bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2022–05–22
eightteen papers selected by
Ralitsa Radostinova Madsen, University College London
  1. The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.
  2. Idelalisib activates AKT via increased recruitment of PI3Kδ/PI3Kβ to BCR signalosome while reducing PDK1 in post-therapy CLL cells.
  3. Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.
  4. Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.
  5. Reflected stemness as a potential driver of the tumour microenvironment.
  6. How to inhibit breast cancer and breast cancer metastasis with Akt inhibitors: Lessons learned from studies in mice.
  7. mTOR substrate phosphorylation in growth control.
  8. Prolonged deprivation of arginine or leucine induces PI3K/Akt-dependent reactivation of mTORC1.
  9. Suppression of nuclear GSK3 signaling promotes serine/one-carbon metabolism and confers metabolic vulnerability in lung cancer cells.
  10. PHGDH heterogeneity potentiates cancer cell dissemination and metastasis.
  11. The phosphatase PTEN links platelets with immune regulatory functions of mouse T follicular helper cells.
  12. Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis.
  13. Deep Visual Proteomics defines single-cell identity and heterogeneity.
  14. Phosphoinositides as membrane organizers.
  15. Visual barcodes for clonal-multiplexing of live microscopy-based assays.
  16. The structural context of posttranslational modifications at a proteome-wide scale.
  17. Reversible lysine-targeted probes reveal residence time-based kinase selectivity.
  18. Universal antigen encoding of T cell activation from high-dimensional cytokine dynamics.
  1. Cell Rep. 2022 May 17. pii: S2211-1247(22)00595-2. [Epub ahead of print]39(7): 110824
    The non-essential TSC complex component TBC1D7 restricts tissue mTORC1 signaling and brain and neuron growth.
    Sandra Schrötter, Christopher J Yuskaitis, Michael R MacArthur, Sarah J Mitchell, Aaron M Hosios, Maria Osipovich, Margaret E Torrence, James R Mitchell, Gerta Hoxhaj, Mustafa Sahin, Brendan D Manning.
      The tuberous sclerosis complex (TSC) 1 and 2 proteins associate with TBC1D7 to form the TSC complex, which is an essential suppressor of mTOR complex 1 (mTORC1), a ubiquitous driver of cell and tissue growth. Loss-of-function mutations in TSC1 or TSC2, but not TBC1D7, give rise to TSC, a pleiotropic disorder with aberrant activation of mTORC1 in various tissues. Here, we characterize mice with genetic deletion of Tbc1d7, which are viable with normal growth and development. Consistent with partial loss of function of the TSC complex, Tbc1d7 knockout (KO) mice display variable increases in tissue mTORC1 signaling with increased muscle fiber size but with strength and motor defects. Their most pronounced phenotype is brain overgrowth due to thickening of the cerebral cortex, with enhanced neuron-intrinsic mTORC1 signaling and growth. Thus, TBC1D7 is required for full TSC complex function in tissues, and the brain is particularly sensitive to its growth-suppressing activities.
    Keywords:  CP: Developmental biology; CP: Neuroscience; TBC1D7; Tsc1; Tsc2; brain; gait; growth; hamartin; mTOR; megalencephaly; mouse model; neurons; rapamycin; tuberin; tuberous sclerosis complex
    DOI:  https://doi.org/10.1016/j.celrep.2022.110824
  2. Leukemia. 2022 May 14.
    Idelalisib activates AKT via increased recruitment of PI3Kδ/PI3Kβ to BCR signalosome while reducing PDK1 in post-therapy CLL cells.
    Murali K Mamidi, Hasan Mahmud, Guru P Maiti, Mariana T Mendez, Stacey M Fernandes, Sara K Vesely, Jennifer Holter-Chakrabarty, Jennifer R Brown, Asish K Ghosh.
