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.

RAS ubiquitylation modulates effector interactions.

RAS proteins function as molecular switches that regulate cellular growth by cycling between active GTP- and inactive GDP bound states. While RAS activity is modulated by factors (guanine nucleotide exchange and GTPase activating proteins) that control levels of active Ras-GTP, RAS proteins also undergo a number of post-translational modifications that regulate their function. One such modification is ubiquitylation. Monoubiquitylation of KRAS at lysine 147 (mUbRAS) enhances Ras activation and promotes signaling through the RAF and Phosphoinositide 3-Kinase (PI3K) signaling pathways. We have previously shown that mUbRAS leads to activation of RAS through a defect in GTPase activating protein (GAP) mediated downregulation, similar to the action of most oncogenic mutations. Consistent with these findings, we now show that mUbRASimpairsRAS binding to the p120 GAP catalytic domain. Mutations in activated G12V RAS that prevent ubiquitylaton at 147 show a decrease in tumorigenesis, suggesting that in addition to activating KRAS, monoubiquitylation at this site may promote downstream signaling and transformation. To investigate whether mUbRAS alters RAS effector interactions, we chemically ubiquitylated KRAS at residue 147 and characterized binding of mUbRAS to RAS binding domains (RBDs) from three distinct downstream effectors that play key roles in RAS-mediated transformation. Results from these studies show a decrease in binding of mUbRAS (7-10-fold) relative to the CRAF RAS Binding Domain (RBD), the catalytic subunit of Phosphoinositide 3-Kinase catalytic gamma (PI3Kcγ) and RALGDS RBD. Intriguingly, we find that mUbRAS shows greatly enhanced (> 40-fold) binding to the CRAF RBD when bound to GDP. These findings, taken together, suggest that mUbRASmay promoteactivation of RAS through a GAP defect, and facilitate RAF association and MAPK signaling in a nucleotide independent manner.

Atypical KRASG12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor.

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)-Golgi secretory pathway. FGF1 is released through a Cu(2+)-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu(2+)-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu(2+) to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu(2+) and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu(2+) in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb(3+) show that there are two Cu(2+)-binding pockets on the C2B domain, and one of these is also a Ca(2+)-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu(2+). The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.

Mental health, help seeking, and stigma and barriers to care among 3- and 12-month postdeployed and never deployed U.S. Army Combat Medics.

U.S. Army Combat Medic serves as both Soldier and provider of combat casualty care, often in the heat of battle and with limited resources. Yet little is known about their help-seeking behavior and perceived stigma and barriers to care. Participants were three groups of U.S. Army Combat Medics surveyed at 3- and 12-months postdeployment from assignment with line units vs. those Medics who had never deployed to combat. The primary data source was surveys of mental health service utilization, perceived stigma and barriers to care, and depression and post-traumatic stress disorder screens. Medics who received help in the past year from a mental health professional ranged from 18% to 30%, with 18% to 30% seeking mental health assistance from other sources. Previously deployed Medics were more likely to obtain assistance than those who never deployed. Those meeting a mental health screening criteria were more likely to report associated stigma and barriers to care. Findings indicate that Medics in need of assistance report greater perceived barriers to mental health care, as well as stigma from seeking treatment, and that depression may be a salient issue for Medics. The longitudinal nature of the ongoing study will help determine the actual trajectory and onset of depression and post-traumatic stress disorder.

Gender differences in combat medic mental health services utilization, barriers, and stigma.

Military health care providers experience considerable stressors related to their exposure to death and traumatic injuries in others. This study used survey data from 799 active duty U.S. Army Combat Medics deployed to Operation Iraqi Freedom/Operation Enduring Freedom. Military experiences, combat exposures, and mental health care seeking of active duty Combat Medics were explored and compared across both genders. Barriers to care were also assessed. Male and female Combat Medics reported surprisingly similar experiences, exposures, and health issues. Overall, results indicate no striking differences in barriers for females compared to their male counterparts, suggesting the barriers to utilization of mental health services may be consistent across gender. Although medics endorsed barriers and stigma related to mental health counseling services, they still sought these health services. Female and male medics who endorsed barriers were more likely to report seeking services than those who did not endorse barriers. This study provides an initial description of utilization of mental health counseling services for U.S. Army Combat Medics, the majority of whom were involved in combat operations in Afghanistan or Iraq. Our findings indicate that comprehensive assessment of the military experiences and combat exposures is needed to appreciate their potential influence on military health care providers.

Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation.

Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associated KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. (1)H-(15)N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.

Training, deployment preparation, and combat experiences of deployed health care personnel: key findings from deployed U.S. Army combat medics assigned to line units.

OBJECTIVE: To describe the perceptions of training and deployment preparation and combat experiences and exposures of U.S. Army combat medics. METHODS: Data were from the first year of a 3-year longitudinal study designed to assess the impact of combat on the behavioral health and resilience of 347 combat medics surveyed 3 to 6 months after returning from a 12-month deployment to Operation Enduring Freedom/Operation Iraqi Freedom theatre and assigned to brigade combat teams. RESULTS: Analyses indicated that combat medics may benefit from better preparation in types of shifts required during deployment, type and intensity of combat likely to be seen and experienced, more adequate training in the area of stress and mental health care management, and easier access to behavioral mental health care. CONCLUSIONS: The military has shown considerable progress in addressing and understanding the mental health care needs of Soldiers. However, challenges remain. Additional emphasis should be placed on reducing the stigma and barriers related to mental health care both in theatre and garrison and on developing an evidence-based, validated program for medics and other Soldiers to recognize stress and mental health issues on the battlefield. For medics, this should be from two perspectives-that of a combat Soldier and that of a medical provider.

Large-scale, hot-filament-assisted synthesis of tungsten oxide and related transition metal oxide nanowires.

A scalable and versatile method for the large-scale synthesis of tungsten trioxide nanowires and their arrays on a variety of substrates, including amorphous quartz and fluorinated tin oxide, is reported. The synthesis involves the chemical-vapor transport of metal oxide vapor-phase species using air or oxygen flow over hot filaments onto substrates kept at a distance. The results show that the density of the nanowires can be varied from 10(6)-10(10) cm(-2) by varying the substrate temperature. The diameter of the nanowires ranges from 100-20 nm. The results also show that variations in oxygen flow and substrate temperature affect the nanowire morphology from straight to bundled to branched nanowires. A thermodynamic model is proposed to show that the condensation of WO(2) species primarily accounts for the nucleation and subsequent growth of the nanowires, which supports the hypothesis that the nucleation of nanowires occurs through condensation of suboxide WO(2) vapor-phase species. This is in contrast to the expected WO(3) vapor-phase species condensation into WO(3) solid phase for nanoparticle formation. The as-synthesized nanowires are shown to form stable dispersions compared to nanoparticles in various organic and inorganic solvents.