bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2024–06–30
twenty-two papers selected by
Ralitsa Radostinova Madsen, MRC-PPU
  1. Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.
  2. Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines.
  3. Myeloid-derived miR-6236 potentiates adipocyte insulin signaling and prevents hyperglycemia during obesity.
  4. Spatiotemporal distribution of PTEN before directed cell migration in monolayers.
  5. protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.
  6. An intermediate Rb-E2F activity state safeguards proliferation commitment.
  7. Functional characterization of PI3K C2 domain mutations detected in breast cancer circulating tumor cells and metastatic cells.
  8. Isoform and pathway-specific regulation of post-transcriptional RNA processing in human cells.
  9. Prioritizing drug targets by perturbing biological network response functions.
  10. cProSite: A web based interactive platform for online proteomics, phosphoproteomics, and genomics data analysis.
  11. ConfluentFUCCI for fully-automated analysis of cell-cycle progression in a highly dense collective of migrating cells.
  12. Multi Omics Applications in Biological Systems.
  13. Systematic mapping of organism-scale gene-regulatory networks in aging using population asynchrony.
  14. RAS-activated PI3K/AKT signaling sustains cellular senescence via P53/P21 axis in experimental models of psoriasis.
  15. A guide to genetically-encoded redox biosensors: state of the art and opportunities.
  16. Pan-cancer proteogenomics expands the landscape of therapeutic targets.
  17. An Update on Physiopathological Roles of Akt in the ReprodAKTive Mammalian Ovary.
  18. Tissue-specific and endogenous protein labeling with split fluorescent proteins.
  19. Automated neuronal reconstruction with super-multicolour Tetbow labelling and threshold-based clustering of colour hues.
  20. Cholesterol inhibits assembly and activation of the EphA2 receptor.
  21. Determining how many cells to average for statistical testing of microscopy experiments.
  22. Multiplexed single-cell characterization of alternative polyadenylation regulators.
  1. Cells. 2024 Jun 19. pii: 1061. [Epub ahead of print]13(12):
    Down-Regulation of AKT Proteins Slows the Growth of Mutant-KRAS Pancreatic Tumors.
    Chuankai Chen, Ya-Ping Jiang, Inchul You, Nathanael S Gray, Richard Z Lin.
      Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinomas (PDACs) harbor activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilized proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. The PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, the inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. The concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions, and the IGF-1 growth stimulation effect was AKT-dependent. The RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth, and the pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.
    Keywords:  AKT; IGF-1; cholesterol metabolism; pancreatic cancer
    DOI:  https://doi.org/10.3390/cells13121061
  2. bioRxiv. 2024 Jun 14. pii: 2024.06.12.598691. [Epub ahead of print]
    Combining quantitative proteomics and interactomics for a deeper insight into molecular differences between human cell lines.
    Anna A Bakhtina, Helisa H Wippel, Juan D Chavez, James E Bruce.
      Cellular functional pathways have evolved through selection based on fitness benefits conferred through protein intra- and inter-molecular interactions that comprise all protein conformational features and protein-protein interactions, collectively referred to as the interactome. While the interactome is regulated by proteome levels, it is also regulated independently by, post translational modification, co-factor, and ligand levels, as well as local protein environmental factors, such as osmolyte concentration, pH, ionic strength, temperature and others. In modern biomedical research, cultivatable cell lines have become an indispensable tool, with selection of optimal cell lines that exhibit specific functional profiles being critical for success in many cases. While it is clear that cell lines derived from different cell types have differential proteome levels, increased understanding of large-scale functional differences requires additional information beyond abundance level measurements, including how protein conformations and interactions are altered in certain cell types to shape functional landscapes. Here, we employed quantitative in vivo protein cross-linking coupled to mass spectrometry to probe large-scale protein conformational and interaction changes among three commonly employed human cell lines, HEK293, MCF-7, and HeLa cells. Isobaric quantitative Protein Interaction Reporter (iqPIR) technologies were used to obtain quantitative values of cross-linked peptides across three cell lines. These data illustrated highly reproducible (R 2 values larger than 0.8 for all biological replicates) quantitative interactome levels across multiple biological replicates. We also measured protein abundance levels in these cells using data independent acquisition quantitative proteomics methods. Combining quantitative interactome and proteomics information allowed visualization of cell type- specific interactome changes mediated by proteome level adaptations as well as independently regulated interactome changes to gain deeper insight into possible drivers of these changes. Among the biggest detected alterations in protein interactions and conformations are changes in cytoskeletal proteins, RNA-binding proteins, chromatin remodeling complexes, mitochondrial proteins, and others. Overall, these data demonstrate the utility and reproducibility of quantitative cross-linking to study systems-level interactome variations. Moreover, these results illustrate how combined quantitative interactomics and proteomics can provide unique insight on cellular functional landscapes.