      Idelalisib targets PI3Kδ in the BCR pathway generating only a partial response in CLL patients, indicating that the leukemic cells may have evolved escape signals. Indeed, we detected increased activation of AKT accompanied by upregulation of MYC/BCL2 in post-therapy CLL cells from patients treated with idelalisib/ofatumumab. To unravel the mechanism of increased AKT-activation, we studied the impact of idelalisib on a CLL-derived cell line, MEC1, as a model. After an initial inhibition, AKT-activation level was restored in idelalisib-treated MEC1 cells in a time-dependent manner. As BCAP (B-cell adaptor for PI3K) and CD19 recruit PI3Kδ to activate AKT upon BCR-stimulation, we examined if idelalisib-treatment altered PI3Kδ-recruitment. Immunoprecipitation of BCAP/CD19 from idelalisib-treated MEC1 cells showed increased recruitment of PI3Kδ in association with PI3Kβ, but not PI3Kα or PI3Kγ and that, targeting both PI3Kδ with PI3Kβ inhibited AKT-reactivation. We detected similar, patient-specific recruitment pattern of PI3K-isoforms by BCAP/CD19 in post-idelalisib CLL cells with increased AKT-activation. Interestingly, a stronger inhibitory effect of idelalisib on P-AKT (T308) than S473 was discernible in idelalisib-treated cells despite increased recruitment of PI3Kδ/PI3Kβ and accumulation of phosphatidylinositol-3,4,5-triphosphate; which could be attributed to reduced PDK1 activity. Thus, administration of isoform-specific inhibitors may prove more effective strategy for treating CLL patients.
    DOI:  https://doi.org/10.1038/s41375-022-01595-0
  3. Biochem J. 2022 May 20. pii: BCJ20220153. [Epub ahead of print]
    Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.
    Xiaowen Duan, Dougall M Norris, Sean Humphrey, Pengyi Yang, Kristen C Cooke, Will P Bultitude, Ben Parker, Olivia J Conway, James G Burchfield, James R Krycer, Frances Brodsky, David James, Daniel J Fazakerley.
      Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.
    Keywords:  Trafficking regulator of GLUT4-1; adipocytes; glucose transport; glycogen synthase kinase; insulin signalling
    DOI:  https://doi.org/10.1042/BCJ20220153
  4. Nat Commun. 2022 May 16. 13(1): 2698
    Purine nucleotide depletion prompts cell migration by stimulating the serine synthesis pathway.
    Mona Hoseini Soflaee, Rushendhiran Kesavan, Umakant Sahu, Alpaslan Tasdogan, Elodie Villa, Zied Djabari, Feng Cai, Diem H Tran, Hieu S Vu, Eunus S Ali, Halie Rion, Brendan P O'Hara, Sherwin Kelekar, James Hughes Hallett, Misty Martin, Thomas P Mathews, Peng Gao, John M Asara, Brendan D Manning, Issam Ben-Sahra, Gerta Hoxhaj.
      Purine nucleotides are necessary for various biological processes related to cell proliferation. Despite their importance in DNA and RNA synthesis, cellular signaling, and energy-dependent reactions, the impact of changes in cellular purine levels on cell physiology remains poorly understood. Here, we find that purine depletion stimulates cell migration, despite effective reduction in cell proliferation. Blocking purine synthesis triggers a shunt of glycolytic carbon into the serine synthesis pathway, which is required for the induction of cell migration upon purine depletion. The stimulation of cell migration upon a reduction in intracellular purines required one-carbon metabolism downstream of de novo serine synthesis. Decreased purine abundance and the subsequent increase in serine synthesis triggers an epithelial-mesenchymal transition (EMT) and, in cancer models, promotes metastatic colonization. Thus, reducing the available pool of intracellular purines re-routes metabolic flux from glycolysis into de novo serine synthesis, a metabolic change that stimulates a program of cell migration.
    DOI:  https://doi.org/10.1038/s41467-022-30362-z
  5. Trends Cell Biol. 2022 May 16. pii: S0962-8924(22)00108-8. [Epub ahead of print]
    Reflected stemness as a potential driver of the tumour microenvironment.
