Saccharomyces cerevisiae senses hyperosmotic conditions via the HOG signaling network that activates the stress-activated protein kinase, Hog1, and modulates metabolic fluxes and gene expression to generate appropriate adaptive responses. The integral control mechanism by which Hog1 modulates glycerol production remains uncharacterized. An additional Hog1-independent mechanism retains intracellular glycerol for adaptation. Candida albicans also adapts to hyperosmolarity via a HOG signaling network. However, it remains unknown whether Hog1 exerts integral or proportional control over glycerol production in C. albicans.S. cerevisiae and C. albicans. We propose a simple ordinary differential equation (ODE) model that highlights the integral control that Hog1 exerts over glycerol biosynthesis in these species. If integral control arises from a separation of time scales (i.e. rapid HOG activation of glycerol production capacity which decays slowly under hyperosmotic conditions), then the model predicts that glycerol production rates elevate upon adaptation to a first stress and this makes the cell adapts faster to a second hyperosmotic stress. It appears as if the cell is able to remember the stress history that is longer than the timescale of signal transduction. This is termed the long-term stress memory. Our experimental data verify this. Like S. cerevisiae, C. albicans mimimizes glycerol efflux during adaptation to hyperosmolarity. Also, transient activation of intermediate kinases in the HOG pathway results in a short-term memory in the signaling pathway. This determines the amplitude of Hog1 phosphorylation under a periodic sequence of stress and non-stressed intervals. Our model suggests that the long-term memory also affects the way a cell responds to periodic stress conditions. Hence, during osmohomeostasis, short-term memory is dependent upon long-term memory. This is relevant in the context of fungal responses to dynamic and changing environments.We combined modeling and experimental approaches to study osmotic stress responses in Our experiments and modeling have provided an example of identifying integral control that arises from time-scale separation in different processes, which is an important functional module in various contexts. Adaptation to dynamically changing environments is required for any life form to survive. Unicellular microorganisms often adapt to environmental changes by modulating their metabolism and reprogramming their gene expression patterns. Generally, microbes are constantly monitoring their environment via specific receptors, and then adapting their cellular physiology accordingly via changes in gene expression and metabolism that are driven by specific signal transduction networks.Saccharomyces cerevisiae is able to adapt to acute changes in extracellular osmolarity. The hyperosmotic signal is amplified and transmitted via the High Osmolarity Glycerol (HOG) signaling network, and this leads to the intracellular accumulation of the osmolyte, glycerol. Increasing intracellular glycerol concentrations decrease the intracellular water potential, restore water influx and consequently restore cell volume and turgor pressure.The yeast S. cerevisiae, the HOG network has two main upstream branches, namely the Sln1 branch [In 1 branch  and Sho11 branch ,4 dτ as the feedback mechanism.The integral controller employs the time integral of the error signal Interestingly, integral control mechanisms have arisen frequently in evolution, presumably because they provide robust means by which systems can adapt effectively to constant environmental stimuli despite stochasticity. Indeed, systems biology studies have revealed a variety of biological processes that employ integral control mechanisms including bacterial chemotaxis , calciumS. cerevisiae following hyperosmotic shock have revealed that nuclear Hog1 levels return to basal levels once the cell has adapted to hyperosmotic conditions, and therefore that the HOG system displays perfect adaptation [S. cerevisiae and C. albicans.Elegant single cell analyses of nuclear Hog1 enrichment in aptation . These aaptation . This veUsing modeling and experimentation, we demonstrated that such integral control arise through a separation of the time scales between rapid signaling events and the relatively slower downstream adaptive processes. In addition, a hyperosmotic shock triggers long-term changes in the physiology of yeast cells that prepares them for subsequent hyperosmotic shocks. It appears as if a yeast cell can “remember” a previous hyperosmotic shock over a period that is longer than the time scale of signal transduction, and therefore adapts quickly to subsequent hyperosmotic shocks. We term this phenomenon a long-term stress memory. Our model suggests the HOG system also has a short-term memory that is engendered in the activation and inactivation of the intermediate kinases during the signal relay. The short-term memory might be affected by the regulation of glycerol channels. Our work highlights the need to study the properties of a signal transduction network in an appropriate biological context.m values), using a total quasi-steady-state approximation to describe the phosphorylation and dephosphorylation processes, it can be demonstrated that the activity of this signaling molecule is mainly determined by the activities of its kinase and phosphatase . An alternative mechanism of saturated enzyme kinetics can give rise to perfect adaptation is discussed in Figure Several possible mechanisms enable integral control Figure . Our aimphatase . Hog1 also increases the activity of a key enzyme (Pfk26) that diverts metabolic flux towards glycerol production [+ symport system that assimilates glycerol from the growth medium [S. cerevisiae the rate of increase in glycerol biosynthetic enzyme levels is proportional to the time integral of Hog1 phosphorylation [S. cerevisiae through a separation of time scales between signaling and glycerol production rate changes was not fully recognized. The connection between Hog1 adaptation and integral control was not reported till later [An alternative mechanism by which integral control can arise is via differential time scales in biological processes , Hog1-dependent intracellular glycerol accumulation (v5 and v7) and an osmolarity-regulated AquaGlyceroPorin activity (v6)  . Thus, the increase in the activity of Glycerol Production Machinery (GPM) will be proportional to the time integral of phosphorylation levels of Hog1. We also note that in the long term in S. cerevisiae, active Hog1 is reported to phosphorylate the aquaglyceroporin to target it for subsequent degradation [S. cerevisiae, and it is not known whether the aquaglyceroporin in C. albicans is regulated in the same way. Hence, the degradation of aquaglyceroporin, despite its potential interest, is not included in our model.In our model we also assume that glycerol productivity does not decrease under hyperosmotic conditions dt due to the integral control. Hence, the time integral of the nuclear enrichment of Hog1, which is highly correlated with the phosphorylation of Hog1, has a linear relationship with ∏ NaCl.The increase in intracellular osmolarity caused by the rise in the intracellular glycerol concentration S. cerevisiae closes the aquaglyceroporin channel, Fps1, to prevent glycerol leakage and help to maintain a high intracellular glycerol concentration [C. albicans appears to have a high restriction over glycerol efflux by an aquaglyceroporin. Our experimental data show that, in the presence of 1 M NaCl, the increase in total glycerol (i.e. the sum of intra- and extra-cellular glycerol) is mainly due to the induction of intracellular glycerol, and extracellular glycerol  does not change significantly . The NaCl concentration at which Hog1 phosphorylation amplitude becomes saturated was used to constrain parameters of the HOG signaling pathway . Secondly, in S. cerevisiaeGPD1 expression was strongly induced at the protein level and this regulation was mediated by Hog1 [C. albicansGPD1 is expressed at high levels even in the absence of hyperosmotic stress, and Gpd1 is not highly induced under hyperosmotic conditions [GPD2 was found to be induced at the mRNA level [C. albicans.Our experimental data revealed two significant differences between the responses of M Figure A, wherea by Hog1 . Howevernditions . HoweverNA level . This inAs mentioned previously, the existence of a long term memory is the key to distinguishing the mode of control that Hog1 exerts upon glycerol production  and to explain its underpinning mechanism . The existence of a long-term cellular memory would imply a mechanism of Hog1 integral control that arises through the separation of time scales between Hog1 signaling events and glycerol production rate changes.C. albicans cells were exposed to an initial hyperosmotic shock (1 M NaCl) until they fully adapted. These cells were then removed from the hyperosmotic condition for about 10 min, and were then exposed to an identical, second hyperosmotic shock. We found that the duration of Hog1 phosphorylation in the second response was much shorter than that for the first stress  typically lasts several minutes [The HOG signaling networks in  minutes ,38.To better understand this phenomenon, we developed a theoretical framework for the basic HOG signaling module, which is represented by a protein that is phosphorylated and dephosphorylated by its kinase and phosphatase, respectively. Employing Michaelis-Menten kinetics, we show that this module is essentially a low-pass filter . In other words, given a constant phosphatase level, the phosphorylation level of the protein faithfully follows a low-frequency activation signal while integrating a high-frequency signal, as reported previously ,37,38. WThese studies were carried out using theoretical frameworks that only modeled the signal transduction pathway itself. We then extended our analyses to include downstream adaptive processes by simulating how the entire system responds to a fluctuating signal. As previously mentioned, the HOG signaling network faithfully monitors low frequency signals. As expected, our simulation results suggest that Hog1 phosphorylation levels follow the sinusoidal signal when its frequency is 0.1 rad/min Figure A. HoweveMeanwhile, the HOG signaling network integrates high frequency signals. It is expected that a cell will respond to a high frequency sinusoidal signal in a similar manner as to a constant hyperosmotic stress with the time-average amplitude of the sine signal. In addition, due to the integral control, Hog1 phosphorylation levels might be expected to return to basal levels once the cell has adapted. However, our simulations predict that Hog1 phosphorylation does not return to the initial condition. Instead the model predicts that, due to the quick release of glycerol in the “off” phase of the signal, the cell does not accumulate intracellular glycerol to the same level as it does under a constant signal Figure B, and thWe also investigated whether the long-term memory, a property of the adaptive process itself, affects the short-term memory of the signaling