    GRAPHICAL ABSTRACT:
    DOI:  https://doi.org/10.1101/2024.06.12.598691
  3. Nat Commun. 2024 Jun 25. 15(1): 5394
    Myeloid-derived miR-6236 potentiates adipocyte insulin signaling and prevents hyperglycemia during obesity.
    Bam D Paneru, Julia Chini, Sam J McCright, Nicole DeMarco, Jessica Miller, Leonel D Joannas, Jorge Henao-Mejia, Paul M Titchenell, David M Merrick, Hee-Woong Lim, Mitchell A Lazar, David A Hill.
      Adipose tissue macrophages (ATMs) influence obesity-associated metabolic dysfunction, but the mechanisms by which they do so are not well understood. We show that miR-6236 is a bona fide miRNA that is secreted by ATMs during obesity. Global or myeloid cell-specific deletion of miR-6236 aggravates obesity-associated adipose tissue insulin resistance, hyperglycemia, hyperinsulinemia, and hyperlipidemia. miR-6236 augments adipocyte insulin sensitivity by inhibiting translation of negative regulators of insulin signaling, including PTEN. The human genome harbors a miR-6236 homolog that is highly expressed in the serum and adipose tissue of obese people. hsa-MIR-6236 expression negatively correlates with hyperglycemia and glucose intolerance, and positively correlates with insulin sensitivity. Together, our findings establish miR-6236 as an ATM-secreted miRNA that potentiates adipocyte insulin signaling and protects against metabolic dysfunction during obesity.
    DOI:  https://doi.org/10.1038/s41467-024-49632-z
  4. In Vitro Cell Dev Biol Anim. 2024 Jun 26.
    Spatiotemporal distribution of PTEN before directed cell migration in monolayers.
    Quanzhi Lu, Saori Sasaki, Toshihiro Sera, Susumu Kudo.
      The intracellular distribution of phosphatase and tensin homolog (PTEN) is closely related to directed cell migration. In single cells, PTEN accumulates at the rear of the cell before and during directed migration; however, the spatiotemporal distribution of PTEN in confluent cell monolayers, particularly before directed migration, remains unclear. In this study, we wounded a cell in confluent fetal rat skin keratinocytes (FRSKs) and examined the dynamics of PTEN in the cells adjacent to the wounded cell. In contrast to single-cell migration, we found that PTEN translocated to the nucleus before the beginning of directed migration. This nuclear translocation of PTEN did not occur in disconnected cells, and it was also suppressed by importin-β inhibitor and actin inhibitor. When the nuclear localization of PTEN was inhibited by an importin-β inhibitor, cell elongation in the direction of migration was also significantly inhibited. Our results indicate that PTEN translocation is induced by the disruption of cell-cell adhesion and requires the involvement of importin-β and actin cytoskeleton signaling. In addition, phosphatidylinositol 3,4,5-triphosphate (PIP3) may regulate PTEN distribution through its localized accumulation at the cell edge. Our findings suggest that the translocation of PTEN is crucial for directed cell migration and for responding to mechanical environmental changes in confluent cell monolayers.
    Keywords:  Cell migration; Phosphatase and tensin homolog; Phosphatidylinositol 3,4,5-triphosphate; Translocation; Wound
    DOI:  https://doi.org/10.1007/s11626-024-00927-x
  5. Nucleic Acids Res. 2024 Jun 26. pii: gkae553. [Epub ahead of print]
    protoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.
