Short Stat5-Interacting Peptide Derived from Phospholipase C-β3 Inhibits Hematopoietic Cell Proliferation and Myeloid Differentiation

Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-β3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-β3-dependent manner. PLC-β3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-β3 (PLC-β3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-β3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-β3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-β3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies

Thermodynamic analysis of high-temperature pumped thermal energy storage systems: Refrigerant selection, performance and limitations

[EN] One of the bottlenecks for a wider implementation of renewable energies is the development of efficient energy storage systems which can compensate for the intermittency of renewable energy sources. Pumped thermal energy storage (PTES) is a very recent technology that can be a promising site-independent alternative to pumped hydro energy storage or compressed air energy storage, without the corresponding geological and environmental restrictions. Accordingly, this paper presents a full thermodynamic analysis of a PTES system consisting of a high-temperature heat pump (HTHP), which drives an organic Rankine cycle (ORC) by means of an intermediate high-temperature thermal energy storage system (HT-TES). The latter combines both latent and sensible heat thermal energy storage sub-systems to maximize the advantage of the refrigerant subcooling. After validating the proposed model, several parametric studies have been carried out to assess the system performance using different refrigerants and configurations, under a wide range of source and sink temperatures. The results show that for a system that employs the same refrigerant in both the HTHP and ORC, and for a latent heat thermal energy storage system at 133 degrees C, R-1233zd(E) and R-1234ze(Z) present the best performance. Among all the cases studied with a latent heat thermal energy storage system at 133 degrees C, the best system performance, also considering the impact on the environment, has been achieved employing R-1233zd(E) in the HTHP and Butene in the ORC. Such a system can theoretically reach a power ratio of 1.34 under HTHP source and ORC sink temperatures of 100 and 25 degrees C, respectively. (C) 2020 Published by Elsevier Ltd.This work has been partially funded by the grant agreement No. 764042 (CHESTER project) of the European Union's Horizon 2020 research and innovation program.Hassan, A.; O'donoghue, L.; Sánchez Canales, V.; Corberán, JM.; Payá-Herrero, J.; Jockenhoefer, H. (2020). Thermodynamic analysis of high-temperature pumped thermal energy storage systems: Refrigerant selection, performance and limitations. Energy Reports. 6(7):147-159. https://doi.org/10.1016/j.egyr.2020.05.010S14715967Abarr, M., Geels, B., Hertzberg, J., & Montoya, L. D. (2017). Pumped thermal energy storage and bottoming system part A: Concept and model. Energy, 120, 320-331. doi:10.1016/j.energy.2016.11.089Abarr, M., Hertzberg, J., & Montoya, L. D. (2017). Pumped Thermal Energy Storage and Bottoming System Part B: Sensitivity analysis and baseline performance. Energy, 119, 601-611. doi:10.1016/j.energy.2016.11.028Aneke, M., & Wang, M. (2016). Energy storage technologies and real life applications – A state of the art review. Applied Energy, 179, 350-377. doi:10.1016/j.apenergy.2016.06.097Arpagaus, C., Bless, F., Uhlmann, M., Schiffmann, J., & Bertsch, S. S. (2018). High temperature heat pumps: Market overview, state of the art, research status, refrigerants, and application potentials. Energy, 152, 985-1010. doi:10.1016/j.energy.2018.03.166BP plc, 2018. BP Statistical Review of World Energy. London.Budt, M., Wolf, D., Span, R., & Yan, J. (2016). A review on compressed air energy storage: Basic principles, past milestones and recent developments. Applied Energy, 170, 250-268. doi:10.1016/j.apenergy.2016.02.108Cheayb, M., Marin Gallego, M., Tazerout, M., & Poncet, S. (2019). Modelling and experimental validation of a small-scale trigenerative compressed air energy storage system. Applied Energy, 239, 1371-1384. doi:10.1016/j.apenergy.2019.01.222Pereira da Cunha, J., & Eames, P. (2016). 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Performance optimization and comparison of pumped thermal and pumped cryogenic electricity storage systems. Energy, 106, 260-269. doi:10.1016/j.energy.2016.03.053Jockenhöfer, H., Steinmann, W.-D., & Bauer, D. (2018). Detailed numerical investigation of a pumped thermal energy storage with low temperature heat integration. Energy, 145, 665-676. doi:10.1016/j.energy.2017.12.087Kusakana, K. (2019). Hydro aeropower for sustainable electricity cost reduction in South African farming applications. Energy Reports, 5, 1645-1650. doi:10.1016/j.egyr.2019.11.023Laughlin, R. B. (2017). Pumped thermal grid storage with heat exchange. Journal of Renewable and Sustainable Energy, 9(4), 044103. doi:10.1063/1.4994054Lecompte, S., Huisseune, H., van den Broek, M., Vanslambrouck, B., & De Paepe, M. (2015). Review of organic Rankine cycle (ORC) architectures for waste heat recovery. Renewable and Sustainable Energy Reviews, 47, 448-461. doi:10.1016/j.rser.2015.03.089Liu, J.-L., & Wang, J.-H. (2016). A comparative research of two adiabatic compressed air energy storage systems. Energy Conversion and Management, 108, 566-578. doi:10.1016/j.enconman.2015.11.049Ma, T., Yang, H., & Lu, L. (2014). Feasibility study and economic analysis of pumped hydro storage and battery storage for a renewable energy powered island. Energy Conversion and Management, 79, 387-397. doi:10.1016/j.enconman.2013.12.047McTigue, J. D., White, A. J., & Markides, C. N. (2015). Parametric studies and optimisation of pumped thermal electricity storage. Applied Energy, 137, 800-811. doi:10.1016/j.apenergy.2014.08.039Navarro-Peris, E., Corberán, J. M., Falco, L., & Martínez-Galván, I. O. (2013). New non-dimensional performance parameters for the characterization of refrigeration compressors. International Journal of Refrigeration, 36(7), 1951-1964. doi:10.1016/j.ijrefrig.2013.07.007Steinmann, W. D. (2014). The CHEST (Compressed Heat Energy STorage) concept for facility scale thermo mechanical energy storage. 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PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