    Felipe S Rodrigues, Francesca D Ciccarelli, Ilaria Malanchi.
      A fundamental requirement for cancer initiation is the activation of developmental programmes by mutant cells. Oncogenic signals often confer an undifferentiated, stem cell-like phenotype that supports the long-term proliferative potential of cancer cells. Although cancer is a genetically driven disease, mutations in cancer-driver genes alone are insufficient for tumour formation, and the proliferation of cells harbouring oncogenic mutations depends on their microenvironment. In this Opinion article we discuss how the reprogrammed status of cancer cells not only represents the essence of their tumorigenicity but triggers 'reflected stemness' in their surrounding normal counterparts. We propose that this reciprocal interaction underpins the establishment of the tumour microenvironment (TME).
    Keywords:  cancer drivers; metastasis; stem cells; tissue regeneration; tumour microenvironment
    DOI:  https://doi.org/10.1016/j.tcb.2022.04.007
  6. J Breast Cancer Res. 2021 ;1(2): 30-33
    How to inhibit breast cancer and breast cancer metastasis with Akt inhibitors: Lessons learned from studies in mice.
    Nissim Hay.
      The PI3K/Akt signaling pathway is frequently hyperactivated in different types of breast cancer. In the past two decades, major efforts have been made to develop inhibitors of this pathway to treat cancer patients. However, the most evolutionarily conserved function of this pathway is in cellular and organismal metabolism, which is hijacked by cancer cells. Thus, adverse metabolic consequences are expected when PI3K or Akt is targeted. These metabolic consequences, particularly hyperinsulinemia, could impede the efficacy of treatment. This review summarizes recent genetic studies in mice that could pave the way to efficient breast cancer and breast cancer metastasis treatment with Akt inhibitors.
  7. Cell. 2022 May 12. pii: S0092-8674(22)00460-3. [Epub ahead of print]
    mTOR substrate phosphorylation in growth control.
    Stefania Battaglioni, Don Benjamin, Matthias Wälchli, Timm Maier, Michael N Hall.
      The target of rapamycin (TOR), discovered 30 years ago, is a highly conserved serine/threonine protein kinase that plays a central role in regulating cell growth and metabolism. It is activated by nutrients, growth factors, and cellular energy. TOR forms two structurally and functionally distinct complexes, TORC1 and TORC2. TOR signaling activates cell growth, defined as an increase in biomass, by stimulating anabolic metabolism while inhibiting catabolic processes. With emphasis on mammalian TOR (mTOR), we comprehensively reviewed the literature and identified all reported direct substrates. In the context of recent structural information, we discuss how mTORC1 and mTORC2, despite having a common catalytic subunit, phosphorylate distinct substrates. We conclude that the two complexes recruit different substrates to phosphorylate a common, minimal motif.
    DOI:  https://doi.org/10.1016/j.cell.2022.04.013
  8. J Biol Chem. 2022 May 13. pii: S0021-9258(22)00470-7. [Epub ahead of print] 102030
    Prolonged deprivation of arginine or leucine induces PI3K/Akt-dependent reactivation of mTORC1.
    Gwen R Buel, Huy Dang, John M Asara, John Blenis, Anders P Mutvei.
