About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anticancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent ...approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. It also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have used the near-diploid hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with WT KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. Epidermal growth factor -induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than WT cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signaling and developing effective anti-KRAS reagents with minimum cytotoxicity on WT cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications ...that occur outside the endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of lysyl hydroxylase 3 (LH3, also known as PLOD3) into newly identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.
Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson’s disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an ...“upstream” oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. Moreover, we previously reported that the yeast DJ-1 homolog Hsp31 localizes to stress granules (SGs) after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. Here, we report that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs, as well as P-bodies, upon induction of either osmotic or oxidative stress. By purifying the mRNA associated with DJ-1 in mammalian cells, we detected several transcripts and found that subpopulations of these localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we find that DJ-1 associates with SGs arising from
N
-methyl-
d
-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-inducing toxins in dopaminergic cell cultures. Thus, our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mutations in the protein DJ-1 cause recessive forms of early onset familial Parkinson’s disease (PD). To date, most of the causative mutations studied destabilize formation of DJ-1 homodimers, which ...appears to be closely linked to its normal function in oxidative stress and other cellular processes. Despite the importance of understanding the dimerization dynamics of this protein, this aspect of DJ-1 biology has not previously been directly studied in living cells. Here, we use bimolecular fluorescence complementation to study DJ-1 dimerization and find not only that DJ-1 forms homodimers in living cells but that most PD causative DJ-1 mutations disrupt this process, including the L166P, M26I, L10P, and P158∆ mutations. Interestingly, the E64D mutant form of DJ-1 retains the ability to form homodimers. However, while wild-type DJ-1 dimers are stabilized under oxidative stress conditions, we find that the E64D mutation blocks this stabilization. Furthermore, our data show that the E64D mutation potentiates the formation of aggresomes containing DJ-1. We also observe that while the widely studied L166P mutation prevents DJ-1 from forming homodimers or heterodimers with wild-type protein, the mutant protein is able to partially disrupt formation of wild-type homodimers. In summary, by investigating DJ-1 dimerization in living cells, we have uncovered several novel properties of PD causative mutations in DJ-1, which may ultimately provide novel insight into PD pathogenesis and possible therapeutic options.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Summary
We have previously demonstrated that increases in cytosolic free Ca2+ are triggered by the self‐incompatibility (SI) response in incompatible Papaver rhoeas (the field poppy) pollen. However, ...one key question that has not been answered is whether extracellular Ca2+ may be involved. To address this question, we have used an ion‐selective vibrating probe to measure changes in extracellular Ca2+ fluxes around poppy pollen tubes. Our data reveal several findings. First, we confirm that there is an oscillating Ca2+ influx directed at the apex of the pollen tube; we also provide evidence that Ca2+ influx also occurs at the shanks of pollen tubes. Second, upon challenge with self‐incompatibility (S) proteins, there is a stimulation of Ca2+ influx along the shank of incompatible pollen tubes, approximately 50 µm behind the pollen tube tip. This demonstration of SI‐induced Ca2+ influx suggests a role for influx of extracellular Ca2+ in the SI response.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The circadian mechanism appears remarkably conserved between Drosophila and mammals, with basic underlying negative and positive feedback loops, cycling gene products, and temporally regulated ...nuclear transport involving a few key proteins. One of these negative regulators is PERIOD, which in Drosophila shows very similar temporal and spatial regulation to TIMELESS. Surprisingly, we observe that in the housefly, Musca domestica, PER does not cycle in Western blots of head extracts, in contrast to the TIM protein. Furthermore, immunocytochemical (ICC) localization using enzymatic staining procedures reveals that PER is not localized to the nucleus of any neurons within the brain at any circadian time, as recently observed for several nondipteran insects. However, with confocal analysis, immunofluorescence reveals a very different picture and provides an initial comparison of PER/TIM-containing cells in Musca and Drosophila, which shows some significant differences, but many similarities. Thus, even in closely related Diptera, there is considerable evolutionary flexibility in the number and spatial organization of clock cells and, indeed, in the expression patterns of clock products in these cells, although the underlying framework is similar.
Bacterial septicaemia is a major cause of mortality, but its pathogenesis remains poorly understood. In experimental pneumococcal murine intravenous infection, an initial reduction of bacteria in the ...blood is followed hours later by a fatal septicaemia. These events represent a population bottleneck driven by efficient clearance of pneumococci by splenic macrophages and neutrophils, but as we show in this study, accompanied by occasional intracellular replication of bacteria that are taken up by a subset of CD169
splenic macrophages. In this model, proliferation of these sequestered bacteria provides a reservoir for dissemination of pneumococci into the bloodstream, as demonstrated by its prevention using an anti-CD169 monoclonal antibody treatment. Intracellular replication of pneumococci within CD169
splenic macrophages was also observed in an ex vivo porcine spleen, where the microanatomy is comparable with humans. We also showed that macrolides, which effectively penetrate macrophages, prevented septicaemia, whereas beta-lactams, with inefficient intracellular penetration, failed to prevent dissemination to the blood. Our findings define a shift in our understanding of the pneumococcus from an exclusively extracellular pathogen to one with an intracellular phase. These findings open the door to the development of treatments that target this early, previously unrecognized intracellular phase of bacterial sepsis.
Circadian rhythms affect the progression and severity of bacterial infections including those caused by
Streptococcus pneumoniae
, but the mechanisms responsible for this phenomenon remain largely ...elusive. Following advances in our understanding of the role of replication of
S. pneumoniae
within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism’s circadian cycle plays a seemingly counterproductive role in the control of invasive infection.
Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs ...it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia.
We used human spleens perfused ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease.
Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease.
We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia.
Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated ...with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections.
In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis.
We show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell SD 1·1). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages.
These findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses.
University of Oxford and a Royal Society Wolfson grant funded biosafety facility.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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