Exact model reduction of combinatorial reaction networks

Receptors and scaffold proteins usually possess a high number of distinct binding domains inducing the formation of large multiprotein signaling complexes. Due to combinatorial reasons the number of distinguishable species grows exponentially with the number of binding domains and can easily reach several millions. Even by including only a limited number of components and binding domains the resulting models are very large and hardly manageable. A novel model reduction technique allows the significant reduction and modularization of these models

Modified Epidermal Growth Factor Receptor (EGFR)-Bearing Liposomes (MRBLs) Are Sensitive to EGF in Solution

Cancers often overexpress EGF and other growth factors to promote cell replication and migration. Previous work has not produced targeted drug carriers sensitive to abnormal amounts of growth factors. This work demonstrates that liposomes bearing EGF receptors covalently crosslinked to p-toluic acid or methyl-PEO4-NHS ester (or, in short, MRBLs) exhibit an increased rate of release of encapsulated drug compounds when EGF is present in solution. Furthermore, the modified EGF receptors retain the abilities to form dimers in the presence of EGF and bind specifically to EGF. These results demonstrate that MRBLs are sensitive to EGF in solution and indicate that MRBL-reconstituted modified EGF receptors, in the presence of EGF in solution, form dimers which increase MRBL permeability to encapsulated compounds

Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs

Trade-Offs and Constraints in Allosteric Sensing

Sensing extracellular changes initiates signal transduction and is the first stage of cellular decision-making. Yet relatively little is known about why one form of sensing biochemistry has been selected over another. To gain insight into this question, we studied the sensing characteristics of one of the biochemically simplest of sensors: the allosteric transcription factor. Such proteins, common in microbes, directly transduce the detection of a sensed molecule to changes in gene regulation. Using the Monod-Wyman-Changeux model, we determined six sensing characteristics – the dynamic range, the Hill number, the intrinsic noise, the information transfer capacity, the static gain, and the mean response time – as a function of the biochemical parameters of individual sensors and of the number of sensors. We found that specifying one characteristic strongly constrains others. For example, a high dynamic range implies a high Hill number and a high capacity, and vice versa. Perhaps surprisingly, these constraints are so strong that most of the space of characteristics is inaccessible given biophysically plausible ranges of parameter values. Within our approximations, we can calculate the probability distribution of the numbers of input molecules that maximizes information transfer and show that a population of one hundred allosteric transcription factors can in principle distinguish between more than four bands of input concentrations. Our results imply that allosteric sensors are unlikely to have been selected for high performance in one sensing characteristic but for a compromise in the performance of many

