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.

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.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Dominant activating RAC2 mutation with lymphopenia, immunodeficiency, and cytoskeletal defects.

Ras-related C3 botulinum toxin substrate 2 (RAC2), through interactions with reduced NAD phosphate oxidase component p67 phox , activates neutrophil superoxide production, whereas interactions with p21-activated kinase are necessary for fMLF-induced actin remodeling. We identified 3 patients with de novo RAC2[E62K] mutations resulting in severe T- and B-cell lymphopenia, myeloid dysfunction, and recurrent respiratory infections. Neutrophils from RAC2[E62K] patients exhibited excessive superoxide production, impaired fMLF-directed chemotaxis, and abnormal macropinocytosis. Cell lines transfected with RAC2[E62K] displayed characteristics of active guanosine triphosphate (GTP)-bound RAC2 including enhanced superoxide production and increased membrane ruffling. Biochemical studies demonstrated that RAC2[E62K] retains intrinsic GTP hydrolysis; however, GTPase-activating protein failed to accelerate hydrolysis resulting in prolonged active GTP-bound RAC2. Rac2+/E62K mice phenocopy the T- and B-cell lymphopenia, increased neutrophil F-actin, and excessive superoxide production seen in patients. This gain-of-function mutation highlights a specific, nonredundant role for RAC2 in hematopoietic cells that discriminates RAC2 from the related, ubiquitous RAC1.

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.

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.

Regulation of large and small G proteins by ubiquitination.

Many sensory and chemical signal inputs are transmitted by intracellular GTP-binding (G) proteins. G proteins make up two major subfamilies: "large" G proteins comprising three subunits and "small" G proteins, such as the proto-oncogene product RAS, which contains a single subunit. Members of both subfamilies are regulated by post-translational modifications, including lipidation, proteolysis, and carboxyl methylation. Emerging studies have shown that these proteins are also modified by ubiquitination. Much of our current understanding of this post-translational modification comes from investigations of the large G-protein α subunit from yeast (Gpa1) and the three RAS isotypes in humans, NRAS, KRAS, and HRAS. Gα undergoes both mono- and polyubiquitination, and these modifications have distinct consequences for determining the sites and mechanisms of its degradation. Genetic and biochemical reconstitution studies have revealed the enzymes and binding partners required for addition and removal of ubiquitin, as well as the delivery and destruction of both the mono- and polyubiquitinated forms of the G protein. Complementary studies of RAS have identified multiple ubiquitination sites, each having distinct consequences for binding to regulatory proteins, shuttling to and from the plasma membrane, and degradation. Here, we review what is currently known about these two well-studied examples, Gpa1 and the human RAS proteins, that have revealed additional mechanisms of signal regulation and dysregulation relevant to human physiology. We also compare and contrast the effects of G-protein ubiquitination with other post-translational modifications of these proteins.

Copper is required for oncogenic BRAF signalling and tumorigenesis.

The BRAF kinase is mutated, typically Val 600→Glu (V600E), to induce an active oncogenic state in a large fraction of melanomas, thyroid cancers, hairy cell leukaemias and, to a smaller extent, a wide spectrum of other cancers. BRAF(V600E) phosphorylates and activates the MEK1 and MEK2 kinases, which in turn phosphorylate and activate the ERK1 and ERK2 kinases, stimulating the mitogen-activated protein kinase (MAPK) pathway to promote cancer. Targeting MEK1/2 is proving to be an important therapeutic strategy, given that a MEK1/2 inhibitor provides a survival advantage in metastatic melanoma, an effect that is increased when administered together with a BRAF(V600E) inhibitor. We previously found that copper (Cu) influx enhances MEK1 phosphorylation of ERK1/2 through a Cu-MEK1 interaction. Here we show decreasing the levels of CTR1 (Cu transporter 1), or mutations in MEK1 that disrupt Cu binding, decreased BRAF(V600E)-driven signalling and tumorigenesis in mice and human cell settings. Conversely, a MEK1-MEK5 chimaera that phosphorylated ERK1/2 independently of Cu or an active ERK2 restored the tumour growth of murine cells lacking Ctr1. Cu chelators used in the treatment of Wilson disease decreased tumour growth of human or murine cells transformed by BRAF(V600E) or engineered to be resistant to BRAF inhibition. Taken together, these results suggest that Cu-chelation therapy could be repurposed to treat cancers containing the BRAF(V600E) mutation.

Can smartphone data identify the local environmental drivers of respiratory disease?

