Role of T198 modification in the regulation of p27(Kip1) protein stability and function

The tumor suppressor gene p27(Kip1) plays a fundamental role in human cancer progression. Its expression and/or functions are altered in almost all the different tumor histotype analyzed so far. Recently, it has been demonstrated that the tumor suppression function of p27 resides not only in the ability to inhibit Cyclins/CDKs complexes through its N-terminal domain but also in the capacity to modulate cell motility through its C-terminal portion. Particular interest has been raised by the last amino-acid, (Threonine 198) in the regulation of both protein stability and cell motility.Here, we describe that the presence of Threonine in position 198 is of primary importance for the regulation of the protein stability and for the control of cell motility. However, while the control of cell motility is dependent on the phosphorylation of T198, the stability of the protein is specifically controlled by the steric hindrance of the last amino acid. The effects of T198 modification on protein stability are not linked to the capacity of p27 to bind Cyclins/CDKs complexes and/or the F-box protein Skp2. Conversely, our results support the hypothesis that conformational changes in the disordered structure of the C-terminal portion of p27 are important in its ability to be degraded via a proteasome-dependent mechanism. On the other hand T198 phosphorylation favors p27/stathmin interaction eventually contributing to the regulation of cell motility, supporting the hypothesis that the presence of T198 is fundamental for the regulation of p27 functions.     

ALK Kinase Domain Mutations in Primary Anaplastic Large Cell Lymphoma: Consequences on NPM-ALK Activity and Sensitivity to Tyrosine Kinase Inhibitors

ALK inhibitor crizotinib has shown potent antitumor activity in children with refractory Anaplastic Large Cell Lymphoma (ALCL) and the opportunity to include ALK inhibitors in first-line therapies is oncoming. However, recent studies suggest that crizotinib-resistance mutations may emerge in ALCL patients. In the present study, we analyzed ALK kinase domain mutational status of 36 paediatric ALCL patients at diagnosis to identify point mutations and gene aberrations that could impact on NPM-ALK gene expression, activity and sensitivity to small-molecule inhibitors. Amplicon ultra-deep sequencing of ALK kinase domain detected 2 single point mutations, R335Q and R291Q, in 2 cases, 2 common deletions of exon 23 and 25 in all the patients, and 7 splicing-related INDELs in a variable number of them. The functional impact of missense mutations and INDELs was evaluated. Point mutations were shown to affect protein kinase activity, signalling output and drug sensitivity. INDELs, instead, generated kinase-dead variants with dominant negative effect on NPM-ALK kinase, in virtue of their capacity of forming non-functional heterocomplexes. Consistently, when co-expressed, INDELs increased crizotinib inhibitory activity on NPM-ALK signal processing, as demonstrated by the significant reduction of STAT3 phosphorylation. Functional changes in ALK kinase activity induced by both point mutations and structural rearrangements were resolved by molecular modelling and dynamic simulation analysis, providing novel insights into ALK kinase domain folding and regulation. Therefore, these data suggest that NPM-ALK pre-therapeutic mutations may be found at low frequency in ALCL patients. These mutations occur randomly within the ALK kinase domain and affect protein activity, while preserving responsiveness to crizotinib.     

Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono‐ and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl‐MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl‐MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12‐oxo‐phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA‐containing galactolipids in the plant kingdom. While acyl‐MGDG was found to be ubiquitous in green tissue of plants ranging from non‐vascular plants to angiosperms, OPDA‐containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non‐oxidized and OPDA‐containing acyl‐MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl‐MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.     

PKCα binds G3BP2 and regulates stress granule formation following cellular stress

Protein kinase C (PKC) isoforms regulate a number of processes crucial for the fate of a cell. In this study we identify previously unrecognized interaction partners of PKCα and a novel role for PKCα in the regulation of stress granule formation during cellular stress. Three RNA-binding proteins, cytoplasmic poly(A)(+) binding protein (PABPC1), IGF-II mRNA binding protein 3 (IGF2BP3), and RasGAP binding protein 2 (G3BP2) all co-precipitate with PKCα. RNase treatment abolished the association with IGF2BP3 and PABPC1 whereas the PKCα-G3BP2 interaction was largely resistant to this. Furthermore, interactions between recombinant PKCα and G3BP2 indicated that the interaction is direct and PKCα can phosphorylate G3BP2 in vitro. The binding is mediated via the regulatory domain of PKCα and the C-terminal RNA-binding domain of G3BP2. Both proteins relocate to and co-localize in stress granules, but not to P-bodies, when cells are subjected to stress. Heat shock-induced stress granule assembly and phosphorylation of eIF2α are suppressed following downregulation of PKCα by siRNA. In conclusion this study identifies novel interaction partners of PKCα and a novel role for PKCα in regulation of stress granules.     