pathway. We performed simulations of a cell pre-stressed in 1 M NaCl for 60 min and observed how it then reacted to a 1 rad/min sinusoidal osmotic signal of a peak-to-peak amplitude of 1 M NaCl centred at 0.5 M NaCl Figure . The modS. cerevisiae[Recently, Gennemark and coworkers reported a simple mathematical model of hyperosmotic responses in erevisiae. Their mS. cerevisiae[C. albicans by constructing a low granularity mathematical model assuming Hog1 integral control. This model integrates the HOG signal transduction pathway with downstream adaptive processes, and encompasses both hyper- and hypo-osmotic conditions. In this model, a signaling or biochemical pathway was concisely represented by an overall reaction  than the ensuing regulatory networks which encompass transcriptional and translational controls (i.e. tens of minutes). It is also common for signals to be converted, via modulation of transcriptional regulators, into the expression of a stable product (i.e. a stable mRNA or protein). Such a separation of time scales could result in an integral control mechanism that would generate an experimentally testable long-term memory. Therefore our experimental approach could be further applied to other signaling networks to examine such a property. In addition, the identification of such a phenomenon allows mathematical simplifications of the regulatory network under study, an important step towards a thorough understanding of signaling networks which may be obscured in exhaustive descriptive models.S. cerevisiae, Hog1 phosphorylation levels become saturated in response to relatively low hyperosmotic signals. In addition, derivative control offers gradual changes in a system, which might be too slow to generate the necessary adaptation to external osmotic challenges. Indeed, derivative control is often found to provide a buffering capacity in cellular systems. For instance, in energy metabolism, phosphorylated molecules, such as phosphocreatine, act as a buffer during high energy demand periods. Phosphocreatine implements a biological derivative control over ATP concentration [The teleological question as to why the yeast cell employs an integral control mechanism remains to be answered. Among the various types of feedback mechanism, proportional control and derivative control mechanisms would allow a system to adapt to a new status that is different from the original condition. However, integral control ensures perfect adaptation of the system, whereby signaling returns to basal levels once adaptation is achieved. This would reduce the potential for cross-talk between signaling pathways. The ability of the cell to restore turgor pressure in the face of an osmotic challenge is crucial for the restoration of growth. Should Hog1 signaling regulate glycerol production via proportional control, a change in osmotic pressure would be reflected by a proportional change in Hog1 activity. In other words, the ability to sense diverse amplitudes of osmotic signal would require high concentrations of HOG signaling proteins, the production of which would demand considerable cellular resources. In contrast, the integral control mechanism converts the error signal into the temporal integral of HOG activity. This avoids the need to express HOG pathway components at high levels. In fact in ntration .Our modeling further suggests that the frequency response of the HOG signaling pathway can be affected by the regulation over the glycerol channels. This prediction is significant because this frequency response is generally thought to be a property of the short-term memory mediated by the regulation of intermediate protein kinases alone. In addition, our simulations show that the long-term memory may also affect the way a cell responds to a high frequency signal. Our simulations suggest that a cell that has previously adapted to hyperosmolarity retains relatively high intracellular glycerol levels for a period. Consequently, the high intracellular osmolarity counterbalances the hyperosmotic signal that the cell perceives. These results highlight the importance of a systems approach to study the signaling networks.C. albicans suggest that, in this pathogen, Hog1 achieves perfect adaptation to a constant hyperosmotic shock through an integral control mechanism that is dependent on separation in time scales between signalling and downstream adaptation. We also predict that Hog1 signalling exhibits a short-term memory that can be revealed by tracking responses to an alternating signal. Furthermore, we predict that this short-term memory depends on the long-term memory. For example, Hog1 phosphorylation levels are predicted to adapt relatively quickly to high frequency signals in cells that have pre-adapted to a constant hyperosmotic stress.Our measurements of Hog1 phosphorylation dynamics in C. albicans strain CA1655 (ura3::λimm434/ura3::λimm434RPS1/rps1::pACT1-FLAG-GFP) was used for the Hog1-phosphorylation assays. This strain is CAI-4 [RPS1 locus. C. albicans CA1655 expresses a FLAG-tagged version of GFP from the ACT1 promoter and functions as an internal loading control for Western blots. C. albicans NGY152 [is CAI-4  containiis CAI-4  integrats NGY152 , which is NGY152 , was use600 of 0.2 in fresh medium and then grown to mid-log phase (OD600 0.8) before any stress was applied [600 of 0.2 in fresh YPD-T with NaCl added to achieve concentrations of 0, 0.3, 0.5, 0.75 or 1 M. Cells were harvested by centrifugation at selected time points and the pellets flash frozen in liquid nitrogen.Cells were grown at 30°C with shaking (200 rpm). To ensure consistency in the starting physiology of the cultures used in our experiments, cells were pre-cultured twice overnight in YPD-T, diluted to an OD applied . A singlFor the experiments involving the imposition of sequential stresses, cells were initially cultured and stressed as described above. At the end of the first stress period, cells were harvested, resuspended in fresh YPD-T with no NaCl and incubated at 30°C for 10 min. NaCl was then added to the cultures and samples were collected after the second stress period. Three biological replicates were prepared for each condition.3VO4 and 50 mM NaF) and sheared with acid-washed glass beads  using a Fast-Prep FP120 machine (Thermo Scientific) . Protein extracts were clarified by centrifugation and protein concentration determined using a Bradford assay [To make protein extracts, flash-frozen cell pellets were resuspended in freshly prepared protein lysis buffer  containing inhibitors . 15 μg protein was loaded in each lane. Proteins were transferred to PVDF membranes, rinsed in phosphate-buffered saline (PBS) and then blocked in PBS-T containing 10 % BSA  BSA) for 30 min at room temperature. Blots were incubated overnight at 4°C in PBS-T containing 5%BSA containing a 1:2000 dilution of anti-phospho-p38 MAPK (Thr180/Tyr182) antibody  and a 1:20000 dilution of anti-FLAG antibody . Blots were washed in PBS-T and then incubated for 1 h at room temperature with Anti-rabbit IgG, HRP-linked antibody . The blots were washed in PBS-T and the signal was detected using LumiGLO™  or SuperSignal®WestFemto (Thermo Scientific) according to manufacturer’s instructions. Phosphorylated Hog1 and GFP-FLAG levels were visualized and quantified using the FluorChem®FC2 (Alpha Innotech) or the FUSION SL™ (Peqlab) systems and the levels of phosphorylated Hog1 were expressed relative to the internal standard (GFP-FLAG).Extracellular and total glycerol concentrations were determined in stressed and unstressed cells at various time points. Cultures were grown and the stress applied as described above. The Free Glycerol Determination Kit (Sigma-Aldrich) was used to determine glycerol concentrations according to the manufacturer’s instructions. To measure extracellular glycerol concentrations, 1.5 ml samples of culture were collected and the cells harvested by centrifugation. Supernatants were transferred to a fresh tube and heated at 100°C for 10 min before their glycerol content was determined. Total glycerol concentrations were measured using 1.5 ml of culture after heating at 100°C for 10 min without prior removal of cells. Cell debris was spun down and the supernatant assayed for glycerol content. Intracellular glycerol concentrations were calculated by deducting the extracellular glycerol concentration from the total glycerol concentration.Supporting data on the following topics are collated in an additional Supplemental data file with the manuscript:Candida albicansBioinformatic analysis of the conservation of proteins in HOG signaling network in Mathematical modelling· Steady-state response of HOG signaling network· Frequency response of HOG signaling network· Model assumptions· The model· Model parameterisation· Frequency response of the systemThe authors declare that they have no competing interests.Model development and analysis: TY, PI. Development of the repeated hyperosmotic shock experiment: APSM, MT, TY, MCR. Experimental data collection: MDJ, EC, MDL, AM. Data analysis: TY. Bioinformatic analysis of signaling component conservation: TT. Frequency response: TY, MCR, APSM, MT. Drafted the paper: TY. Also participated in paper writing: PI, MDJ, APSM, MCR, MDL, NARG, KH, AJPB. Supervision of modeling: CG, JS. Supervision of experiments: AJPB, KH, NARG. Conceived the research: JS, CG, MS, KH, AJPB. All authors approved the final manuscript.A Systems Biology analysis of long and short-term memories of osmotic stress responses in fungi.Click here for file