      The mechanistic target of rapamycin complex 1 (mTORC1) is a serine/threonine kinase complex that promotes anabolic processes including protein, lipid, and nucleotide synthesis, while suppressing catabolic processes such as macroautophagy. mTORC1 activity is regulated by growth factors and amino acids which signal through distinct but integrated molecular pathways: growth factors largely signal through the PI3K/Akt-dependent pathway, whereas the availabilities of amino acids leucine and arginine are communicated to mTORC1 by the Rag-GTPase pathway. While it is relatively well described how acute changes in leucine and arginine levels affect mTORC1 signaling, the effects of prolonged amino acid deprivation remain less well understood. Here, we demonstrate that prolonged deprivation of arginine and/or leucine leads to reactivation of mTORC1 activity, which reaches activation levels similar to those observed in nutrient-rich conditions. Surprisingly, we find that this reactivation is independent of the regeneration of amino acids by canonical autophagy or proteasomal degradation, but is dependent on PI3K/Akt signaling. Together, our data identify a novel crosstalk between the amino acid and PI3K/Akt signaling pathways upstream of mTORC1. These observations extend our understanding of the role of mTORC1 in growth-related diseases and indicate that dietary intervention by removal of leucine and/or arginine may be an ineffective therapeutic approach.
    DOI:  https://doi.org/10.1016/j.jbc.2022.102030
  9. Sci Adv. 2022 May 20. 8(20): eabm8786
    Suppression of nuclear GSK3 signaling promotes serine/one-carbon metabolism and confers metabolic vulnerability in lung cancer cells.
    Long He, Jennifer Endress, Sungyun Cho, Zhongchi Li, Yuxiang Zheng, John M Asara, John Blenis.
      Serine/one-carbon metabolism provides critical resources for nucleotide biosynthesis and epigenetic maintenance and is thus necessary in cancer cell growth, although the detailed regulatory mechanisms remain unclear. We uncover a critical role of glycogen synthase kinase 3 (GSK3) in regulating the expression of serine/one-carbon metabolic enzymes. Nuclear enrichment of GSK3 significantly suppresses genes that mediate de novo serine synthesis, including PHGDH, PSAT1, PSPH, and one-carbon metabolism, including SHMT2 and MTHFD2. FRAT1 promotes nuclear exclusion of GSK3, enhances serine/one-carbon metabolism, and, as a result, confers cell vulnerability to inhibitors that target this metabolic process such as SHIN1, a specific SHMT1/2 inhibitor. Furthermore, pharmacological or genetic suppression of GSK3 promotes serine/one-carbon metabolism and exhibits a significant synergistic effect in combination with SHIN1 in suppressing cancer cell proliferation in cultured cells and in vivo. Our observations indicate that inhibition of nuclear GSK3 signaling creates a vulnerability, which results in enhanced efficacy of serine/one-carbon metabolism inhibitors for the treatment of cancer.
    DOI:  https://doi.org/10.1126/sciadv.abm8786
  10. Nature. 2022 May 18.
    PHGDH heterogeneity potentiates cancer cell dissemination and metastasis.
    Matteo Rossi, Patricia Altea-Manzano, Margherita Demicco, Ginevra Doglioni, Laura Bornes, Marina Fukano, Anke Vandekeere, Alejandro M Cuadros, Juan Fernández-García, Carla Riera-Domingo, Cristina Jauset, Mélanie Planque, H Furkan Alkan, David Nittner, Dongmei Zuo, Lindsay A Broadfield, Sweta Parik, Antonino Alejandro Pane, Francesca Rizzollo, Gianmarco Rinaldi, Tao Zhang, Shao Thing Teoh, Arin B Aurora, Panagiotis Karras, Ines Vermeire, Dorien Broekaert, Joke Van Elsen, Maximilian M L Knott, Martin F Orth, Sofie Demeyer, Guy Eelen, Lacey E Dobrolecki, Ayse Bassez, Thomas Van Brussel, Karl Sotlar, Michael T Lewis, Harald Bartsch, Manfred Wuhrer, Peter van Veelen, Peter Carmeliet, Jan Cools, Sean J Morrison, Jean-Christophe Marine, Diether Lambrechts, Massimiliano Mazzone, Gregory J Hannon, Sophia Y Lunt, Thomas G P Grünewald, Morag Park, Jacco van Rheenen, Sarah-Maria Fendt.
