by Keyword: Akt

Molina-Fernandez, R, Picon-Pages, P, Barranco-Almohalla, A, Crepin, G, Herrera-Fernandez, V, Garcia-Elias, A, Fanlo-Ucar, H, Fernandez-Busquets, X, Garcia-Ojalvo, J, Oliva, B, Munoz, FJ, (2022). Differential regulation of insulin signalling by monomeric and oligomeric amyloid beta-peptide Brain Commun 4, fcac243

Alzheimer's disease and Type 2 diabetes are pathological processes associated to ageing. Moreover, there are evidences supporting a mechanistic link between Alzheimer's disease and insulin resistance (one of the first hallmarks of Type 2 diabetes). Regarding Alzheimer's disease, amyloid beta-peptide aggregation into beta-sheets is the main hallmark of Alzheimer's disease. At monomeric state, amyloid beta-peptide is not toxic but its function in brain, if any, is unknown. Here we show, by in silico study, that monomeric amyloid beta-peptide 1-40 shares the tertiary structure with insulin and is thereby able to bind and activate insulin receptor. We validated this prediction experimentally by treating human neuroblastoma cells with increasing concentrations of monomeric amyloid. beta-peptide 1-40. Our results confirm that monomeric amyloid beta-peptide 1-40 activates insulin receptor autophosphorylation, triggering downstream enzyme phosphorylarions and the glucose Transporter 4 translocation to the membrane. On the other hand, neuronal insulin resistance is known to be associated to Alzheimer's disease since early stages. We thus modelled the docking of oligomeric amyloid peptide 1-40 to insulin receptor. We found that oligomeric amyloid. beta-peptide 1-40 blocks insulin receptor, impairing its activation. It was confirmed in vitro by observing the lack of insulin receptor autophosphorylation, and also the impairment of insulin-induced intracellular enzyme activations and the glucose Transporter 4 translocation to the membrane. By biological system analysis, we have carried out a mathematical model recapitulating the process that turns amyloid beta-peptide binding to insulin receptor from the physiological to the pathophysiological regime. Our results suggest that monomeric amyloid beta-peptide 1-40 contributes to mimic insulin effects in the brain, which could be good when neurons have an extra requirement of energy beside the well-known protective effects on insulin intracellular signalling, while its accumulation and subsequent oligomerization blocks the insulin receptor producing insulin resistance and compromising neuronal metabolism and protective pathways.

JTD Keywords: A-beta, Aggregation, Akt, Alzheimer's disease, Alzheimers-disease, Amyloid beta-peptide, Brain, Design, Insulin, Insulin resistance, Precursor protein, Protein-protein docking, Receptor, Resistance, Site

Zañudo JGT, Mao P, Alcon C, Kowalski K, Johnson GN, Xu G, Baselga J, Scaltriti M, Letai A, Montero J, Albert R, Wagle N, (2021). Cell line-specific network models of er breast cancer identify potential pi3kainhibitor resistance mechanisms and drug combinations Cancer Research 81, 4603-4617

Durable control of invasive solid tumors necessitates identifying therapeutic resistance mechanisms and effective drug combinations. In this work, we used a network-based mathematical model to identify sensitivity regulators and drug combinations for the PI3Ka inhibitor alpelisib in estrogen receptor positive (ER) PIK3CAmutant breast cancer. The model-predicted efficacious combination of alpelisib and BH3 mimetics, for example, MCL1 inhibitors, was experimentally validated in ER breast cancer cell lines. Consistent with the model, FOXO3 downregulation reduced sensitivity to alpelisib, revealing a novel potential resistance mechanism. Cell line-specific sensitivity to combinations of alpelisib and BH3 mimetics depended on which BCL2 family members were highly expressed. On the basis of these results, newly developed cell line-specific network models were able to recapitulate the observed differential response to alpelisib and BH3 mimetics. This approach illustrates how network-based mathematical models can contribute to overcoming the challenge of cancer drug resistance.