The importance of scavenging reactive oxygen species in anti‐aging medicine

In a pilot study, we had reported on the beneficial effects of Ginkgo biloba (EGb 761) on arteriosclerotic nanoplaque formation and size in cardiovascular high‐risk patients who had undergone an aortocoronary bypass operation. Briefly, nanoplaque formation and size, the ratio oxLDL/LDL, and the highly atherothrombotic lipoprotein(a) concentration were substantially reduced, while superoxide dismutase activity and the blood concentration of the vasodilating substances cAMP and cGMP were upregulated. Since the arteriosclerosis prophylactic and well‐aging promotive impact of Ginkgo extract has been proven in this pilot study, we wanted to confirm these beneficial effects through a second observational clinical trial. The measurable variables formerly used were additionally supplemented by a wide, novel biomarker spectrum, through which the latest parameters and markers of plaque stability and progression, oxidative stress, and inflammation were available. In eleven patients with metabolic syndrome in the initial stage, the reduction of arteriosclerotic nanoplaque formation amounted to 14.3±2.9% (p<0.0077) and of nanoplaque size to 23.4±3.7% (p<0.0004), respectively, after 2‐months of treatment with Ginkgo biloba extract. Additionally, superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were upregulated by 19.6±10.0% (p<0.0785) and 11.6±2.3% (p<0.001), respectively, the quotient oxLDL/LDL lowered by 21.0±4.3% (p<0.002), and lipoprotein(a) concentration decreased by 26.3±4.8% (p<0.001) in the patients' blood. The concentration of cAMP and cGMP was augmented by 43.5±12.0% (p<0.001) and 32.9±10.4% (p<0.001), respectively. Surprisingly, we found a lowering of the serum Ca²⁺ concentration by 5.4±1.6% (p<0.0076) from 2.37±0.03 to 2.24±0.04 mmol/L (p<0.0069). Apart from an additional vasodilatory effect, the lowered extracellular Ca²⁺ concentration affects nanoplaque formation restrictively, since this is a Ca²⁺ driven process. Furthermore, we could show a favourable development of the biomarkers 8‐iso‐PGF_2α, oxLDL/LDL, SOD, GPx (oxidative stress), hs‐CRP, MPO, TNFα, TGFβ₁ (inflammatory status) and MMP‐9 (plaque stability). The markers selected here are suited to provide a comprehensive risk profile for the prevention of arteriosclerosis. Finally, a multiple regression analysis reveals a basis for a mechanistic explanation of nanoplaque reduction under Ginkgo treatment. The arteriosclerosis inhibiting effect is due to an attenuation of the risk factors oxLDL/LDL, Lp(a), and [Ca²⁺]_o as well as to a significant increase in the vasodilator cAMP and cGMP concentration. Thus, Ginkgo with its pleiotropic effects should be assigned a fixed rank among the anti‐aging medical therapeutics as a prophylactic measure, especially in patients with early‐stage metabolic syndrome.     

Increase in Acid Tolerance of Campylobacter jejuni through Coincubation with Amoebae

