educational, patient self-management and diagnostic tool. For government and peak bodies, apps can add value by addressing environmental health literacy and monitoring and forecasting gaps, as well as supporting advocacy efforts and public health surveillance. We conclude that environmental health smartphone apps can support a range of stakeholders to achieve shared goals and priorities related to individual and public health outcomes. Further research is needed to better understand how apps could be embedded into clinical practice and policy settings.

Molecular basis and cellular functions of vinculin-actin directional catch bonding.

The ability of cells and tissues to respond differentially to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding remain unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of the variants did not affect Vcn activation but reduced Vcn loading and altered exchange dynamics, consistent with the loss of directional catch bonding. Expression of Vcn variants perturbed the coordination of subcellular structures and cell migration, establishing key cellular functions for Vcn directional catch bonding.

Methods of Assessing Health Care Costs in a Changing Climate: A Case Study of Heatwaves and Ambulance Dispatches in Tasmania, Australia.

Anthropogenic climate change is causing a rise in global temperatures, with this trend projected to increase into the future. Rising temperatures result in an increase in the frequency and severity of heatwave events, with an associated increase in poor health outcomes for vulnerable individuals. This places an increasing strain on health care services. However, methods calculating future health care costs associated with this trend are poorly understood. We calculated health care costs attributable to heatwave events in Tasmania 2009-2019, using ambulance dispatches as a case study. We also modeled the expected health and economic burden for projected heatwave frequencies between 2010 and 2089. We developed our models based on two possible approaches to describing population adaptation to heatwaves-an adapted population calculated by determining heatwave episodes using a rolling baseline, and a non-adapted population calculated by determining heatwave episodes using a static baseline. Using a rolling baseline calculation for 2010 to 2089, we estimated additional ambulance costs averaging AUD$57,147 per year and totaling AUD$4,571,788. For the same period using a static baseline, we estimated additional ambulance costs averaging AUD$517,342 per year and totaling AUD$41,387,349. While this method is suitable for estimating the health care costs associated with heatwaves, it could be utilized for estimating health care costs related to other climate-related extreme events. Different methods of estimating heatwaves, modeling an adapted versus non-adapted population, provide substantial differences in projected costs. There is potential for considerable health system cost savings when a population is supported to adapt to extreme heat.

Catalytic site mutations confer multiple states of G protein activation.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

Using the live assessment to discriminate between Autism Spectrum Disorder and Disinhibited Social Engagement Disorder.

BACKGROUND: Children with Autism Spectrum Disorder (ASD) and children with Disinhibited Social Engagement Disorder (DSED) may present with similar social problems, despite differing aetiologies, resulting in diagnostic conundrums. METHODS: Thirty children: 10 with ASD, no maltreatment history, 10 with DSED and 10 typically developing children were assessed via 'gold standard' ASD assessments, including the Autism Diagnostic Observational Schedule (ADOS) and a unique unstructured observation known as the Live assessment. Live utilises a triadic interaction (2 assessors and child), playful teasing and social 'stress' scenarios to increase the social challenge. RESULTS: The ADOS supported discrimination of DSED from ASD to a degree. Where additional neurodevelopmental problems created ambiguity, the Live assessment was more supportive than the ADOS for unpicking the underlying nature of the social problems. CONCLUSION: Live supported differentiation between ASD, DSED and other neurodevelopmental problems. The greater social challenge presented by Live exacerbated core problems of ASD and, in DSED, core social skills stood out.

Elucidation of the Molecular Basis and Cellular Functions of Vinculin-Actin Directional Catch Bonding.

The ability of cells and tissues to differentially resist or adapt to mechanical forces applied in distinct directions is mediated by the ability of load-bearing proteins to preferentially maintain physical linkages in certain directions. However, the molecular basis and biological consequences of directional force-sensitive binding are unclear. Vinculin (Vcn) is a load-bearing linker protein that exhibits directional catch bonding due to interactions between the Vcn tail domain (Vt) and filamentous (F)-actin. We developed a computational approach to predict Vcn residues involved in directional catch bonding and produced a set of associated Vcn variants with unaltered Vt structure, actin binding, or phospholipid interactions. Incorporation of these variants into Vcn biosensors did not perturb Vcn conformation, but reduced Vcn loading consistent with loss of directional catch bonding. Expression of Vcn variants perturbed the coalignment of FAs and F-actin and directed cell migration, establishing key cellular functions for Vcn directional catch bonding.

Evaluating User Preferences, Comprehension, and Trust in Apps for Environmental Health Hazards: Qualitative Case Study.