Recent studies predict that the Arctic Ocean will have ice-free summers within the next 30 years. This poses a significant challenge for the marine organisms associated with the Arctic sea ice, such as marine mammals and, not least, the ice-associated crustaceans generally considered to spend their entire life on the underside of the Arctic sea ice. Based upon unique samples collected within the Arctic Ocean during the polar night, we provide a new conceptual understanding of an intimate connection between these under-ice crustaceans and the deep Arctic Ocean currents. We suggest that downwards vertical migrations, followed by polewards transport in deep ocean currents, are an adaptive trait of ice fauna that both increases survival during ice-free periods of the year and enables re-colonization of sea ice when they ascend within the Arctic Ocean. From an evolutionary perspective, this may have been an adaptation allowing success in a seasonally ice-covered Arctic. Our findings may ultimately change the perception of ice fauna as a biota imminently threatened by the predicted disappearance of perennial sea ice. Water depth at location was 2200 m. Apherusa glacialis were present in all net hauls taken, but owing to challenging ice and wind conditions, only a total of four hauls were possible. The depth interval 0–200 m did not contain any individuals in any of the hauls. Haul 1 contained A. glacialis in depth intervals of 2000–600 m and 600–200 m , haul 2 contained A. glacialis in depth intervals of 1200–600 m, 600–400 m and 400–200 m , haul 3 contained A. glacialis in depth intervals of 1200–900 m and 900–600 m . One additional net haul with a WP3 net contained A. glacialis . The total lipid of individual specimens was extracted in chloroform: methanol following the method of Folch et al. [All samples were collected using a multi plankton sampler , similar to those previously published from the Arctic sea ice [A polar night campaign in the Arctic Ocean in early January 2012 provided a special opportunity to collect samples from a location and time of the year otherwise very poorly known –20. Sampd 2000 m . Abundan sea ice ,12. Ovig sea ice , but mor sea ice  winter rA. glacialis with the deep Atlantic current flowing in the opposite direction from the transpolar ice drift within the Eurasian part of the Arctic. The model assumes a so-far undocumented ascent within the Arctic Ocean, but the well-documented recruitment of amphipods into newly frozen sea ice [A. glacialis and potentially other ice fauna that migrate to the depth will be transported by these return currents against the drift of the pack ice from areas of their release from the melting ice back to the Arctic Ocean. This regional-scale, annual migration strategy is analogous to the diel/tidal migrations performed by other organisms to retain their positions in estuaries [Calanus hyperboreus, enabling it to retain its distribution within the central Arctic Ocean [A. glacialis collected from the ice [Calanus spp. [Apherusa spp., have long been known to perform even extensive diel vertical migrations [Apherusa glacialis, similar to many herbivorous zooplankton of high latitude oceans [Based upon the oceanography of the Arctic and our unique, although limited, mid-winter observations, we propose a conceptual model  that lin sea ice  provides sea ice ) are cha sea ice ,21,22, bstuaries . Also, i the ice  and for grations . We there oceans . It shouApherusa glacialis has previously been recorded from deep plankton nets during the polar night [ar night  within tar night ,26,27. Iar night . Rather,ar night ,16, and A. glacialis’ life history and annual routine, developed as an adaptation to a seasonally ice-covered Arctic Ocean. In combination with the timing of reproduction, this behaviour both prevents export into the North Atlantic/Pacific [We argue that a pelagic phase may be an important part of /Pacific  and enhaA. glacialis are less threatened by the predicted disappearance of Arctic summer sea ice than previously assumed. However, at the same time, it highlights the need for a more comprehensive understanding of processes governing the Arctic Ocean in general, and during the polar night, including energetic costs related to vertical migration during winter in particular.From a food availability perspective, the polar night offers the best time for the organisms to detach from the sea-ice habitat. Mid-winter sea-ice holds a very limited amount of algal food; hence, the pelagic stage does not cause lost feeding opportunities. Furthermore, the very low light levels during winter cause low exposure to visual predators. The predation risk would therefore be low if the pelagic stage takes place during the polar night. Return-current speeds of 9 km per day  suggest


Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO2 levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO2 signaling in mammals, diptera and nematodes.Elevated CO Under physiological conditions, the alveolar epithelium provides optimal gas exchange by minimizing fluid in the alveolar space through active vectorial Na2 in mammalian neurons have been described decades ago 2 concentrations 1 is phylogenetically highly conserved with orthologs in Drosophila and C. elegans1 at the Ser-129 residue by protein kinase C (PKC) has been shown to be required for maximal JNK induction While chemoreception of CO2 changes. For example, CO2 avoidance, which is mediated by specific neurons, has been demonstrated in both C. elegans and Drosophila2 levels also exhibit specific, non-neural effects in both C. elegans and Drosophila, which appear to be independent of any previously indentified stress adaptation pathways 2 levels lead to rapid endocytosis of the Na,K-ATPase in Drosophila S2 cells by a yet unidentified mechanism 2 levels might be mediated by JNK in mammals, Drosophila and C. elegans indicating that JNK may play a central, evolutionary conserved role in CO2 signaling and adaptation to hypercapnia.Notably, not only mammalian cells sense and adapt to CO2 activates JNK in the alveolar epithelium, we assessed JNK phosphorylation at residues Thr-183/Tyr-185, which reflects the activation status of JNK 2 levels (60–120 mmHg at an extracellular pH (pHe) of 7.4) led to a concentration- and time-dependent phosphorylation of JNK (2 levels (40 mmHg at a pHe 7.2), JNK phosphorylation was not observed (To determine whether elevated COn of JNK . Importaobserved .1-GFP), while hypercapnia-induced Na,K-ATPase endocytosis was preserved in ATII cells infected with a null (Ad-null) virus  virus . Similarl) virus  or siRNAl) virus , Na,K-ATSP600125  without SP600125 , suggest2 sensing 2 levels for 10 min, was prevented when ATII cells were infected with an adenovirus expressing a dominant-negative variant of AMPK-α1  is an important element of COrylation ; thus, A2-induced JNK activation  prevented the hypercapnia-induced phosphorylation of the JNK downstream target c-Jun (1-HA (as opposed to wild-type JNK) prevented endocytosis of the Na,K-ATPase during hypercapnia  completely prevented the CO2-induced Na,K-ATPase endocytosis  or kgb-2(gk361) null mutations , another member of the MAPK superfamily, has been also shown to play an important role in CO1-subunit at the Ser-11 or Ser-18 (depending on the species) thereby promoting its endocytosis 1-subunit prevents the CO2-induced endocytosis of the Na,K-ATPase upon hypercapnia 2-induced signaling pattern. These possibilities are currently under investigation in our laboratory.Once activated, PKC-ζ phosphorylates the Na,K-ATPase αIt is well established that JNK activation requires its phosphorylation at the TPY motif by MAPKK 2 and that some of those responses are highly similar Drosophila S2 cells reduce their surface Na,K-ATPase levels in elevated CO2 conditions Drosophila cells, strongly supporting the hypothesis that at least some responses to CO2 are evolutionary conserved. Furthermore, downregulation of the C. elegans JNK homologs jnk-1 or kgb-2 significantly rescued the reduction in fertility but not the pharyngeal pumping rate defects caused by elevated CO2 levels, suggesting the existence of multiple pathways that can mediate CO2 responses and highlighting the specificity of JNK action.Recently, it has become increasingly clear that cells and organisms respond to CO2 responses in mammals, Drosophila melanogaster and C. elegans. Further, we identify mammalian PKC-ζ as a novel upstream kinase responsible for JNK phosphorylation at Ser-129, leading to downregulation of the Na,K-ATPase and thus alveolar epithelial dysfunction.In summary, we provide evidence that JNK activation is an evolutionary conserved mediator of CO2 media, cell surface biotinylation and isolated-perfused lung preparation have been described in detail previously The COAnimals were handled according to National Institutes of Health guidelines and the experimental protocol for the use of rats (2010–2177) was approved by the Animal Care and Use Committee at Northwestern University.Day 2 ATII cells, plated on 60-mm cell culture dishes were incubated with null adenovirus  or with adenovirus expressing a dominant-negative, JNK1 tagged with GFP (Ad-DN JNK1-GFP) as previously described 1-subunit  1 (WT-JNK1-HA) and a mutant JNK1 variant in which Ser-129 was replaced by an alanine (S129A-JNK1-HA) have been described previously The expression vectors encoding an HA-tagged wild-type JNK5Drosophila S2 cells  were incubated with 7.5 µg double-stranded RNA for 30 min in serum-free medium in 6-well plates, and thereafter grown in 10% FBS-containing medium for 5 days. CO2 treatments were performed at 15% CO2 using pre-equilibrated media buffered to pH equal to air condition, with S2 cells attached to 6-well plates. Cell surface biotinylation was performed as previously described after 1 h in 15% CO2For RNA knockdown, 5×10jnk-1(gk7) and kgb-2(gk361) null mutations were allowed to lay eggs for 6 hours at 20°C in either 19% CO2 or in control air condition. After 6 hours adult worms were removed, progeny was scored as described previously 2.Gravid worms of wild type (N2), p value<0.05 was considered significant.Data are expressed as mean ± SEM. Data were compared using analysis of variance adjusted for multiple comparisons with the Dunnet test. When comparisons were performed between two groups of values, significance was evaluated by Student’s test. A Figure S12 with a pHe of 7.4 or to 40 mmHg CO2 with a pHe of 7.2 for 10 min and the phosphorylation of JNK at Thr-183/Tyr-185 (p-JNK) and the total amount of JNK (JNK) was measured by Western blot analysis.ATII cells were exposed to 40 mmHg CO Top: Graph represents the p-JNK/JNK ratio. Values are expressed as mean ± SEM, n = 3. Bottom: Representative Western blots of p-JNK and total JNK. pHe: extracellular pH.(TIF)Click here for additional data file.Figure S21 (siRNA - JNK1) or scrambled siRNA (scr siRNA) as described in the Supplemental methods. Twenty four hours after transfection cells were exposed to 40 or 120 mmHg CO2 (pHe 7.4) for 10 min. Representative Western blots of p-c-Jun, JNK1 and JNK2 as well as actin (loading control) from A549 whole cell lysates (WCL) are shown. (B) Twenty four hours after transfection A549 were exposed to 40 (open bars) or 120 (closed bars) mmHg CO2 (pHe 7.4) for 30 min. Na,K-ATPase at the plasma membrane was determined by biotin-streptavidin pull down and subsequent Western blot analysis. Bars represent the mean ± SEM, n = 3, **, p<0.01. Representative Western blots of Na,K-ATPase α1-subunit and E-cadherin (E-cad) at the plasma membrane (PM) are shown. n.s.: non-specific band.(A) A549 cells were transfected with siRNA against JNK(TIF)Click here for additional data file.Figure S3ATII cells were treated with 2 mM AICAR or its vehicle for 30 or 60 min and phosphorylation of AMPK, acetyl-CoA carboxylase (ACC) and JNK  and the amount of total AMPK and JNK were determined by Western blot. Representative Western blots are shown.(TIF)Click here for additional data file.Figure S42 (pHe 7.4) for 10 min in the presence or absence of bisindolylmaleimide I . p-JNK and total JNK were determined by Western blot. Graph represents the p-JNK/JNK ratio, values are expressed as mean ± SEM, n = 3. **, p<0.01. Representative Western blots of p-JNK and total JNK are shown. (B) ATII cells were exposed to 40 (open bars) or 120 (closed bars) mmHg CO2 (pHe 7.4) for 10 min in the presence or absence of Gö6976  or PMA . p-JNK and total JNK were determined by Western blot. Graph represents the p-JNK/JNK ratio, values are expressed as mean ± SEM, n = 3. **, p<0.01. Representative Western blots of p-JNK and total JNK are shown.(A) ATII cells were exposed to 40 (open bars) or 120 (closed bars) mmHg CO(TIF)Click here for additional data file.Methods S1Supporting Methods.(PDF)Click here for additional data file.