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.Campylobacter jejuni is a major cause of human bacterial enteritis, with an incidence exceeding that of Salmonella spp. or Escherichia coli O157 (6, 28). Most infections are associated with consumption of contaminated food, primarily undercooked chicken meat, but unchlorinated water and unpasteurized milk can also be sources of Campylobacter infection (reviewed in reference 13). Apart from food-borne sources, additional risk factors include close contact with pets or farm animals and activities in recreational waters (reviewed in reference 13). C. jejuni is widely distributed in many animals and has also been reported to be isolated from surface waters (15) and, occasionally, even from groundwater (31). However, the bacterium has been shown to be relatively sensitive to environmental stress outside its hosts, including heating, disinfectants, oxygen exposure, osmotic stress, desiccation, and acidity (5, 9, 19, 35).Several hygiene practices have been implemented in broiler production facilities to reduce C. jejuni carriage in live birds. Such measures include hygiene barriers such as changing clothes before entering the broiler houses and disinfection of the interior of the building with acid between flock rotations (20). Such efforts may reduce the number of C. jejuni organisms, but the bacterium is still difficult to eradicate from contaminated farms, and subsequent outbreaks at the same farm are not rare (11). Contradictory to its fragility in different in vitro settings, C. jejuni seems to be well adapted to survive the acidic milieu of the human stomach during the passage to the lower intestinal tract, where infection is established. This is illustrated by the very low infectious dose for both broiler chickens (7) and humans (4) and indicates that the bacterium has developed strategies to avoid or withstand low pH in order to survive the transit. The gastric acid is the first line of defense against ingested pathogens. During fasting conditions in healthy humans, the luminal pH in the stomach is usually around 2.0, but it may range from 1.5 to 5.5 depending on food intake, such as a diet with a high pH, or the use of proton pump inhibitors (36). Laboratory studies have demonstrated that C. jejuni in solution survives a maximum of 30 min at pH levels below pH 2.5 and for up to 60 min at pH 3 (5, 23). When the bacterium is mixed with food, it seems to be protected, and it has been shown that C. jejuni inoculated onto ground beef survived at pH 2.5 for 2 h at 37°C (37).In the last few years, laboratory studies have identified a new potential epidemiological pathway for C. jejuni in which the bacterium colonizes unicellular eukaryotic organisms (protozoa) and thereby acquires protection from adverse environmental conditions (2, 17, 29). C. jejuni can colonize protozoa and survive longer in its protozoan host than as a free-living bacterium, and given the right temperature, the bacterium can also replicate intracellularly (1, 2). Protozoa, especially amoebae, serve as natural reservoirs or vehicles for the dissemination of several other pathogenic bacteria, including Legionella pneumophila (25), Vibrio cholerae (34), and Helicobacter pylori (38). Amoebae are abundant in virtually all natural water systems and can be found grazing on biofilms in water supply systems (14). In their trophozoite form, amoebae are naturally resistant to many environmental factors that are lethal to Campylobacter, and they can multiply at pHs ranging from 4 to 12 (16). Moreover, amoebae can enter a cyst form when challenged with unfavorable conditions. These cysts generally have a double cell wall that might explain their capability to survive chlorination, antimicrobials, and changes in pH and osmotic pressure. This resistance feature of amoebae makes them suitable hosts for other, less-resistant microorganisms (16, 32).In this study, we built on the advances gained in protozoa-Campylobacter research and investigated whether internalization of C. jejuni into Acanthamoeba affects bacterial tolerance to hydrochloric acid. Using an in vitro setup, we found that C. jejuni survived better in an acidic environment when it was coincubated with amoebae than when it was incubated as bacteria in solution. Furthermore, we show that bacterial motility and adhesion to and internalization into amoeba are trigged by moderately acidic conditions. The implications of these findings for the survival of C. jejuni in food production as well as in transit through the human stomach are discussed.     

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 Å. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 Å and 2.6 Å, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.     

Protein kinase Cepsilon binds peripherin and induces its aggregation, which is accompanied by apoptosis of neuroblastoma cells

A hallmark of the afflicted nervous tissue in amyotrophic lateral sclerosis is the presence of protein aggregates, which to a large extent contain the intermediate filament protein peripherin. Here we show that activation of protein kinase C (PKC) or overexpression of PKCepsilon induces the aggregation of peripherin in cultured neuroblastoma cells with elevated amounts of peripherin. The formation of aggregates was coupled to an increased apoptosis, suggesting a functional link between these events. Both induction of aggregates and apoptosis were suppressed in cells that had been transfected with small interfering RNAs targeting PKCepsilon. PKCepsilon and peripherin associate as shown by co-immunoprecipitation, and the interaction is dependent on and mediated by the C1b domain of PKCepsilon. The interaction was specific for PKCepsilon since corresponding structures from other isoforms did not co-precipitate peripherin, with the exception for PKCeta and -, which pulled down minute amounts. PKCepsilon interacts with vimentin through the same structures but does not induce its aggregation. When the PKCepsilon C1b domain is expressed in neuroblastoma cells together with peripherin, both phorbol ester-induced peripherin aggregation and apoptosis are abolished, supporting a model in which PKCepsilon through its interaction with peripherin facilitates its aggregation and subsequent cell death. These events may be prevented by expressing molecules that bind peripherin at the same site as PKCepsilon.     

Automated solid-phase extraction method for measuring urinary polycyclic aromatic hydrocarbon metabolites in human biomonitoring using isotope-dilution gas chromatography high-resolution mass spectrometry

In order to perform comprehensive epidemiological studies where multiple metabolites of several PAHs are measured and compared in low-dose urine samples, fast and robust methods are needed to measure many analytes in the same sample. We have modified a previous method used for measuring polycyclic aromatic hydrocarbon (PAH) metabolites by automating the solid-phase extraction (SPE) and including an additional eight metabolites. We also added seven new carbon-13 labeled standards, which improves the use of isotope-dilution calibration. Our method included enzyme hydrolysis, automated SPE and derivatization with a silylating reagent followed by gas chromatography (GC), coupled with high-resolution mass spectrometry (HRMS). Using this method, we measured 23 metabolites, representing 9 parent PAHs, with detection limits in the low pg/mL range. All steps in the clean-up procedure were optimized individually, resulting in a method that gives good recoveries (69-93%), reproducibility (coefficient of variation for two quality control pools ranged between 4.6 and 17.1%, N>156), and the necessary specificity. We used the method to analyze nearly 3000 urine samples in the fifth National Health and Nutrition Examination Survey (NHANES 2001-2002).