Detection of Hepatocyte Growth Factor (HGF) Ligand-c-MET Receptor Activation in Formalin-Fixed Paraffin Embedded Specimens by a Novel Proximity Assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels o

Interleukin-4 Alters Early Phagosome Phenotype by Modulating Class I PI3K Dependent Lipid Remodeling and Protein Recruitment

Phagocytosis is a complex process that involves membranelipid remodeling and the attraction and retention of key effector proteins. Phagosome phenotype depends on the type of receptor engaged and can be influenced by extracellular signals. Interleukin 4 (IL-4) is a cytokine that induces the alternative activation of macrophages (MΦs) upon prolonged exposure, triggering a different cell phenotype that has an altered phagocytic capacity. In contrast, the direct effects of IL-4 during phagocytosis remain unknown. Here, we investigate the impact of short-term IL-4 exposure (1 hour) during phagocytosis of IgG-opsonized yeast particles by MΦs. By time-lapse confocal microscopy of GFP-tagged lipid-sensing probes, we show that IL-4 increases the negative charge of the phagosomal membrane by prolonging the presence of the negatively charged second messenger PI(3,4,5)P3. Biochemical assays reveal an enhanced PI3K/Akt activity upon phagocytosis in the presence of IL-4. Blocking the specific class I PI3K after the onset of phagocytosis completely abrogates the IL-4-induced changes in lipid remodeling and concomitant membrane charge. Finally, we show that IL-4 direct signaling leads to a significantly prolonged retention profile of the signaling molecules Rac1 and Rab5 to the phagosomal membrane in a PI3K-dependent manner. This protracted early phagosome phenotype suggests an altered maturation, which is supported by the delayed phagosome acidification measured in the presence of IL-4. Our findings reveal that molecular differences in IL-4 levels, in the extracellular microenvironment, influence the coordination of lipid remodeling and protein recruitment, which determine phagosome phenotype and, eventually, fate. Endosomal and phagosomal membranes provide topological constraints to signaling molecules. Therefore, changes in the phagosome phenotype modulated by extracellular factors may represent an additional mechanism that regulates the outcome of phagocytosis and could have significant impact on the net biochemical output of a cell

The Lipid Transfer Protein CERT Interacts with the Chlamydia Inclusion Protein IncD and Participates to ER-Chlamydia Inclusion Membrane Contact Sites

Bacterial pathogens that reside in membrane bound compartment manipulate the host cell machinery to establish and maintain their intracellular niche. The hijacking of inter-organelle vesicular trafficking through the targeting of small GTPases or SNARE proteins has been well established. Here, we show that intracellular pathogens also establish direct membrane contact sites with organelles and exploit non-vesicular transport machinery. We identified the ER-to-Golgi ceramide transfer protein CERT as a host cell factor specifically recruited to the inclusion, a membrane-bound compartment harboring the obligate intracellular pathogen Chlamydia trachomatis. We further showed that CERT recruitment to the inclusion correlated with the recruitment of VAPA/B-positive tubules in close proximity of the inclusion membrane, suggesting that ER-Inclusion membrane contact sites are formed upon C. trachomatis infection. Moreover, we identified the C. trachomatis effector protein IncD as a specific binding partner for CERT. Finally we showed that depletion of either CERT or the VAP proteins impaired bacterial development. We propose that the presence of IncD, CERT, VAPA/B, and potentially additional host and/or bacterial factors, at points of contact between the ER and the inclusion membrane provides a specialized metabolic and/or signaling microenvironment favorable to bacterial development