Allergy is an increasing autoimmune disease in the developed world. Current consensus is that allergy is the result of a misdirected immune response directed towards ‘seemingly innocuous antigens’. Many of the allergens have similarities with parasite proteins and it is generally accepted that the IgE (Immunoglobulin E) response is a normal defense mechanism against parasitic infestations. The conventional treatment of allergy is through pharmacotherapy where corticosteroids are used to reduce the expression of inflammatory proteins; although effective, they do not combat the underlying cause of allergy. Novel immunotherapeutic treatments have been developed that immunize sufferers with anti-IgE antibodies, but this passive immunization strategy is only temporary, and patients need to return for follow-up injections.2-3, derived from human IgE, was injected into rats, and followed up by a boost with a chimeric human-dog-human IgE, in an attempt to direct the immune system and develop polyclonal antibodies that target a certain epitope in the IgE antibody which would lead to the removal of serum and FcεRI receptor bound IgE.This project reports the development of an active anti-allergy immunotherapy. The synthetic peptide 2Fcε2-3 peptide, this serum was also found to target native human IgE, as well as the dog and the horse native IgE antibody. Further analysis showed that the serum antibodies cross linked receptor bound IgE and resulted in cell degranulation and mediator release.The experiment resulted in a rat serum with strong antibody titer targeting the 2FcεThe immunization strategy was successful but did not fully work as predicted. The study does, however, lay significant foundations for future potential anti-allergy vaccine designs.


In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.− ILT7− CD1c+; pDC: CD19− ILT7+ CD1c−). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.Following informed consent of subjects , peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC. Severe viral respiratory infections early in life are associated with increased risk of asthma and atopic disease One hallmark of our model is that murine cDC and pDC do not express FcεRI except during severe viral infections, when cognate interactions with a specific neutrophil subset drives FcεRI expression Our study documents expression patterns of FcεRI on peripheral blood cDC and pDC in young children. While the receptors are present early in life, we found a marked differential effect of low and high IgE on cDC FcεRI expression. This finding implies that cDC expressed FcεRI has disparate effects on cDC function depending on serum level of IgE. Further, our data suggest that IgE control of FcεRI may be different in children compared to adults.A cross sectional time analysis was employed. Our primary goal was to determine the age at which human cDC and pDC expressed FcεRI. Our secondary goal was to determine if sensitization to any food or inhalant allergen was associated with FcεRI and IgE expression.This study was approved by the Children's Hospital of Wisconsin IRB. Parental written consent and subject verbal assent was obtained before enrollment of subjects into this study.Once consent and assent was obtained, an abbreviated atopic history was collected and venipuncture performed. Eligible patients were children aged 1–15 years. Exclusion criteria were: atopic dermatitis, persistent controlled asthma, uncontrolled asthma, immunodeficiency, or any co-morbid disease; use of any corticosteroids within the last month; current therapy with any prescribed medication; use of any investigational agent in the last 30 days; women of childbearing age not on contraception or women breastfeeding.D. farinae, D. pteronyssinus, cockroach, cat dander, dog dander, mouse epithelium, rat epithelium, guinea pig epithelium, mouse serum protein, rat serum protein, mouse urine protein, rat urine protein, June grass, Alternaria tenuis, Cladosporium herbarum, Aspergillis fumigatus, common ragweed, oak, birch, L. destructor) were determined by ImmunoCAP (Quest Dignostics/IBT Laboratories). Sensitization was defined as elevated serum specific IgE (>0.35 kU/L) to one or more allergens tested.Total IgE and specific IgE levels to foods  and aeroallergens  was performed with 5×105 to 1×106 total cellular events collected on each stained specimen. cDC  were identified as CD19−/ILT7−/CD1c+ and pDC identified as CD19−/ILT7+/CD1c−. This type of phenotyping strategy has been used before, as ILT7 is a specific marker for pDC and CD19−CD1c+ cells have been shown to be essentially all cDC Immunophenotyping for evaluation of pDC and cDC was performed within 6 hours of specimen collection. 100 µl of heparinized whole blood was stained with antibodies to human CD19 , ILT7 , and CD1c , as well as either antibodies against FcεRIα  or IgE  for 15 min. at room temperature. Erythrocytes were lysed with 2 ml FACSLyse (BD Biosciences) and stained cells were washed with PBS/0.1% NaNhttp://salford-systems.com/). Regression trees are a non-parametric approach where the data are split recursively into two groups based on an optimizing function, until the specified limits for subgroups are reached. A least absolute deviance from the median was used as the optimizing function, with a limit of 10 in any group to be split and 5 minimally needed for any group. Linear regression was used to fit the data with linear or a polynomial trend with normality of the error term checked with a normal probability plot. cDC IgE was fit to serum IgE using a non-linear regression for a sigmoidal type curve of the form 1/(1+M/serum IgE)m, where M is the (fitted) median value of cDC IgE and m is the slope of the curve at that value. We fit linear regression using SAS 9.2 and non-linear regression using JMP 9.0. Given the relatively small sample, we did not use a bootstrap approach or a holdout sample to validate the model results. Therefore, these results will need to be validated on another sample.Data were expressed as frequency and percent for categorical variables and median and interquartile range for continuous variables. Since the data were skewed, continuous variables were compared using a Mann-Whitney test while doing 2 group comparisons, and continuous variables were examined using scatter plots and Pearson correlations. To investigate the inter-related effects of variables on the outcomes of interest, a regression tree analysis was done using Salford Systems' CART software , with about 25% of the variation of IgE explained by the linear relationship with age. However, much of this relationship occurred in the non-sensitized group (r = 0.56) compared to the sensitized group (r = 0.29). This is not surprising, since serum IgE levels can fluctuate, such as with viral infections, and our data were obtained at only a single time point per subject Comparing the relative frequencies of conventional (cDC) and plasmacytoid dendritic cells (pDC) amongst all of our subjects, we found 1.26 (0.66–2.55) (median (IQR)) fold more cDC than pDC. This ratio was not significantly different between sensitized and non-sensitized subjects, nor did it correlate with age (data not shown). While this ratio of cDC to pDC is lower than what has been reported in older children, it is similar to what has been reported in adult studies using a dendritic cell phenotyping strategy similar to ours We initially hypothesized that similar to the rodent system, peripheral blood cDC would not express FcεRI early in life. However, as shown in In human mast cells and basophils, IgE modulates FcεRI expression Sensitization status did not differentiate level of expression of FcεRI on cDC . HoweverSince bound IgE modulates expression of FcεRI on human mast cells and basophils, we examined the level of IgE on peripheral blood cDC and correlated this with FcεRI expression. As shown in By overlaying serum IgE levels, we found only subjects with a serum IgE above 34–42 kU/L had a cDC fold IgE expression of 12 or greater . These dUnlike peripheral blood cDC, pDC expression of FcεRI was minimal (median (IQR) = 15.82 (12.74–21.05) versus 2.47 (1.47–3.25) on cDC and pDC, respectively). In fact, 6 of the 27 subjects appeared to have no expression of FcεRI on their pDC . As shown in Unlike with cDC , serum IOther studies have attempted to describe relationships between serum IgE, cell-bound IgE, and FcεRI expression on dendritic cells Interestingly, we found no association between sensitization status and FcεRI expression. We cannot equate sensitization with atopy because we did not challenge our subjects with the respective allergens. Further, it is quite possible that those subjects we identified as non-sensitized were indeed making specific IgE—just against antigens that were not included in our panels. For these reasons our data may be different from two studies in adults where atopic subjects were noted to have significantly higher FcεRI expression on cDC and pDC The most intriguing finding of our study is the major difference in the role of IgE in receptor expression. Whereas a direct relationship between FcεRI expression and IgE on both cDC and pDC have been reported by others, we found that only pDC FcεRI expression correlated in a linear fashion with bound IgE Unlike pDC, expression of FcεRI on cDC correlated directly with serum IgE levels. However, the relationship between bound IgE and FcεRI was more complex. When cell bound IgE was below 12 fold MFI there was little effect of bound IgE on FcεRI expression. Once bound IgE levels crossed the threshold, a direct relationship developed. At low levels of serum IgE (<34–42 kU/L) there is rapid accumulation of bound IgE with little effect on FcεRI expression. At these low levels of IgE, a difference in correlation of bound IgE and FcεRI exists between sensitized and non-sensitized subjects. Both responses are parabolic, however, non-sensitized subjects tend to have an initial increase in FcεRI expression with increasing bound IgE (below 12 fold MFI). Sensitized subjects had the exact opposite effect. Once serum IgE levels reached 34–42 kU/L, corresponding to bound IgE levels of 12 fold MFI, both sensitized and non-sensitized subjects exhibited a direct correlation between IgE and FcεRI.We have found a threshold effect of bound IgE on cDC FcεRI. The functional implications for this threshold effect are unclear; it is inviting to speculate that FcεRI has disparate functions at low versus high IgE levels. Perhaps at lower levels FcεRI acts as a rheostat, similar to what is seen in rodents, where the receptor functions as a mechanism for cellular recruitment and less for antigen-uptake Our study had several limitations. First and foremost, we utilized statistical modeling to evaluate our data, and, as a result, it is important that these findings be validated in a second cohort. Further, our sample size was relatively small and likely limited our ability to detect subtle differences. We did not enroll infants younger than 12 months of age, and we were unable to determine if viral infection relates to induction of FcεRI. Finally, defining sensitization status by selected ImmunoCAP may not be ideal—especially from a clinical perspective. However, given that our results were mostly independent of sensitization status, we believe this is not a major defect. Indeed, most of the limitations of our study would only lessen our ability to define statistically significant differences. Since we were able to document clear relationships between IgE (serum and cell bound) and cDC and pDC expression of FcεRI despite these limitations, their presence only strengthen the importance of our findings.In summary, our results demonstrate the presence of FcεRI on peripheral blood cDC and pDC in children as young as 1 year old, which is in contrast to what is seen in the rodent system. Our data also provide evidence of differential regulation of FcεRI on cDC and pDC in children. With cDC a complex inter-relationship between serum IgE, cell bound IgE, and expression of FcεRI exists that appears to hinge on an IgE level of 34–42 kU/L. The functional significance of this relationship is unclear, and we do not yet understand the mechanisms responsible for this relationship. Future studies need to be undertaken to examine expression of FcεRI on cDC and how IgE modulates this receptor and cDC function.