JTD Keywords: activation, akt, feedback, foxo, leads, p27(kip1), phosphorylation, reveals, transcription factors, Dependent kinase inhibitor

Selfa IL, Gallo M, Montserrat N, Garreta E, (2021). Directed Differentiation of Human Pluripotent Stem Cells for the Generation of High-Order Kidney Organoids Methods In Molecular Biology 2258, 171-192

© 2021, The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature. Our understanding in the inherent properties of human pluripotent stem cells (hPSCs) have made possible the development of differentiation procedures to generate three-dimensional tissue-like cultures, so-called organoids. Here we detail a stepwise methodology to generate kidney organoids from hPSCs. This is achieved through direct differentiation of hPSCs in two-dimensional monolayer culture toward the posterior primitive streak fate, followed by induction of intermediate mesoderm-committed cells, which are further aggregated and cultured in three-dimensions to generate kidney organoids containing segmented nephron-like structures in a process that lasts 20 days. We also provide a concise description on how to assess renal commitment during the time course of kidney organoid generation. This includes the use of flow cytometry and immunocytochemistry analyses for the detection of specific renal differentiation markers.

JTD Keywords: 2d monolayer, 3d organotypic culture, differentiation, flow cytometry, human pluripotent stem cells, immunocytochemistry, intermediate mesoderm, kidney organoid, nephron progenitor cells, nephrons, primitive streak, 2d monolayer, 3d organotypic culture, Differentiation, Flow cytometry, Human pluripotent stem cells, Immunocytochemistry, Intermediate mesoderm, Kidney organoid, Nephron progenitor cells, Nephrons, Primitive streak, Tissue

Ikonomov, O. C., Altankov, G., Sbrissa, D., Shisheva, A., (2018). PIKfyve inhibitor cytotoxicity requires AKT suppression and excessive cytoplasmic vacuolation Toxicology and Applied Pharmacology 356, 151-158

PIKfyve phosphoinositide kinase produces PtdIns(3,5)P2 and PtdIns5P and governs a myriad of cellular processes including cytoskeleton rearrangements and cell proliferation. The latter entails rigorous investigation since the cytotoxicity of PIKfyve inhibition is a potential therapeutic modality for cancer. Here we report the effects of two PIKfyve-specific inhibitors on the attachment/spreading and viability of mouse embryonic fibroblasts (MEFs) and C2C12 myoblasts. Importantly, 18-h treatment of adherent cells with YM201636 (800 nM) and apilimod (20 nM) in serum-containing culture media did not affect cell viability despite the presence of multiple cytoplasmic vacuoles, a hallmark of PIKfyve inhibition. Strikingly, at the same dose and duration the inhibitors caused excessive cytoplasmic vacuolation, initial suppression of cell attachment/spreading and subsequent marked detachment/death in serum-deprived cells. The remaining adherent cells under serum-deprived conditions had smaller surface area, lacked vinculin/actin-positive focal adhesions and displayed vacuoles occupying the entire cytoplasm. Serum or growth factors protected against PIKfyve inhibitor cytotoxicity. This protection required Akt activation evidenced by the abrogated beneficial effect of serum upon treatment with the clinically-relevant Akt inhibitor MK-2206. Moreover, Akt inhibition triggered cell detachment/death even in serum-fed adherent MEFs treated with apilimod. Intriguingly, BafilomycinA1 (H+-vacuolar ATPase inhibitor), which prevents the cytoplasmic vacuolation under PIKfyve perturbations, rescued all defects in attaching/spreading as well as in adherent cells under serum-starved or serum-fed conditions, respectively. Together, the results indicate that the cytotoxicity of PIKfyve inhibitors in MEFs and C2C12 myoblasts requires Akt suppression and excessive cytoplasmic vacuolation.

JTD Keywords: AKT, Cytotoxicity, MK-2206, PIKfyve, Ppilimod, YM201636

Redondo, L., Giannotti, M. I., Sanz, F., (2012). Stability of lipid bilayers as model membranes: Atomic force microscopy and spectroscopy approach Atomic force microscopy in liquid (ed. Baró, A. M., Reifenberger, R. G.), Wiley-VCH Verlag GmbH & Co.KGaA (Weinheim, Germany) Part I: General Atomic Force Microscopy, 259-284