      Cancer metastasis requires the transient activation of cellular programs enabling dissemination and seeding in distant organs1. Genetic, transcriptional and translational heterogeneity contributes to this dynamic process2,3. Metabolic heterogeneity has also been observed4, yet its role in cancer progression is less explored. Here we find that the loss of phosphoglycerate dehydrogenase (PHGDH) potentiates metastatic dissemination. Specifically, we find that heterogeneous or low PHGDH expression in primary tumours of patients with breast cancer is associated with decreased metastasis-free survival time. In mice, circulating tumour cells and early metastatic lesions are enriched with Phgdhlow cancer cells, and silencing Phgdh in primary tumours increases metastasis formation. Mechanistically, Phgdh interacts with the glycolytic enzyme phosphofructokinase, and the loss of this interaction activates the hexosamine-sialic acid pathway, which provides precursors for protein glycosylation. As a consequence, aberrant protein glycosylation occurs, including increased sialylation of integrin αvβ3, which potentiates cell migration and invasion. Inhibition of sialylation counteracts the metastatic ability of Phgdhlow cancer cells. In conclusion, although the catalytic activity of PHGDH supports cancer cell proliferation, low PHGDH protein expression non-catalytically potentiates cancer dissemination and metastasis formation. Thus, the presence of PHDGH heterogeneity in primary tumours could be considered a sign of tumour aggressiveness.
    DOI:  https://doi.org/10.1038/s41586-022-04758-2
  11. Nat Commun. 2022 May 19. 13(1): 2762
    The phosphatase PTEN links platelets with immune regulatory functions of mouse T follicular helper cells.
    Xue Chen, Yanyan Xu, Qidi Chen, Heng Zhang, Yu Zeng, Yan Geng, Lei Shen, Fubin Li, Lei Chen, Guo-Qiang Chen, Chuanxin Huang, Junling Liu.
      Beyond a function in hemostasis and thrombosis, platelets can regulate innate and adaptive immune responses. Hyperactive platelets are frequently associated with multiple human autoimmune diseases, yet their pathogenic functions in these diseases have not been fully established. Emerging studies show an essential function of the phosphatase and tensin homolog (PTEN) in maintenance of immune homeostasis. Here, we show that mice with platelet-specific deletion of Pten, develop age-related lymphoproliferative diseases and humoral autoimmunity not seen in wildtype animals. Platelet-specific Pten-deficient mice have aberrant T cell activation, excessive T follicular helper (Tfh) cell responses and accumulation of platelet aggregates in lymph nodes. Transferred Pten-deficient platelets are able to infiltrate into the peripheral lymphoid tissues and form more aggregates. Moreover, Pten-deficient platelets are hyperactive and overproduce multiple Tfh-promoting cytokines via activation of the PDK1/mTORC2-AKT-SNAP23 pathway. Pten-deficient platelets show enhanced interaction with CD4+ T cells and promote conversion of CD4+ T cells into Tfh cells. Our results implicate PTEN in platelet-mediated immune homeostasis, and provide evidence that hyperactive platelets function as an important mediator in autoimmune diseases using mouse models.
    DOI:  https://doi.org/10.1038/s41467-022-30444-y
  12. Dev Cell. 2022 May 11. pii: S1534-5807(22)00286-6. [Epub ahead of print]
    Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis.
    Roxana E Oberkersch, Giovanna Pontarin, Matteo Astone, Marianna Spizzotin, Liaisan Arslanbaeva, Giovanni Tosi, Emiliano Panieri, Sara Ricciardi, Maria Francesca Allega, Alessia Brossa, Paolo Grumati, Benedetta Bussolati, Stefano Biffo, Saverio Tardito, Massimo M Santoro.