BACKGROUND: Climate change is projected to increase environmental health hazard risks through fire-related air pollution and increased airborne pollen levels. To protect vulnerable populations, it is imperative that evidence-based and accessible interventions are available. The environmental health app, AirRater, was developed in 2015 in Australia to provide information on multiple atmospheric health hazards in near real time. The app allows users to view local environmental conditions, and input and track their personal symptoms to enable behaviors that protect health in response to environmental hazards. OBJECTIVE: This study aimed to develop insights into users' perceptions of engagement, comprehension, and trust in AirRater to inform the future development of environmental health apps. Specifically, this study explored which AirRater features users engaged with, what additional features or functionality needs users felt they required, users' self-perception of understanding app information, and their level of trust in the information provided. METHODS: A total of 42 adult AirRater users were recruited from 3 locations in Australia to participate in semistructured interviews to capture location- or context-specific experiences. Participants were notified of the recruitment opportunity through multiple avenues including newsletter articles and social media. Informed consent was obtained before participation, and the participants were remunerated for their time and perspectives. A preinterview questionnaire collected data including age range, any preexisting conditions, and location (postcode). All participant data were deidentified. Interviews were recorded, transcribed, and analyzed using thematic analysis in NVivo 12 (QSR International). RESULTS: Participants discussed app features and functionality, as well as their understanding of, and trust in, the information provided by the app. Most (26/42, 62%) participants used and valued visual environmental hazard features, especially maps, location settings, and hazard alerts. Most (33/42, 78%) found information in the app easy to understand and support their needs, irrespective of their self-reported literacy levels. Many (21/42, 50%) users reported that they did not question the accuracy of the data presented in the app. Suggested enhancements include the provision of meteorological information (eg, wind speed or direction, air pressure, UV rating, and humidity), functionality enhancements (eg, forecasting, additional alerts, and the inclusion of health advice), and clarification of existing information (eg, symptom triggers), including the capacity to download personal summary data for a specified period. CONCLUSIONS: Participants' perspectives can inform the future development of environmental health apps. Specifically, participants' insights support the identification of key elements for the optimal development of environmental health app design, including streamlining, capacity for users to customize, use of real time data, visual cues, credibility, and accuracy of data. The results also suggest that, in the future, iterative collaboration between developers, environmental agencies, and users will likely promote better functional design, user trust in the data, and ultimately better population health outcomes.

Functional and biological heterogeneity of KRASQ61 mutations.

Missense mutations at the three hotspots in the guanosine triphosphatase (GTPase) RAS-Gly12, Gly13, and Gln61 (commonly known as G12, G13, and Q61, respectively)-occur differentially among the three RAS isoforms. Q61 mutations in KRAS are infrequent and differ markedly in occurrence. Q61H is the predominant mutant (at 57%), followed by Q61R/L/K (collectively 40%), and Q61P and Q61E are the rarest (2 and 1%, respectively). Probability analysis suggested that mutational susceptibility to different DNA base changes cannot account for this distribution. Therefore, we investigated whether these frequencies might be explained by differences in the biochemical, structural, and biological properties of KRASQ61 mutants. Expression of KRASQ61 mutants in NIH 3T3 fibroblasts and RIE-1 epithelial cells caused various alterations in morphology, growth transformation, effector signaling, and metabolism. The relatively rare KRASQ61E mutant stimulated actin stress fiber formation, a phenotype distinct from that of KRASQ61H/R/L/P, which disrupted actin cytoskeletal organization. The crystal structure of KRASQ61E was unexpectedly similar to that of wild-type KRAS, a potential basis for its weak oncogenicity. KRASQ61H/L/R-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines exhibited KRAS-dependent growth and, as observed with KRASG12-mutant PDAC, were susceptible to concurrent inhibition of ERK-MAPK signaling and of autophagy. Our results uncover phenotypic heterogeneity among KRASQ61 mutants and support the potential utility of therapeutic strategies that target KRASQ61 mutant-specific signaling and cellular output.

Enhanced BRAF engagement by NRAS mutants capable of promoting melanoma initiation.

A distinct profile of NRAS mutants is observed in each tumor type. It is unclear whether these profiles are determined by mutagenic events or functional differences between NRAS oncoproteins. Here, we establish functional hallmarks of NRAS mutants enriched in human melanoma. We generate eight conditional, knock-in mouse models and show that rare melanoma mutants (NRAS G12D, G13D, G13R, Q61H, and Q61P) are poor drivers of spontaneous melanoma formation, whereas common melanoma mutants (NRAS Q61R, Q61K, or Q61L) induce rapid tumor onset with high penetrance. Molecular dynamics simulations, combined with cell-based protein-protein interaction studies, reveal that melanomagenic NRAS mutants form intramolecular contacts that enhance BRAF binding affinity, BRAF-CRAF heterodimer formation, and MAPK > ERK signaling. Along with the allelic series of conditional mouse models we describe, these results establish a mechanistic basis for the enrichment of specific NRAS mutants in human melanoma.