    Duo Peng, Madhuri Vangipuram, Joan Wong, Manuel D Leonetti.
      CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.
    DOI:  https://doi.org/10.1093/nar/gkae553
  6. Nature. 2024 Jun 26.
    An intermediate Rb-E2F activity state safeguards proliferation commitment.
    Yumi Konagaya, David Rosenthal, Nalin Ratnayeke, Yilin Fan, Tobias Meyer.
      Tissue repair, immune defence and cancer progression rely on a vital cellular decision between quiescence and proliferation1,2. Mammalian cells proliferate by triggering a positive feedback mechanism3,4. The transcription factor E2F activates cyclin-dependent kinase 2 (CDK2), which in turn phosphorylates and inactivates the E2F inhibitor protein retinoblastoma (Rb). This action further increases E2F activity to express genes needed for proliferation. Given that positive feedback can inadvertently amplify small signals, understanding how cells keep this positive feedback in check remains a puzzle. Here we measured E2F and CDK2 signal changes in single cells and found that the positive feedback mechanism engages only late in G1 phase. Cells spend variable and often extended times in a reversible state of intermediate E2F activity before committing to proliferate. This intermediate E2F activity is proportional to the amount of phosphorylation of a conserved T373 residue in Rb that is mediated by CDK2 or CDK4/CDK6. Such T373-phosphorylated Rb remains bound on chromatin but dissociates from it once Rb is hyperphosphorylated at many sites, which fully activates E2F. The preferential initial phosphorylation of T373 can be explained by its relatively slower rate of dephosphorylation. Together, our study identifies a primed state of intermediate E2F activation whereby cells sense external and internal signals and decide whether to reverse and exit to quiescence or trigger the positive feedback mechanism that initiates cell proliferation.
    DOI:  https://doi.org/10.1038/s41586-024-07554-2
  7. Cell Signal. 2024 Jun 21. pii: S0898-6568(24)00238-9. [Epub ahead of print] 111270
    Functional characterization of PI3K C2 domain mutations detected in breast cancer circulating tumor cells and metastatic cells.
    Daniel J Smit, Helena Brauer, Stefan Horn, Gökhan Yigit, Marie-Therese Haider, Vivian Pogenberg, Udo Schumacher, Klaus Pantel, Manfred Jücker.
       BACKGROUND: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor.
    METHODS: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging.
    RESULTS: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction.
    CONCLUSIONS: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.
    Keywords:  Breast cancer; C2 domain; Circulating tumor cells; PI3K
    DOI:  https://doi.org/10.1016/j.cellsig.2024.111270
  8. bioRxiv. 2024 Jun 12. pii: 2024.06.12.598705. [Epub ahead of print]
    Isoform and pathway-specific regulation of post-transcriptional RNA processing in human cells.
    Karan Bedi, Brian Magnuson, Ishwarya Venkata Narayanan, Ariel McShane, Mario Ashaka, Michelle T Paulsen, Thomas E Wilson, Mats Ljungman.
      Steady-state levels of RNA transcripts are controlled by their rates of synthesis and degradation. Here we used nascent RNA Bru-seq and BruChase-seq to profile RNA dynamics across 16 human cell lines as part of ENCODE4 Deeply Profiled Cell Lines collection. We show that RNA turnover dynamics differ widely between transcripts of different genes and between different classes of RNA. Gene set enrichment analysis (GSEA) revealed that transcripts encoding proteins belonging to the same pathway often show similar turnover dynamics. Furthermore, transcript isoforms show distinct dynamics suggesting that RNA turnover is important in regulating mRNA isoform choice. Finally, splicing across newly made transcripts appears to be cooperative with either all or none type splicing. These data sets generated as part of ENCODE4 illustrate the intricate and coordinated regulation of RNA dynamics in controlling gene expression to allow for the precise coordination of cellular functions.
    DOI:  https://doi.org/10.1101/2024.06.12.598705
  9. PLoS Comput Biol. 2024 Jun 27. 20(6): e1012195
    Prioritizing drug targets by perturbing biological network response functions.