      Angiogenesis, the active formation of new blood vessels from pre-existing ones, is a complex and demanding biological process that plays an important role in physiological as well as pathological settings. Recent evidence supports cell metabolism as a critical regulator of angiogenesis. However, whether and how cell metabolism regulates endothelial growth factor receptor levels and nucleotide synthesis remains elusive. We here shown in both human cell lines and mouse models that during developmental and pathological angiogenesis, endothelial cells (ECs) use glutaminolysis-derived glutamate to produce aspartate (Asp) via aspartate aminotransferase (AST/GOT). Asp leads to mTORC1 activation which, in turn, regulates endothelial translation machinery for VEGFR2 and FGFR1 synthesis. Asp-dependent mTORC1 pathway activation also regulates de novo pyrimidine synthesis in angiogenic ECs. These findings identify glutaminolysis-derived Asp as a regulator of mTORC1-dependent endothelial translation and pyrimidine synthesis. Our studies may help overcome anti-VEGF therapy resistance by targeting endothelial growth factor receptor translation.
    Keywords:  angiogenesis; aspartate metabolism; endothelial metabolism; mTOR signalling; tumor angiogenesis
    DOI:  https://doi.org/10.1016/j.devcel.2022.04.018
  13. Nat Biotechnol. 2022 May 19.
    Deep Visual Proteomics defines single-cell identity and heterogeneity.
    Andreas Mund, Fabian Coscia, András Kriston, Réka Hollandi, Ferenc Kovács, Andreas-David Brunner, Ede Migh, Lisa Schweizer, Alberto Santos, Michael Bzorek, Soraya Naimy, Lise Mette Rahbek-Gjerdrum, Beatrice Dyring-Andersen, Jutta Bulkescher, Claudia Lukas, Mark Adam Eckert, Ernst Lengyel, Christian Gnann, Emma Lundberg, Peter Horvath, Matthias Mann.
      Despite the availabilty of imaging-based and mass-spectrometry-based methods for spatial proteomics, a key challenge remains connecting images with single-cell-resolution protein abundance measurements. Here, we introduce Deep Visual Proteomics (DVP), which combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultra-high-sensitivity mass spectrometry. DVP links protein abundance to complex cellular or subcellular phenotypes while preserving spatial context. By individually excising nuclei from cell culture, we classified distinct cell states with proteomic profiles defined by known and uncharacterized proteins. In an archived primary melanoma tissue, DVP identified spatially resolved proteome changes as normal melanocytes transition to fully invasive melanoma, revealing pathways that change in a spatial manner as cancer progresses, such as mRNA splicing dysregulation in metastatic vertical growth that coincides with reduced interferon signaling and antigen presentation. The ability of DVP to retain precise spatial proteomic information in the tissue context has implications for the molecular profiling of clinical samples.
    DOI:  https://doi.org/10.1038/s41587-022-01302-5
  14. Nat Rev Mol Cell Biol. 2022 May 19.
    Phosphoinositides as membrane organizers.
    York Posor, Wonyul Jang, Volker Haucke.
      Phosphoinositides are signalling lipids derived from phosphatidylinositol, a ubiquitous phospholipid in the cytoplasmic leaflet of eukaryotic membranes. Initially discovered for their roles in cell signalling, phosphoinositides are now widely recognized as key integrators of membrane dynamics that broadly impact on all aspects of cell physiology and on disease. The past decade has witnessed a vast expansion of our knowledge of phosphoinositide biology. On the endocytic and exocytic routes, phosphoinositides direct the inward and outward flow of membrane as vesicular traffic is coupled to the conversion of phosphoinositides. Moreover, recent findings on the roles of phosphoinositides in autophagy and the endolysosomal system challenge our view of lysosome biology. The non-vesicular exchange of lipids, ions and metabolites at membrane contact sites in between organelles has also been found to depend on phosphoinositides. Here we review our current understanding of how phosphoinositides shape and direct membrane dynamics to impact on cell physiology, and provide an overview of emerging concepts in phosphoinositide regulation.
    DOI:  https://doi.org/10.1038/s41580-022-00490-x
  15. Nat Commun. 2022 May 18. 13(1): 2725
    Visual barcodes for clonal-multiplexing of live microscopy-based assays.