N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of  Vinca -colchicine. Furthermore, the study being performed on living cells, it provides information on the ability of the drug to cross cell membranes, to diffuse through the cytosol and to react with dynamically functional microtubules that are in contact with several cytoskeleton-associated proteins, information that are unavailable when using only purified tubulins in a in vitro setting.To circumvent these limitations, we have developed an inexpensive and simple detection technique to assess the binding of antimicrotubule agents acting on the colchicine-binding site. This method is based on the property of ing site . Luduenaing site ,12. In ting site , we founIn this study, we assessed the robustness of the method using: 1) drugs binding to the three main binding sites found on the α-,β-tubulin heterodimer, namely the taxol-, the colchicine-, and the vinca-binding sites, 2) drugs unrelated to tubulins and the cytoskeleton such as daunorubicin and verapamil, and (3) four cell lines found in most laboratories. As shown in Table 1, in Additional file  drugs bi drugs un50), a semi-quantitative assessment of the affinity of the drug for the binding site is possible. Moreover, the purity of the sodium dodecyl sulfate (SDS) used to perform the experiments is crucial, since SDS at 65% significantly improved the separation between native β-tubulin and the EBI: β-tubulin adduct.As illustrated in Figure v/v) defined bovine calf serum . Cells were maintained in a moisture-saturated atmosphere at 37°C in 5% of CO2. Biochemicals and the monoclonal anti-β-tubulin antibody (clone TUB 2.1) were purchased from Sigma-Aldrich  and used as received unless specified otherwise. SDS at 98.5% and SDS at 65% of purity were purchased from Sigma-Aldrich. The enhanced chemiluminescence Western blotting detection reagent kit was provided by Amersham Canada . Mixtures are expressed as volume/volume ratios unless indicated otherwise. Daunorubicin was purchased from Rhône-Poulenc  and combretastatin A-4 was synthesized according to Petit et al. [Human breast carcinoma MDA-MB-231, human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7 cells were obtained from the American Type Culture Collection . Cells were grown in high glucose Dulbecco's Modified Eagle Medium supplemented with 5%  for 1.5 h at 37°C without changing the culture medium, which contains the drugs. The control cells were treated with 0.5% dimethylsulfoxide. Afterward, floating and adherent cells were harvested using a rubber policeman and centrifuged for 3 min at 8,000 rpm. The pellets were washed with 500 μL of cold phosphate-buffered saline and stored at -80°C until use.Prior to drug exposition, 6-well plates were seeded with either MDA-MB-231, HT-29, M21, or MCF-7 cells at 7 × 10v/v) Tween-20 with 2.5% fat-free dry milk) for 1 h at room temperature and then with the anti-β-tubulin primary antibody in TBSMT (1:500) for 16 h at 4°C. Membranes were washed with TBST  Tween-20) and incubated with peroxidase-conjugated anti-mouse immunoglobulin in TBSMT  for 2.5 h at room temperature. After washing the membranes with TBST, detection of the immunoblot was carried out with an enhanced chemiluminescence detection reagent kit.The cells pellets were lysed by addition of 100 μL of Laemmli sample buffer 5× . Cell extracts were boiled for 5 min. The protein concentration was determined using the Bradford method. Twenty micrograms of proteins from the protein extracts were subjected to electrophoresis using 0.1% SDS (98.5% or 65% of purity) and 10% polyacrylamide gels. The proteins were transferred onto nitrocellulose membranes that were incubated with TBSMT 6. Transfer of the proteins onto nitrocellulose membrane 7. Incubation of the nitrocellulose membrane with TBSMT for 1 h8. Incubation of the nitrocellulose membrane with the primary antibody anti-β-tubulin for 16 h at 4°C then washing five times with TBST9. Incubation of the nitrocellulose membrane with the peroxidase-conjugated anti-mouse immunoglobulin for 2.5 h at room temperature and then washing five times with TBST10. Detection of β-tubulin bands using an enhanced chemiluminescence detection reagent kitConcentration and duration of the incubation of the tested drug with the cells may change from one class of compound to another. However, all drugs tested so far have produced good signals when using the aforementioned experimental conditions. The purity of the SDS used for the electrophoresis seems critical for optimal separation of the β-tubulin and the EBI: β-tubulin adduct. In our hand, the SDS at 65% of purity supplied by Sigma-Aldrich was optimal Figure .Click here for fileClick here for file


Previous studies in our laboratory were the first to report the presence of IgE anti-varicella zoster virus (VZV) in an adolescent patient with shingles. However, the presence and long term persistence of IgE anti VZV antibodies has not been studied in adults. The presence of serum IgE in addition to IgE and IgG anti-VZV antibody in sera were studied in children (N=12) (0–16 y/o) and adults (N=9) (32–76 y/o) with either a past history of (wild type) chicken pox  or 5 years after vaccination with varicella zoster (N=2 children) , as well as in non-infected subjects (N=3 children). Of the patients who had a positive history of chicken pox 13 of 16 (81%) contained IgE anti-VZV antibodies; they were both serum IgEHi (>100 IU/ml) and IgELo (<100 IU/ml). Of the patients who were vaccinated, IgE anti-VZV antibodies were undetected. In contrast, serum from the patients without a history of chicken pox or vaccination did not make either IgE or IgG anti-VZV antibodies. This is the first demonstration of the existence of IgE anti-VZV antibodies, and its long-term persistence in serum of previously infected subjects. Future studies regarding the functional role of anti-viral IgE and its relationship to VZV are warranted.The production of IgE specific to different viruses , and the ability for IgE anti-HIV-1 to suppress HIV-1 production  B. Burgdorferi) and its persistence one year later in serum of lyme-infected children  , 8, paraThis is the first description of the presence of IgE anti-varicella zoster virus antibodies and its long term persistence in serum of previously infected adults or pediatric subjects. The exact role of IgE in VZV infection remains unknown; however, the presence of IgE anti VZV antibodies in infection 10–40+ years after chicken pox infection, warrants further investigation of the biological significance, if any, of these antibodies.Peripheral blood  was obtained from both pediatric (N=12) (0–16 yrs old) and adult (N=9)  subjects from the SUNY Downstate Allergy Clinic, who were atopic and non atopic. Atopic subjects were skin prick positive (N=4) for environmental  or food allergens. Exclusion criteria included patients with milk allergies. At the time of study, the subjects had not received allergy therapy, and were not being treated with any medication. Subjects did not have a past history of parasite infection. Approval was obtained from the SUNY Downstate Institutional Review Board, and the procedures followed were in accordance with institutional guidelines involving human subjects.Subjects were interviewed about past history of wild type chicken pox infection (WT) or whether they had been vaccinated . All adults had wild type varicella infection prior to the advent of the Varicella virus vaccine (1997). Time post infection for chicken pox infection in children was 10–14 years; in adults 15–40 yrs. Past history of wild type chicken pox infection was confirmed by positive titers for IgG varicella zoster virus by Quest Diagnostics Inc. .For studies of serum immunoglobulins (Ig), blood was collected into red top monoject tubes  and sent to Quest Disagnostics, Inc. for Ig determinations.Total serum immunoglobulin E (IgE) was determined by The Clinical Immunology Laboratory at SUNY Downstate  according to standard procedure . Reference range for healthy adult or child serum: IgE: 20–100 IU/mL.IgG. Serum IgG to varicella zoster virus were determined by enzyme immunoassay (EIA) performed by Quest Diagnostics Inc.  according to standard procedure. Data are reported as ratio report. .IgE. The presence of IgE anti-varicella zoster virus antibodies was determined by immunoblot (dot blot), as previously described .Serum IgE levels and IgE and IgG anti-varicella zoster virus were studied in children N=12) (m/f 0–16 y/o) and adults (N=9) (32–76 y/o) with either a past history of (wild type) chicken pox  or 5 years after vaccination with varicella zoster (N=2 children) , as well as non-infected children (N=3).   had reduced serum IgE levels <100 IU/ml); and eight  had elevated serum IgE (110–346 IU/ml). Vaccinated children, and children with no history of chicken pox had serum IgE levels which were low . In contrast, serum from non-infected (control) subjects did not make IgG anti-VZV antibodies (data not shown).IgE. Serum obtained from subjects who had wild type chicken pox 13 of 16 (81%) had positive dot blots for IgE anti VZV antibodies; they had high and low levels of serum IgE . VZV is a member of the Herpesviridae family, a group of large DNA viruses that replicate in the cytoplasm of virus-infected cells . Chickenet al.  have shoin vitro . Furtherin vitro . Earlierin vitro , 21. Howin vitro . Althougin vitro , 21 may VZV vaccination has been recommended by the Center for Disease Control (CDC) for all health care workers  as well Future studies are necessary to elucidate the role of IgE in VZV infection and to determine whether IgE possesses any functional or immunomodulatory role in this disease, and whether it might have use as a potential biomarker for chicken pox infection.