Asthma and allergic rhinitis (or hay fever) are ubiquitous, chronic health conditions that seasonally affect a sizeable proportion of the population. Both are commonly triggered or exacerbated by environmental conditions including aeroallergens, air quality and weather. Smartphone technology offers new opportunities to identify environmental drivers by allowing large-scale, real-time collection of day-to-day symptoms. As yet, however, few studies have explored the potential of this technology to provide useful epidemiological data on environment-symptom relationships. Here, we use data from the smartphone app 'AirRater' to examine relationships between asthma and allergic rhinitis symptoms and weather, air quality and pollen loads in Hobart, Tasmania, Australia. We draw on symptom data logged by app users over a three-year period and use time-series analysis to assess the relationship between symptoms and environmental co-variates. Symptoms are associated with particulate matter (IRR 1.06, 95% CI: 1.04-1.08), maximum temperature (IRR 1.28, 95% CI: 1.13-1.44) and pollen taxa including Betula (IRR 1.04, 95% CI: 1.02-1.07), Cupressaceae (IRR 1.02, 95% CI: 1.01-1.04), Myrtaceae (IRR 1.06, 95% CI: 1.02-1.10) and Poaceae (IRR 1.05, 95% CI: 1.01-1.09). The importance of these pollen taxa varies seasonally and more taxa are associated with allergic rhinitis (eye/nose) than asthma (lung) symptoms. Our results are congruent with established epidemiological evidence, while providing important local insights including the association between symptoms and Myrtaceae pollen. We conclude that smartphone-sourced data can be a useful tool in environmental epidemiology.

Amino acid metabolites that regulate G protein signaling during osmotic stress.

All cells respond to osmotic stress by implementing molecular signaling events to protect the organism. Failure to properly adapt can lead to pathologies such as hypertension and ischemia-reperfusion injury. Mitogen-activated protein kinases (MAPKs) are activated in response to osmotic stress, as well as by signals acting through G protein-coupled receptors (GPCRs). For proper adaptation, the action of these kinases must be coordinated. To identify second messengers of stress adaptation, we conducted a mass spectrometry-based global metabolomics profiling analysis, quantifying nearly 300 metabolites in the yeast S. cerevisiae. We show that three branched-chain amino acid (BCAA) metabolites increase in response to osmotic stress and require the MAPK Hog1. Ectopic addition of these BCAA derivatives promotes phosphorylation of the G protein α subunit and dampens G protein-dependent transcription, similar to that seen in response to osmotic stress. Conversely, genetic ablation of Hog1 activity or the BCAA-regulatory enzymes leads to diminished phosphorylation of Gα and increased transcription. Taken together, our results define a new class of candidate second messengers that mediate cross talk between osmotic stress and GPCR signaling pathways.

Macrophage origin limits functional plasticity in helminth-bacterial co-infection.

Rapid reprogramming of the macrophage activation phenotype is considered important in the defense against consecutive infection with diverse infectious agents. However, in the setting of persistent, chronic infection the functional importance of macrophage-intrinsic adaptation to changing environments vs. recruitment of new macrophages remains unclear. Here we show that resident peritoneal macrophages expanded by infection with the nematode Heligmosomoides polygyrus bakeri altered their activation phenotype in response to infection with Salmonella enterica ser. Typhimurium in vitro and in vivo. The nematode-expanded resident F4/80high macrophages efficiently upregulated bacterial induced effector molecules (e.g. MHC-II, NOS2) similarly to newly recruited monocyte-derived macrophages. Nonetheless, recruitment of blood monocyte-derived macrophages to Salmonella infection occurred with equal magnitude in co-infected animals and caused displacement of the nematode-expanded, tissue resident-derived macrophages from the peritoneal cavity. Global gene expression analysis revealed that although nematode-expanded resident F4/80high macrophages made an anti-bacterial response, this was muted as compared to newly recruited F4/80low macrophages. However, the F4/80high macrophages adopted unique functional characteristics that included enhanced neutrophil-stimulating chemokine production. Thus, our data provide important evidence that plastic adaptation of MΦ activation does occur in vivo, but that cellular plasticity is outweighed by functional capabilities specific to the tissue origin of the cell.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.

Dyadic interdependence of psychosocial outcomes among haematological cancer survivors and their support persons.