Oncogenic KRAS G12C: Kinetic and redox characterization of covalent inhibition.

The recent development of mutant-selective inhibitors for the oncogenic KRASG12C allele has generated considerable excitement. These inhibitors covalently engage the mutant C12 thiol located within the phosphoryl binding loop of RAS, locking the KRASG12C protein in an inactive state. While clinical trials of these inhibitors have been promising, mechanistic questions regarding the reactivity of this thiol remain. Here, we show by NMR and an independent biochemical assay that the pKa of the C12 thiol is depressed (pKa ∼7.6), consistent with susceptibility to chemical ligation. Using a validated fluorescent KRASY137W variant amenable to stopped-flow spectroscopy, we characterized the kinetics of KRASG12C fluorescence changes upon addition of ARS-853 or AMG 510, noting that at low temperatures, ARS-853 addition elicited both a rapid first phase of fluorescence change (attributed to binding, Kd = 36.0 ± 0.7 µM) and a second, slower pH-dependent phase, taken to represent covalent ligation. Consistent with the lower pKa of the C12 thiol, we found that reversible and irreversible oxidation of KRASG12C occurred readily both in vitro and in the cellular environment, preventing the covalent binding of ARS-853. Moreover, we found that oxidation of the KRASG12C Cys12 to a sulfinate altered RAS conformation and dynamics to be more similar to KRASG12D in comparison to the unmodified protein, as assessed by molecular dynamics simulations. Taken together, these findings provide insight for future KRASG12C drug discovery efforts, and identify the occurrence of G12C oxidation with currently unknown biological ramifications.

Post-translational modification of RAS proteins.

Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.

Ambulance dispatches and heatwaves in Tasmania, Australia: A case-crossover analysis.

BACKGROUND: Climate change is causing an increase in the frequency and severity of heatwave events, with a corresponding negative impact on human health. Health service utilisation during a heatwave is increased, with a greater risk of poor health outcomes identified for specific population groups. In this study, we examined the impact of heatwave events on ambulance dispatches in Tasmania, Australia from 2008 to 2019 to explore health service utilisation and identify the most vulnerable populations at a local level. METHODS: We used a time-stratified case-crossover analysis with conditional logistic regression to examine the association between ambulance dispatches and three levels of heatwave events (extreme, severe, and low-intensity). We examined the relationship for the whole study population, and by age, gender, socio-economic advantage and clinical diagnostic group. RESULTS: We found that ambulance dispatches increase by 34% (OR 1.34, 95% CI 1.18-1.52) during extreme heatwaves, by 10% (OR 1.10, 95% CI 1.05-1.15) during severe heatwaves and by 4% (OR 1.04, 95% CI 1.02-1.06) during low-intensity heatwaves. We found significant associations for the elderly (over 65), the young (5 and under) and for regions with the greatest socio-economic disadvantage. CONCLUSION: Heatwaves were associated with increased demands on ambulance services in Tasmania. In subgroups of people aged over 65 or under 5 years of age, and those from areas of higher disadvantage, we generally observed greater effect sizes than for the population as a whole.

Divergent Mechanisms Activating RAS and Small GTPases Through Post-translational Modification.

RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound "OFF" state and GTP-bound "ON" state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound "ON" state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.

Characterising non-linear associations between airborne pollen counts and respiratory symptoms from the AirRater smartphone app in Tasmania, Australia: A case time series approach.

Pollen is a well-established trigger of asthma and allergic rhinitis, yet concentration-response relationships, lagged effects, and interactions with other environmental factors remain poorly understood. Smartphone technology offers an opportunity to address these challenges using large, multi-year datasets that capture individual symptoms and exposures in real time. We aimed to characterise associations between six pollen types and respiratory symptoms logged by users of the AirRater smartphone app in Tasmania, Australia. We analyzed 44,820 symptom reports logged by 2272 AirRater app users in Tasmania over four years (2015-2019). With these data we evaluated associations between daily respiratory symptoms and atmospheric pollen concentrations. We implemented Poisson regression models, using the case time series approach designed for app-sourced data. We assessed potentially non-linear and lagged associations with (a) total pollen and (b) six individual pollen taxa. We adjusted for seasonality and meteorology and tested for interactions with particulate air pollution (PM2.5). We found evidence of non-linear associations between total pollen and respiratory symptoms for up to three days following exposure. For total pollen, the same-day relative risk (RR) increased to 1.31 (95% CI: 1.26-1.37) at a concentration of 50 grains/m3 before plateauing. Associations with individual pollen taxa were also non-linear with some diversity in shapes. For all pollen taxa the same-day RR was highest. The interaction between total pollen and PM2.5 was positive, with risks associated with pollen significantly higher in the presence of high concentrations of PM2.5. Our results support a non-linear response between airborne pollen and respiratory symptoms. The association was strongest on the day of exposure and synergistic with particulate air pollution. The associations found with Dodonaea and Myrtaceae highlight the need to further investigate the role of Australian native pollen types in allergic respiratory disease.