We evaluated here whether MAPKs are involved in this effect.The endogenous, cholestatic metabolite estradiol 17ß-S-glutathione, respectively. Protein kinase participation in E217G-induced secretory failure was assessed by co-administering selective inhibitors. Internalization of Bsep/Mrp2 was assessed by confocal microscopy and image analysis.ERK1/2, JNK1/2, and p38 MAPK activation was assessed by the increase in their phosphorylation status. Hepatocanalicular function was evaluated in isolated rat hepatocyte couplets (IRHCs) by quantifying the apical secretion of fluorescent Bsep and Mrp2 substrates, and in isolated, perfused rat livers (IPRLs), using taurocholate and 2,4-dinitrophenyl-217G activated all kinds of MAPKs. The PI3K inhibitor wortmannin prevented ERK1/2 activation, whereas the cPKC inhibitor Gö6976 prevented p38 activation, suggesting that ERK1/2 and p38 are downstream of PI3K and cPKC, respectively. The p38 inhibitor SB203580 and the ERK1/2 inhibitor PD98059, but not the JNK1/2 inhibitor SP600125, partially prevented E217G-induced changes in transporter activity and localization in IRHCs. p38 and ERK1/2 co-inhibition resulted in additive protection, suggesting complementary involvement of these MAPKs. In IPRLs, E217G induced endocytosis of canalicular transporters and a rapid and sustained decrease in bile flow and biliary excretion of Bsep/Mrp2 substrates. p38 inhibition prevented this initial decay, and the internalization of Bsep/Mrp2. Contrarily, ERK1/2 inhibition accelerated the recovery of biliary secretion and the canalicular reinsertion of Bsep/Mrp2.E217G-induced cholestasis, through internalization and sustained intracellular retention of canalicular transporters, respectively.cPKC/p38 MAPK and PI3K/ERK1/2 signalling pathways participate complementarily in E Bile formation is a highly regulated process that depends upon the coordinated transport activity of basolateral and canalicular carriers on the hepatocyte. Complex signalling pathways regulate the density and activity of these carriers, and its imbalance impairs secretory function, leading to retention of biliary components both in liver and blood 217G) is an endogenous metabolite of 17ß-estradiol that induces an acute, reversible cholestasis in rats Estradiol 17ß-D-glucuronide  protein kinase C (cPKC) isoforms The mechanisms involved in ESince the biological effects of cPKC and PI3K are often mediated through downstream protein kinases, we speculated that other signalling mediators are involved, provided they can crosstalk with cPKC or PI3K. Likely candidates are the mitogen activated protein kinases (MAPKs). Both cPKC e.g., Ras, Rap) and two protein kinases (MAPKKK and MAPKK), which act as dual-specificity enzymes that activate a selective MAPK type. Non-canonical activation of MAPKs involves MAPK autophosphorylation, or direct MAPK phosphorylation by alternative protein kinases, such as Src or ZAP70 The main vertebrate MAPKs are the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 and 2 (JNK1/2), and p38 kinase (isoforms α and β in hepatocytes). ERK1/2 is preferentially activated by growth factors, while JNK1/2 and p38 are more responsive to stress stimuli 217G-induced cholestasis, 17ß-estradiol, a E217G precursor which evokes in hepatocyte-derived cell lines most signalling pathways activated by E217G  and in the isolated, perfused rat liver (IPRL). Our results demonstrate that both p38 and ERK1/2 contribute to the impairment of localization and function of Bsep and Mrp2 induced by E217G, by acting in a complementary manner, and downstream of cPKC and PI3K, respectively.These findings have prompted us to study the role for MAPKs in E217G, collagenase type A (from Clostridium histolyticum), bovine serum albumin (BSA), trypan blue, L-15 culture medium, dimethyl sulfoxide (DMSO), Triton X-100, ethylene glycol tetraacetic acid, sodium dodecyl sulfate, tetramethylethylenediamine, dithiothreitol, ammonium persulfate, urethane, protease inhibitor cocktail, β nicotinamide adenine dinucleotide hydrate and, 1-chloro-2,4-dinitrobenzene (CDNB) were from Sigma Chemical Co. . 5-Chloromethylfluorescein diacetate (CMFDA) was from Molecular Probes . Gö6976, SB203580, PD980589, SP 600125, and sodium taurocholate were from Calbiochem . Wortmannin (WM) was from Fluka AG . Cell lysis buffer was from Cell Signaling Technology . The chemiluminescence reagent, and Hyperfilm ECL were from Thermo Fisher Scientific, Inc. . All the other reagents were of analytical-grade.Cholyl-glycylamido-fluorescein (CGamF) was a generous gift from Prof. Alan Hofmann . EAll animals received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” Eight Edition . Experimental procedures were carried out according to the local Guideline for the Use of Laboratory Animals (Resolution N° 6109/012) established by the institutional Bioethical Committee for the Management of Laboratory Animals and approved by the Faculty of Biochemical and Pharmaceutical Sciences of the National University of Rosario. A system of local committee-based regulatory control offers an equivalent level of regulatory control to that exercised by the systems in Canada. The guidelines for the use of laboratory animals has been approved by our Faculty in 2002, based on CCAC  guidelines documents. The document has been updated since then according to current and emerging issues for the research community and to advances in laboratory animal care.Adult female Wistar rats weighing 250–300 g and bred in our animal house as described 5 U/mL in L-15 culture medium, and cultured for 5 h.To obtain a preparation enriched in IRHCs, livers were perfused according to the two-step collagenase perfusion procedure, and further enriched by centrifugal elutriation 217G (200 µM) for 20 min. To evaluate the role of MAPKs in E217G-induced hepatocanalicular dysfunction, IRHCs were preincubated with SB203580 (1 µM), a p38α/ß inhibitor, PD980589 (5 µM), an inhibitor of the MAPKK upstream of ERK1/2 (MEK1/2), or SP600125 (1 µM), a JNK1/2 inhibitor, for 15 min., followed by addition of E217G for another 20-min period. Co-inhibition of cPKC and PI3K was carried out by co-administering their specific inhibitors Gö6976 (1 µM) and WM (100 nM), respectively.IRHCs were exposed to the vehicle  or EFunctional changes in Bsep and Mrp2 were evaluated by assessing the canalicular vacuolar accumulation (cVA) of CGamF, a fluorescent Bsep substrate For transport studies, cells were washed twice with L-15 and exposed to 0.3 µM CGamF Colocalization studies of Mrp2 and Bsep with Rab11a were performed as previously described 6 cells/mL. After 5 h of culture, cells were exposed to E217G (200 µM) for 10–60 min, washed with cold phosphate-buffered saline, and finally resuspended in cell lysis buffer and a protease inhibitor cocktail. Aliquots containing an equivalent total protein content, as determined by the Lowry procedure with BSA as the standard Activation of p38, ERK1/2, and JNK1/2 was assessed by evaluating by Western blotting the phosphorylation status of these MAPKs in lysates of primary-cultured rat hepatocytes. Briefly, isolated rat hepatocytes were obtained by collagenase perfusion, as described in situ, as described elsewhere S-glutathione (DNP-SG) and taurocholate excretion, respectively. For this purpose, sodium taurocholate (2 µM) and CDNB (0.5 µM) were added to the perfusion medium.Livers from bile duct-cannulated rats  were perfused 217G , or its solvent in controls [DMSO/10% BSA in saline (4∶96)]. Bile was then collected at 5-min intervals for another 30-min period. Experiments were considered valid only if the initial bile flow was greater than 30 µL/min/kg of body weight. Liver viability was evaluated by monitoring lactate dehydrogenase release into the perfusate outflow After a 20-min equilibration period, the p38 inhibitor SB203580 (250 nM) or the ERK1/2 inhibitor PD98059 (5 µM) or their solvent  were added to the reservoir. Fifteen min later, a 5-min basal bile sample was collected, followed by administration of EDNP-SG content in bile was measured by high-performance liquid chromatography, as described At the end of the perfusion period, a liver lobe was excised, frozen in isopentane precooled in liquid nitrogen, and stored at −80°C for further immunofluorescence and confocal microscopy analysis of Mrp2 and Bsep intracellular localization. F-actin staining was carried out to demarcate the limits of the canaliculi, as described A, right panel), p-ERK1/2 (B) and p-JNK1/2 (C) showed that E217G increased the amount of each p-MAPK in a time-dependent manner, with increments becoming apparent as soon as 10 min after E217G administration. Pretreatment with the cPKC inhibitor Gö6976 (1 µM) or the PI3K inhibitor WM (100 nM) selectively prevented the increase in p-p38 and p-ERK1/2, respectively -p38 . Besides, the pretreatment of IRHCs with these same combinations of inhibitors markedly prevented the E217G-induced internalization of canalicular carriers  217G induced colocalization of canalicular carriers with Rab11a (yellow/orange merge). IRHCs pretreated with either SB203580 or PD98059 showed lack of colocalization, as in controls; this indicates that either or both the carrier internalization towards ARE was prevented or the carrier reinsertion from ARE has been accelerated.In E217G-induced cholestasis, which differentially depends upon the cPKC/p38 and PI3K/ERK1/2 pathways (see above). The nature of these two cholestatic mechanisms cannot however be ascertained by using this approach. Using the IPRL model, which unlike IRHCs allow for the dissection of mechanisms occurring separately in time, we had shown a differential role for cPKC and PI3K in E217G-induced cholestasis: whereas cPKC is involved in the initial reduction in bile flow due to transporter endocytosis, PI3K (via Akt) blocks the otherwise spontaneous reinsertion of the endocytosed transporters The IRHC model revealed the existence of two complementary mechanisms accounting for E217G induced a 61% decrease in bile flow within 10 min, which did not recover throughout the perfusion period .The bolus administration of E217G by SB203580 and PD98059 was accompanied by the recovery of the normal canalicular localization of Bsep and Mrp2 at the end of the perfusion period ; this indicates endocytic internalization of the carriers. Whereas SB203580 and PD98059 itself did not induce any changes in transporter localization (data not shown), both inhibitors extensively prevented the internalization of Bsep and Mrp2, as illustrated by a control-like pattern of the Bsep and Mrp2 distribution profiles in livers pretreated with the inhibitors, as compared with the E217G-treated group . This supports our contention that p38 contributes to E217G-induced cholestasis by retrieving canalicular carriers from their membrane domain in a cPKC-dependent manner. On the other hand, ERK1/2 would hinder the spontaneous retargeting of the endocytosed