PURPOSE: This study aimed to explore the dyadic relationships between unmet need, depression, and anxiety in people diagnosed with haematological cancer and their support persons. METHODS: Adult survivors (18 years+) who had been diagnosed with a haematological cancer were recruited to a cross-sectional mailed survey via five state cancer registries in Australia. Participating survivors invited a support person to also complete a survey. Structural equation modelling was used to explore the relationships among survivor and support person self-reported depression, anxiety, and unmet needs. RESULTS: Of the 4299 eligible haematological cancer survivors contacted by the registries, 1511 (35%) returned a completed survey as did 1004 support persons. There were 787 dyads with complete data. After adjusting for age, gender, rurality, cancer type, and whether the support person was a relative, positive correlations were found between survivor and support person scores for depression (p = 0.0029) and unmet needs (p < 0.001), but not anxiety scores (p = 0.075). Survivor unmet needs were significantly related to support person depression (p = 0.0036). Support person unmet needs were significantly related to a higher depression score for survivors (p = 0.0067). Greater support person unmet needs were significantly related to a higher anxiety score for survivors (p = 0.0083). Survivor unmet needs did not have a significant relationship to support person anxiety (p = 0.78). CONCLUSION: Unmet needs may mediate the interdependence of psychosocial experiences for survivors and support persons, although a longitudinal study is required to confirm causality. Addressing unmet needs may be a potential target for improving outcomes for both groups.

The Voice of Experience: Diet and Weight Change in Women with Breast Cancer Associate with Psychosocial and Treatment-Related Challenges.

PURPOSE: This study investigated relationships between psychosocial and treatment-related factors, diet, and weight change in women treated with chemotherapy for early-stage breast cancer. METHODS: Comprehensive qualitative interviews were conducted with 28 women who were within 12 months of completing chemotherapy treatment. RESULTS: Changes in food intake and eating patterns were universal over the course of chemotherapy, with broad variability in treatment effects and associated dietary responses linked to weight change. Increased appetite, food cravings, and intake of energy-dense comfort foods were more common among women who gained weight during treatment (n = 11). Changes in taste, nausea, and emotional distress were central in promoting these dietary responses. Women who lost weight during treatment (n = 6) tended to report more severe and persistent side effects of treatment leading to poor appetite and lower food intake, and they were more likely to live alone. CONCLUSIONS: While the etiology of weight change in this population is complex, this study suggests that changes in food intake related to treatment and psychosocial challenges may play an important role for some women. These findings may help to identify women who are most at risk of weight change during treatment and may inform the development of tailored dietary interventions.

Reduction in Cell Viability and in Homeobox Protein Levels Following in Vitro Exposure to δ-tocopherol in Acute Myeloid Leukemia.

δ-Tocopherol (δ-T), the least prevalent tocopherol in our diet, was described to have a more potent anticancer activity in solid tumors compared to the other tocopherols. δ-T induces tumor cell death through peroxisome proliferator-activated receptor γ (PPAR-γ) induction, cyclin-D1 inhibition, and modulation of redox balance. Nevertheless, the role of δ-T in preventing or treating hematologic malignancies has not been studied. In this study, we screened the efficacy of δ-T against six cell lines representing a wide spectrum of hematologic malignancies: Jurkat (acute T-cell leukemia), K-562 (chronic myeloid leukemia), KG-1 [acute myeloid leukemia (AML)], THP-1 (acute monocytic leukemia), TOM-1 (acute lymphoblastic leukemia), and UMCL01-101 (AIDS-associated diffuse large B-cell lymphoma). Interestingly, the AML cell line KG-1 was the only one to be significantly affected at concentrations of δ-T as low as 20 µM. The antileukemic activity of δ-T in AML was verified in a set of primary cells collected from patients newly diagnosed with AML. Apoptotic induction and cell cycle arrest explained the efficacy of δ-T against KG-1 cells. The mechanism of cell growth inhibition of δ-T was through downregulation of cyclin-D1 and a set of homeobox proteins (HOXA9, PBX1, and Cdx2) that have a well-documented role in the pathobiology of AML.

Phosphorylation at Y1065 in vinculin mediates actin bundling, cell spreading, and mechanical responses to force.

Vinculin is an essential structural adaptor protein that localizes to sites of adhesion and is involved in a number of cell processes including adhesion, spreading, motility, force transduction, and cell survival. The C-terminal vinculin tail domain (Vt) contains the necessary structural components to bind and cross-link actin filaments. Actin binding to Vt induces a conformational change that promotes dimerization through the C-terminal hairpin of Vt and enables actin filament cross-linking. Here we show that Src phosphorylation of Y1065 within the C-terminal hairpin regulates Vt-mediated actin bundling and provide a detailed characterization of Y1065 mutations. Furthermore, we show that phosphorylation at Y1065 plays a role in cell spreading and the response to the application of mechanical force.

Differences in the regulation of K-Ras and H-Ras isoforms by monoubiquitination.

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.

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 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.