Monoubiquitination of KRAS at Lysine104 and Lysine147 Modulates Its Dynamics and Interaction with Partner Proteins.

KRAS, a 21 kDa guanine nucleotide-binding protein that functions as a molecular switch, plays a key role in regulating cellular growth. Dysregulation of this key signaling node leads to uncontrolled cell growth, a hallmark of cancer cells. KRAS undergoes post-translational modification by monoubiquitination at various locations, including at lysine104 (K104) and lysine147 (K147). Previous studies have suggested that K104 stabilizes helix-2/helix-3 interactions and K147 is involved in nucleotide binding. However, the impact of monoubiquitination at these residues on the overall structure, dynamics, or function of KRAS is not fully understood. In this study, we examined KRAS monoubiquitination at these sites using data from extensive (12 µs aggregate time) molecular dynamics simulations complemented by nuclear magnetic resonance spectroscopy data. We found that ubiquitin forms dynamic nonspecific interactions with various regions of KRAS and that ubiquitination at both sites modulates conformational fluctuations. In both cases, ubiquitin samples a broad range of conformational space and does not form long-lasting noncovalent contacts with KRAS but it adopts several preferred orientations relative to KRAS. To examine the functional impact of these preferred orientations, we performed a systematic comparison of the dominant configurations of the ubiquitin/KRAS simulated complex with experimental structures of KRAS bound to regulatory and effector proteins as well as a model membrane. Results from these analyses suggest that conformational selection and population shift may minimize the deleterious effects of KRAS ubiquitination at K104 and K147 on binding to some but not all interaction partners. Our findings thus provide new insights into the steric effects of ubiquitin and suggest a potential avenue for therapeutic targeting.

Environmental Hazards and Behavior Change: User Perspectives on the Usability and Effectiveness of the AirRater Smartphone App.

AirRater is a free smartphone app developed in 2015, supporting individuals to protect their health from environmental hazards. It does this by providing (i) location-specific and near real-time air quality, pollen and temperature information and (ii) personal symptom tracking functionality. This research sought to evaluate user perceptions of AirRater's usability and effectiveness. We collected demographic data and completed semi-structured interviews with 42 AirRater users, identified emergent themes, and used two frameworks designed to understand and support behavior change-the Behavior Change Wheel (BCW) and the Protective Action Decision Model (PADM)-to interpret results. Of the 42 participants, almost half indicated that experiencing symptoms acted as a prompt for app use. Information provided by the app supported a majority of the 42 participants to make decisions and implement behaviors to protect their health irrespective of their location or context. The majority of participants also indicated that they shared information provided by the app with family, friends and/or colleagues. The evaluation also identified opportunities to improve the app. Several study limitations were identified, which impacts the generalizability of results beyond the populations studied. Despite these limitations, findings facilitated new insights into motivations for behavior change, and contribute to the existing literature investigating the potential for smartphone apps to support health protection from environmental hazards in a changing climate.

A universal allosteric mechanism for G protein activation.

G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.

Biophysical and Structural Characterization of Novel RAS-Binding Domains (RBDs) of PI3Kα and PI3Kγ.

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate phosphatidylinositol 4,5-bisphosphate to generate a key lipid second messenger, phosphatidylinositol 3,4,5-bisphosphate. PI3Kα and PI3Kγ require activation by RAS proteins to stimulate signaling pathways that control cellular growth, differentiation, motility and survival. Intriguingly, RAS binding to PI3K isoforms likely differ, as RAS mutations have been identified that discriminate between PI3Kα and PI3Kγ, consistent with low sequence homology (23%) between their RAS binding domains (RBDs). As disruption of the RAS/PI3Kα interaction reduces tumor growth in mice with RAS- and epidermal growth factor receptor driven skin and lung cancers, compounds that interfere with this key interaction may prove useful as anti-cancer agents. However, a structure of PI3Kα bound to RAS is lacking, limiting drug discovery efforts. Expression of full-length PI3K isoforms in insect cells has resulted in low yield and variable activity, limiting biophysical and structural studies of RAS/PI3K interactions. This led us to generate the first RBDs from PI3Kα and PI3Kγ that can be expressed at high yield in bacteria and bind to RAS with similar affinity to full-length PI3K. We also solved a 2.31 Å X-ray crystal structure of the PI3Kα-RBD, which aligns well to full-length PI3Kα. Structural differences between the PI3Kα and PI3Kγ RBDs are consistent with differences in thermal stability and may underly differential RAS recognition and RAS-mediated PI3K activation. These high expression, functional PI3K RBDs will aid in interrogating RAS interactions and could aid in identifying inhibitors of this key interaction.