transporters by acting downstream PI3K.Prevention of the biliary secretory failure induced by En period . In cont217G-induced cholestasis.This study provides further insights into the intracellular signalling pathways involved in E217G activates the main MAPK types, i.e., p38, ERK1/2 and JNK1/2  1) Bsep and Mrp2 colocalize with the ARE marker Rab11a (see 2) Mrp2 and Bsep colocalize on the same vesicles with both the microtubule motor dynein and polymeric immunoglobulin receptor (pIgR), a surface protein that is transcytosed via ARE from the basolateral to the apical membrane 3) insertion of Bsep back to the canalicular membrane from ARE is a microtubule-dependent process 4) ARE is interconnected in a microtubule-dependent manner with apical early endosomes (AEE), the first station on the endocytic pathway involved in apical endocytosis Our results also show that the PI3K/ERK1/2 pathway is involved in the intracellular retention of the endocytosed transporters, which otherwise would rapidly return to the canalicular membrane in a microtubule-dependent manner b11a see , 2) Mrp2217G-induced cholestasis on both p38 and ERK1/2 results somewhat paradoxical with respect to other reports showing that both MAPK types are involved in choleretic phenomena, such as those induced by the bile salt tauroursodeoxycholate (TUDC) 217G may be limited to this early endosomal pathway, whereas the influence on Golgi/post-Golgi pathway would require further localization of specific MAPK scaffold proteins at this site 217G and TUDC may activate different signalling pathways apart from MAPKs that, by operating in concert with MAPKs, result in opposite final effects. For example, TUDC-induced acceleration of the Golgi/post-Golgi exocytic pathway depends upon dual activation of p38 and novel PKC isoforms 217G does not activate the novel PKC isoform ε 217G-induced internalization of canalicular carriers 217G, and whether cAMP reverses this decrease as part of its anticholestatic action.The dependency of EWhile further studies will be required to assess the specific molecular mechanisms mediated by ERK1/2 and p38 to impair the localization status of canalicular transporters in estrogen-induced cholestasis, our results strengthen the idea that there is a clear interplay between signalling cascades and intracellular trafficking in this cholestasis, and that both MAPKs are key players in its ethiology. Drugs that inhibit selectively MAPKs have been used in preclinical and clinical studies with reasonable success


Phosphorylation-triggered degradation is a common strategy for elimination of regulatory proteins in many important cell signaling processes. Interesting examples include cyclin-dependent kinase inhibitors such as p27 in human and Sic1 in yeast, which play crucial roles during the G1/S transition in the cell cycle. In this work, we have modeled and analyzed the dynamics of multisite-phosphorylation-triggered protein degradation systematically. Inspired by experimental observations on the Sic1 protein and a previous intriguing theoretical conjecture, we develop a model to examine in detail the degradation dynamics of a protein featuring multiple phosphorylation sites and a threshold site number for elimination in response to a kinase signal. Our model explains the role of multiple phosphorylation sites, compared to a single site, in the regulation of protein degradation. A single-site protein cannot convert a graded input of kinase increase to much sharper output, whereas multisite phosphorylation is capable of generating a highly switch-like temporal profile of the substrate protein with two characteristics: a temporal threshold and rapid decrease beyond the threshold. We introduce a measure termed temporal response coefficient to quantify the extent to which a response in the time domain is switch-like and further investigate how this property is determined by various factors including the kinase input, the total number of sites, the threshold site number for elimination, the order of phosphorylation, the kinetic parameters, and site preference. Some interesting and experimentally verifiable predictions include that the non-degradable fraction of the substrate protein exhibits a more switch-like temporal profile; a sequential system is more switch-like, while a random system has the advantage of increased robustness; all the parameters, including the total number of sites, the threshold site number for elimination and the kinetic parameters synergistically determine the exact extent to which the degradation profile is switch-like. Our results suggest design principles for protein degradation switches which might be a widespread mechanism for precise regulation of cellular processes such as cell cycle progression. One third of all proteins in eukaryotic cells are phosphorylated at any time Xenopus protein Wee1A are responsible for the mitotic inactivation of this protein, while at least two others regulate its proteolysis during interphase Saccharomyces cerevisiae.The number of phosphorylation sites observed in one protein can vary from 1 to over 100 et al. investigated how multisite phosphorylation of Sic 1 regulates its ubiquitination and degradation in vitro and revived the cells in vivo. These experiments clearly revealed that there is a threshold number of phosphorylated sites required to render binding of Sic1 with Cdc4. The “counting” mechanism underlying this multisite-dependent digital interaction between phosphorylated Sic1 and Cdc4 has been studied both theoretically and experimentally. Mathematical modeling suggested that cooperative interactions between a disordered multi-phosphorylated Sic1 and a single-site receptor Cdc4 can explain the observed phosphorylation threshold Sic1 inhibits Clb5,6-Cdc28 kinase required for DNA replication and is believed to provide precise timing for the G1 to S transition by undergoing switch-like proteasome-mediated degradation upon phosphorylation by the Cln2-Cdc28 kinase complex. Yeast strains lacking Sic1 initiate DNA replication earlier and show extended S phase et al. hypothesized that this phosphorylation threshold eventually causes Sic1 to degrade in a switch-like manner during the G1/S transition Upon their remarkable discovery that Sic1 requires at least six sites phosphorylated to bind to Cdc4 for subsequent ubiquitination and degradation, Nash in vitro experimental assays, on how switch-like behaviors arise. Nevertheless, what is crucial for many systems, particularly in vivo processes, is the transient stimulus-response curve such as the temporal profile of Sic1 during the G1/S transition, whereas switch-like responses in the temporal domain have been investigated very limitedly. It should be noted that the cell cycle signaling network in yeast has been examined very extensively through mathematical modeling Switch-like behaviors have been studied extensively in the steady state domain, where the response of a biological system (e.g. the amount of oxygen bound by the hemoglobin protein in response to the change of oxygen concentration) exhibits the very intriguing property of buffering fluctuations in the stimulus below a threshold and amplifying drastically the change of stimulus above the threshold. This type of switch-like response in the steady state domain has been termed “ultrasensitivity” in the literature and a number of mechanisms have been proposed to account for its sources, including ligand cooperativity In a previous work, we showed that multisite phosphorylation is a potential source of switch-like steady-state responses; most importantly, a large number of total sites combined with an intermediate threshold number of sites for changing substrate functionality account for the switch-like behavior (manuscript in revision). Here, extending our previous studies, we investigate switch-like responses in the time domain when protein stability depends on the degree of phosphorylation. We will present a model to analyze phosphorylation-triggered elimination of the substrate protein in response to the rise of kinase activity. We will show quantitatively how different parameters affect the protein elimination dynamics when degradation occurs above a threshold number of phosphorylations. In particular, we will explore how the extent to which the degradation dynamics is switch-like is affected by the type of kinase stimuli, the number of phosphorylation sites, the order of phosphorylation reactions, and kinetic parameters. We have developed the model mainly based on the Sic1 system to reveal the role of its existing multiple sites in regulating the protein's switch-like destruction during the G1/S transition. However, multisite phosphorylation is potentially a widespread source of switch-like protein degradation and the design principles revealed by our model might be applicable to many other multisite regulatory proteins.Mammalia, is phosphorylated by kinase Gsk3 on three sites sequentially during the G2/M-G1 transition During proteolysis, once a substrate protein is phosphorylated properly, it is then ubiquitinated by a constitutively active SCF ubiquitin ligase in vitro experiments. For in vivo systems, we do not know to what extent these two conditions would cover all circumstances, because of scarce kinetic information of multi-step phosphorylation/dephosphorylation reactions. Nevertheless, considering what is generally believed with regard to the rate-limiting step in enzymatic reactions, we believe that either of these conditions can be satisfied for a wide range of systems. Hence the model presented here potentially represents a phosphorylation-triggered degradation process that captures common features of many multisite proteins. Under the aforementioned two conditions, the enzyme-substrate complexes evolve in a much faster time scale compared with the free substrates and can be assumed to operate at quasi steady states. Consequently, they can be neglected in the model and the governing ODEs reduce to simplified forms as described below.Our interest is to investigate the temporal change of the substrate protein as the kinase level increases converting the substrate to degradable forms and causing it to be eliminated from the system. An ordinary differential equation (ODE) based approach was employed to examine this dynamic process. According to the Michaelis-Menten formalism for enzymatic reactions, the substrate reacts with the kinase to form a substrate-kinase complex that can in turn dissociate to form either the enzyme and substrate, or the enzyme and a product which has one more site phosphorylated. Similarly, the phosphorylated protein and the phosphatase react to form the substrate-phosphatase complex, which dissociates to form either the phosphatase and phosphorylated protein or the phosphatase and a product that has one less site phosphorylated. A complete mechanistic model with these elementary steps would