    Tom Kaufman, Erez Nitzan, Nir Firestein, Miriam Bracha Ginzberg, Seshu Iyengar, Nish Patel, Rotem Ben-Hamo, Ziv Porat, Jaryd Hunter, Andreas Hilfinger, Varda Rotter, Ran Kafri, Ravid Straussman.
      While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins that can be deconvoluted using common microscopy equipment. To address this limitation, we develop visual barcodes that discriminate the clonal identity of single cells by different fluorescent proteins that are targeted to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then use visual barcodes to generate 'Signalome' cell-lines by mixing 12 clones of different live reporters into a single population, allowing simultaneous monitoring of the activity in 12 branches of signaling, at clonal resolution, over time. Using the 'Signalome' we identify two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological systems.
    DOI:  https://doi.org/10.1038/s41467-022-30008-0
  16. PLoS Biol. 2022 May 16. 20(5): e3001636
    The structural context of posttranslational modifications at a proteome-wide scale.
    Isabell Bludau, Sander Willems, Wen-Feng Zeng, Maximilian T Strauss, Fynn M Hansen, Maria C Tanzer, Ozge Karayel, Brenda A Schulman, Matthias Mann.
      The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.
    DOI:  https://doi.org/10.1371/journal.pbio.3001636
  17. Nat Chem Biol. 2022 May 19.
    Reversible lysine-targeted probes reveal residence time-based kinase selectivity.
    Tangpo Yang, Adolfo Cuesta, Xiaobo Wan, Gregory B Craven, Brad Hirakawa, Penney Khamphavong, Jeffrey R May, John C Kath, John D Lapek, Sherry Niessen, Alma L Burlingame, Jordan D Carelli, Jack Taunton.
      The expansion of the target landscape of covalent inhibitors requires the engagement of nucleophiles beyond cysteine. Although the conserved catalytic lysine in protein kinases is an attractive candidate for a covalent approach, selectivity remains an obvious challenge. Moreover, few covalent inhibitors have been shown to engage the kinase catalytic lysine in animals. We hypothesized that reversible, lysine-targeted inhibitors could provide sustained kinase engagement in vivo, with selectivity driven in part by differences in residence time. By strategically linking benzaldehydes to a promiscuous kinase binding scaffold, we developed chemoproteomic probes that reversibly and covalently engage >200 protein kinases in cells and mice. Probe-kinase residence time was dramatically enhanced by a hydroxyl group ortho to the aldehyde. Remarkably, only a few kinases, including Aurora A, showed sustained, quasi-irreversible occupancy in vivo, the structural basis for which was revealed by X-ray crystallography. We anticipate broad application of salicylaldehyde-based probes to proteins that lack a druggable cysteine.
    DOI:  https://doi.org/10.1038/s41589-022-01019-1
  18. Science. 2022 May 20. 376(6595): 880-884
    Universal antigen encoding of T cell activation from high-dimensional cytokine dynamics.
    Sooraj R Achar, François X P Bourassa, Thomas J Rademaker, Angela Lee, Taisuke Kondo, Emanuel Salazar-Cavazos, John S Davies, Naomi Taylor, Paul François, Grégoire Altan-Bonnet.
      Systems immunology lacks a framework with which to derive theoretical understanding from high-dimensional datasets. We combined a robotic platform with machine learning to experimentally measure and theoretically model CD8+ T cell activation. High-dimensional cytokine dynamics could be compressed onto a low-dimensional latent space in an antigen-specific manner (so-called "antigen encoding"). We used antigen encoding to model and reconstruct patterns of T cell immune activation. The model delineated six classes of antigens eliciting distinct T cell responses. We generalized antigen encoding to multiple immune settings, including drug perturbations and activation of chimeric antigen receptor T cells. Such universal antigen encoding for T cell activation may enable further modeling of immune responses and their rational manipulation to optimize immunotherapies.
    DOI:  https://doi.org/10.1126/science.abl5311
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