    Matthew C Perrone, Michael G Lerner, Matthew Dunworth, Andrew J Ewald, Joel S Bader.
      Therapeutic interventions are designed to perturb the function of a biological system. However, there are many types of proteins that cannot be targeted with conventional small molecule drugs. Accordingly, many identified gene-regulatory drivers and downstream effectors are currently undruggable. Drivers and effectors are often connected by druggable signaling and regulatory intermediates. Methods to identify druggable intermediates therefore have general value in expanding the set of targets available for hypothesis-driven validation. Here we identify and prioritize potential druggable intermediates by developing a network perturbation theory, termed NetPert, for response functions of biological networks. Dynamics are defined by a network structure in which vertices represent genes and proteins, and edges represent gene-regulatory interactions and protein-protein interactions. Perturbation theory for network dynamics prioritizes targets that interfere with signaling from driver to response genes. Applications to organoid models for metastatic breast cancer demonstrate the ability of this mathematical framework to identify and prioritize druggable intermediates. While the short-time limit of the perturbation theory resembles betweenness centrality, NetPert is superior in generating target rankings that correlate with previous wet-lab assays and are more robust to incomplete or noisy network data. NetPert also performs better than a related graph diffusion approach. Wet-lab assays demonstrate that drugs for targets identified by NetPert, including targets that are not themselves differentially expressed, are active in suppressing additional metastatic phenotypes.
    DOI:  https://doi.org/10.1371/journal.pcbi.1012195
  10. J Biotechnol Biomed. 2023 ;6(4): 573-578
    cProSite: A web based interactive platform for online proteomics, phosphoproteomics, and genomics data analysis.
    Dunrui Wang, Xiaolan Qian, Yi-Chieh Nancy Du, Beatriz Sanchez-Solana, Kailing Chen, Madhu Kanigicherla, Lisa M Jenkins, Jason Luo, Sarah Eng, Brian Park, Ben Chen, Xiaozheng Yao, Thuong Nguyen, Brajendra K Tripathi, Marian E Durkin, Douglas R Lowy.
      We developed cProSite, a website that provides online genomics, proteomics, and phosphoproteomics analysis for the data of The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC). This tool focuses on comparisons and correlations between different proteins and mRNAs of tumors and normal tissues. Our website is designed with biologists and clinicians in mind, with a user-friendly environment and fast search engine. The search results of cProSite can be used for clinical data validation and provide useful strategic information to identify drug targets at proteomic, phosphoproteomic, or genomic levels. The site is available at http://cprosite.ccr.cancer.gov.
    DOI:  https://doi.org/10.26502/jbb.2642-91280119
  11. PLoS One. 2024 ;19(6): e0305491
    ConfluentFUCCI for fully-automated analysis of cell-cycle progression in a highly dense collective of migrating cells.
    Leo Goldstien, Yael Lavi, Lior Atia.
      Understanding mechanisms underlying various physiological and pathological processes often requires accurate and fully automated analysis of dense cell populations that collectively migrate. In such multicellular systems, there is a rising interest in the relations between biophysical and cell cycle progression aspects. A seminal tool that led to a leap in real-time study of cell cycle is the fluorescent ubiquitination-based cell cycle indicator (FUCCI). Here, we introduce ConfluentFUCCI, an open-source graphical user interface-based framework that is designed, unlike previous tools, for fully automated analysis of cell cycle progression, cellular dynamics, and cellular morphology, in highly dense migrating cell collectives. We integrated into ConfluentFUCCI's pipeline state-of-the-art tools such as Cellpose, TrackMate, and Napari, some of which incorporate deep learning, and we wrap the entire tool into an isolated computational environment termed container. This provides an easy installation and workflow that is independent of any specific operation system. ConfluentFUCCI offers accurate nuclear segmentation and tracking using FUCCI tags, enabling comprehensive investigation of cell cycle progression at both the tissue and single-cell levels. We compare ConfluentFUCCI to the most recent relevant tool, showcasing its accuracy and efficiency in handling large datasets. Furthermore, we demonstrate the ability of ConfluentFUCCI to monitor cell cycle transitions, dynamics, and morphology within densely packed epithelial cell populations, enabling insights into mechanotransductive regulation of cell cycle progression. The presented tool provides a robust approach for investigating cell cycle-related phenomena in complex biological systems, offering potential applications in cancer research and other fields.