consist of With the assumption that ATP is abundant, the concentration of each specific substrate state with a unique combination of phosphorylated and nonphosphorylated sites (termed phospho-state in this work) depends on the concentration of other phospho-states that are upstream or downstream in the phosphorylation-dephosphorylation network, the concentration of the kinase and phosphatase, as well as the kinetic parameters. For easiness of understanding, we illustrate first the equations for a sequential system which involves For a random system, there are To simulate phosphorylation-triggered proteolysis, we assume that a basal activity of the phosphatase is always present in the system and the substrate exists in the unphosphorylated state initially. The kinase is then introduced into the system and increases gradually (detailed discussions will follow in the next section), converting the substrate to more and more phosphorylated states. As the substrate becomes sufficiently phosphorylated  starting from the time the kinase concentration increases, there is no appreciable change in the substrate concentration until a critical moment , and ii) the substrate concentration decreases immediately after passing the temporal threshold. Such a in vitro experiments when the kinase is added instantaneously to a phosphorylation-dependent degradation system. Under in vivo conditions, the rise of the kinase can only occur gradually, either in a linear or nonlinear fashion. For nonlinear stimuli, we use a sigmoid function with the general formula of The stimulus in our model is the increase of kinase level, but very little is known concerning exactly how a kinase rises in phosphorylation-triggered protein degradation. We started out by considering three types of stimuli that differ in how the level of active kinase changes from zero to the same maximum value within the same period of time: step-function increase; linear increase; and nonlinear increase. cant see . AccordiAnother relevant property of the stimulus is its duration. It is very likely that the kinase concentration will not be maintained permanently after it reaches the maximum. Instead, it will decrease gradually in a period of time because of degradation or other reasons. This is the case for many cyclin-dependent kinases, which rise and fall periodically during the cell cycle. For example, in the Sic1 system, the concentration of Cln2, which activates the Cdc28 kinase for Sic1 phosphorylation, increases gradually in the G1 phase, reaches its maximum before budding (beginning of the S phase), and then decreases With well-defined kinase stimulus, we now turn to the main puzzle of whether a multisite protein is switch-like in its degradation, most importantly, why multiple sites may help to achieve this property. In addition, we want to address the question of how the extent to which the degradation dynamics is switch-like is determined quantitatively.As described above, it is assumed that a protein with As shown in We can quantify the nonlinearity of the kinase stimulus with a stimulus coefficient similar to the response coefficient: S. cerevisiae phosphorylation on at least six out of nine sites is required for Sic1 degradation. According to our model, this design would enable the Sic1 protein to respond to the kinase signal in a highly switch-like manner: the non-degradable fraction of this CDK inhibitor does not change appreciably before a temporal threshold even though kinase has risen, then once the temporal threshold is passed, the non-degradable fraction decreases very rapidly. We hypothesize that this switch-like degradation is crucial for Sic1's regulatory function during the G1/S transition of the cell cycle. Otherwise, if Sic1 could bind to Cdc4 for ubiquitination and subsequent degradation, either as soon as it was phosphorylated on a single site , and thus the kinetic parameters may change along the phosphorylation/dephosphorylation chain. Here, we consider the case where the phosphorylation/dephosphorylation rate constants might increase or decrease after passing the threshold for degradation. As the comparison of five cases shows in As described above, our model consolidates ubiquitination and destruction by proteasome into one single degradation reaction. Relatively little is known about the kinetics of ubiquitination in general. Fortunately, a recent study has revealed the kinetics of polyubiquitylation of Sic1 rylation ; in the rylation . It is f of  see . It remaOur model of multisite phosphorylation triggered protein degradation is subject to many sources of uncertainty, including lack/inaccuracy of data and biological fluctuations. To investigate how variations in kinetic parameters affect the model output (i.e. the response coefficient of non-degradable substrate) we conducted sensitivity analysis. As a demonstration, here we focus on a sequential system with First, we examine how perturbation of each parameter affects the model output. We choose to change the parameter within 20% of its nominal value which correspond to the base scenario discussed above  is 1.81 and the standard deviation is 0.04, about 2.2% of the mean. We were also interested in how each parameter correlates with the model output in this context and calculated the partial rank correlation coefficient (PRCC). PRCC is a robust measure of sensitivity for nonlinear but monotonic relationships between a certain input (a parameter in our context) and the output as long as little or no correlation exists between the inputs The above analysis assumes simultaneous and independent perturbation of all the parameters which accounts for certain types of uncertainty such as stochastic fluctuations of kinetics. There might also exist uncertainty associated with the enzymes that affects a whole set of parameters. Thus, in another analysis, we considered a total of three parameters in our model  and then perturbed them in a similar manner. Results from 1000 samplings and simulations led to a mean response coefficient of 1.81 and a standard deviation of 0.09 (i.e. 5% of the mean).These results clearly show that the switch-like behavior of a multisite protein's degradation is very robust against random fluctuations of kinetic parameters.Finally, we will consider the effect of site preference on the degradation dynamics. In the completely random system described above, no site is preferred over other sites in term of the affinity for the kinase. However, for real proteins, due to effect of neighborhood amino acids, usually some sites are more likely to associate with the kinase and become phosphorylated. Consequently some pathways in the phosphorylation/dephosphorylation network are more dominant than others. A well-studied example is the extracellular-signal-regulated protein kinase ERK of the MAPK cascade. ERK contains two phosphorylable sites, Thr188 and Tyr190, for MEK kinase. The estimated kinetic parameters indicate that phosphorylation of Tyr is much faster than phosphorylation of Thr When site preference exists, as expected, our model predicts that deletion of a preferred site has a stronger effect in both increasing the half-life of the protein and reducing the sharpness of the response curve, compared to deletion of ordinary sites see . HoweverS. cerevisiae, during its degradation at the G1/S transition of the cell cycle. Most importantly, the protein becomes degradable upon phosphorylation on a critical number of sites In this paper, we have analyzed systematically a general model for multisite-phosphorylation-triggered protein degradation processes. The model has been developed largely based on what were revealed experimentally for Sic1, a nine-site protein in Schizosaccharomyces pombe, might also involve up to eight phosphorylation sites Saccharomyces cerevisiaeArabidopsis thalianaGiven the mostly unknown parameters involved in our model, it remains to be tested whether the exact predictions agree with actual degradation of multisite proteins such as Sic1. On the other hand, multisite phosphorylation has emerged as a recurring theme as researchers dissect the proteasome-dependent degradation pathway of various proteins in diverse species. de novo synthesis The work presented here for phosphorylation-triggered degradation also has certain limitations. First of all, we used a simplified kinetic model, as explained above and in in vitro experiments and under specific conditions which may not reflect biological realities. Another difficulty is the lack of qualitative knowledge on the phosphorylation process and degradation dynamics. Even the exact number of phosphorylation sites for a given protein is usually not provided in the literature. Of the proteins listed in The biggest obstacle in theoretical studies of protein phosphorylation and dephosphorylation is that biochemical parameters are not known for most biosystems; even for well-studied systems, parameters are obtained through A main challenge in quantitative understanding of phosphorylation and dephosphorylation processes is the identification and quantification of phosphorylated protein species. Mass spectrometry is becoming widely used as a fast, sensitive and high-throughput measurement method in the identification and quantification of protein phosphorylation. For instance, Olsen and colleagues detected and quantified phosphorylation of 6,600 sites on 2,244 proteins in response to a stimulus in mammalian cells through stable isotope labeling by amino acids in cell culture In summary, in this paper, we investigated the capability of a single protein with multiple phosphorylation sites in converting a graded input to a switch-like output signal in the time domain. We would like to point out that besides phosphorylation, a dominant post-translational modification for regulating protein stability and activity, other types of modification such as ubiquitination, methylation and glycosilation have also been shown to be involved in tuning the stability, activity and translocation of macromolecules. The model presented here or its variations can potentially explain and predict the behavior of these multisite modification systems as well. Finally, as have been demonstrated by a synthetic single-molecule signaling switch using multiple autoinhibitory domains http://www.engin.umich.edu/dept/che/research/lin/downloads.html.Systems of ordinary differential equations were formulated and solved with MATLAB 7.1. The codes are available at our website The events integrated in Note S1Model simplification.(0.06 MB PDF)Click here for additional data file.Note S2Non-dimensionalization.(0.04 MB PDF)Click here for additional data file.Note S3Effect of the sharpness of the stimulus profile.(0.05 MB PDF)Click here for additional data file.Figure S1Change of response coefficient for non-degradable substrate upon single-parameter perturbation.(20.24 MB TIF)Click here for additional data file.