    DOI:  https://doi.org/10.1371/journal.pone.0305491
  12. Curr Issues Mol Biol. 2024 Jun 11. 46(6): 5777-5793
    Multi Omics Applications in Biological Systems.
    Cristian D Gutierrez Reyes, Gerardo Alejo-Jacuinde, Benjamin Perez Sanchez, Jesus Chavez Reyes, Sherifdeen Onigbinde, Damir Mogut, Irma Hernández-Jasso, Denisse Calderón-Vallejo, J Luis Quintanar, Yehia Mechref.
      Traditional methodologies often fall short in addressing the complexity of biological systems. In this regard, system biology omics have brought invaluable tools for conducting comprehensive analysis. Current sequencing capabilities have revolutionized genetics and genomics studies, as well as the characterization of transcriptional profiling and dynamics of several species and sample types. Biological systems experience complex biochemical processes involving thousands of molecules. These processes occur at different levels that can be studied using mass spectrometry-based (MS-based) analysis, enabling high-throughput proteomics, glycoproteomics, glycomics, metabolomics, and lipidomics analysis. Here, we present the most up-to-date techniques utilized in the completion of omics analysis. Additionally, we include some interesting examples of the applicability of multi omics to a variety of biological systems.
    Keywords:  foodomics; genomics; glycomics; glycoproteomics; lipidomics; metabolomics; omics; proteomics; systems biology; transcriptomics
    DOI:  https://doi.org/10.3390/cimb46060345
  13. Cell. 2024 Jun 21. pii: S0092-8674(24)00594-4. [Epub ahead of print]
    Systematic mapping of organism-scale gene-regulatory networks in aging using population asynchrony.
    Matthias Eder, Olivier M F Martin, Natasha Oswal, Lucia Sedlackova, Cátia Moutinho, Andrea Del Carmen-Fabregat, Simon Menendez Bravo, Arnau Sebé-Pedrós, Holger Heyn, Nicholas Stroustrup.
      In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.
    Keywords:  Caenorhabditis elegans; aging; complex systems; computational biology; gene regulation; individual variation; non-genetic individuality; quantitative biology; statistical modeling
    DOI:  https://doi.org/10.1016/j.cell.2024.05.050
  14. J Dermatol Sci. 2024 Mar 07. pii: S0923-1811(24)00034-3. [Epub ahead of print]
    RAS-activated PI3K/AKT signaling sustains cellular senescence via P53/P21 axis in experimental models of psoriasis.
    Laura Mercurio, Jacob Bailey, Adam Bleier Glick, Elena Dellambra, Claudia Scarponi, Sabatino Pallotta, Cristina Albanesi, Stefania Madonna.
       BACKGROUND: Psoriasis is a chronic immune-mediated skin disease in which upper epidermal keratinocytes exhibit a senescent-like phenotype. In psoriatic skin, a variety of inflammatory cytokines can activate intracellular pathways including phosphatidylinositol 3-kinase (PI3K)/AKT signaling and RAS effectors. AKT and RAS participate to cellular senescence, but currently their role in senescence responses occurring in psoriasis have not yet been investigated.
    OBJECTIVE: The role of AKT molecular axis and RAS activation was evaluated in the context of cellular senescence in psoriasis disease.
    METHODS: RAS/AKT involvement in senescence was analyzed in psoriatic keratinocytes cultures subjected to multiple passages to promote senescence in vitro, as well as in skin lesions of patients affected by psoriasis. The impact of pharmacological inhibition of PI3K/AKT pathway on senescence and inflammation responses was tested in senescent psoriatic keratinocytes and in a psoriasiform dermatitis murine model induced by RAS overexpression in the upper epidermis of mice.
    RESULTS: We found AKT hyperactivation associated to the upregulation of senescence markers, in senescent psoriatic keratinocyte cultures, as well as in skin lesions of psoriatic patients. AKT-induced senescence was sustained by constitutive RAS activation, and down-stream responses were mediated by P53/P21 axis. PI3K/AKT inhibition contrasted senescence processes induced by cytokines in psoriatic keratinocytes. Additionally, RAS-induced psoriasis-like dermatitis in mice was accompanied by AKT upregulation, increase of senescence marker expression and by skin inflammation. In this model, both senescence and inflammation were significantly reduced by selective AKT inhibition.
    CONCLUSION: Therefore, targeting RAS-AKT pathway could be a promising novel strategy to counteract multiple psoriasis symptoms.
    Keywords:  AKT; Keratinocytes; Psoriasis; RAS; Senescence
    DOI:  https://doi.org/10.1016/j.jdermsci.2024.03.002
  15. Arch Biochem Biophys. 2024 Jun 20. pii: S0003-9861(24)00188-7. [Epub ahead of print] 110067
    A guide to genetically-encoded redox biosensors: state of the art and opportunities.
    Brandán Pedre.
      Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.
    Keywords:  FAST; FRET; Fluorescent protein; HaloTag; NADH; NADPH; UnaG; fluorescence anisotropy; fluorescence lifetime imaging microscopy; genetically-encoded biosensors; glutaredoxin; glutathione; hydrogen peroxide; roGFP2; thioredoxins
    DOI:  https://doi.org/10.1016/j.abb.2024.110067
  16. Cell. 2024 Jun 17. pii: S0092-8674(24)00583-X. [Epub ahead of print]
    Pan-cancer proteogenomics expands the landscape of therapeutic targets.
    Sara R Savage, Xinpei Yi, Jonathan T Lei, Bo Wen, Hongwei Zhao, Yuxing Liao, Eric J Jaehnig, Lauren K Somes, Paul W Shafer, Tobie D Lee, Zile Fu, Yongchao Dou, Zhiao Shi, Daming Gao, Valentina Hoyos, Qiang Gao, Bing Zhang.
      Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.
    Keywords:  pan-cancer, proteogenomics, drug targets, proteomics, synthetic lethality, neoantigens, tumor antigens, data integration
    DOI:  https://doi.org/10.1016/j.cell.2024.05.039
  17. Life (Basel). 2024 Jun 02. pii: 722. [Epub ahead of print]14(6):
    An Update on Physiopathological Roles of Akt in the ReprodAKTive Mammalian Ovary.
    Carlo Giaccari, Sevastiani Antonouli, George Anifandis, Sandra Cecconi, Valentina Di Nisio.
      The phosphoinositide 3-kinase (PI3K)/Akt pathway is a key signaling cascade responsible for the regulation of cell survival, proliferation, and metabolism in the ovarian microenvironment. The optimal finetuning of this pathway is essential for physiological processes concerning oogenesis, folliculogenesis, oocyte maturation, and embryo development. The dysregulation of PI3K/Akt can impair molecular and structural mechanisms that will lead to follicle atresia, or the inability of embryos to reach later stages of development. Due to its pivotal role in the control of cell proliferation, apoptosis, and survival mechanisms, the dysregulation of this molecular pathway can trigger the onset of pathological conditions. Among these, we will focus on diseases that can harm female fertility, such as polycystic ovary syndrome and premature ovarian failure, or women's general health, such as ovarian cancer. In this review, we report the functions of the PI3K/Akt pathway in both its physiological and pathological roles, and we address the existing application of inhibitors and activators for the balancing of the molecular cascade in ovarian pathological environments.
    Keywords:  PCOS; PI3K/Akt pathway; POF; folliculogenesis; ovarian cancer; ovary
    DOI:  https://doi.org/10.3390/life14060722
  18. Dev Biol. 2024 Jun 21. pii: S0012-1606(24)00161-1. [Epub ahead of print]514 109-116
    Tissue-specific and endogenous protein labeling with split fluorescent proteins.
    Gloria D Ligunas, German F Paniagua, Jesselynn LaBelle, Adela Ramos-Martinez, Kyle Shen, Emma H Gerlt, Kaddy Aguilar, Ngoc Nguyen, Stefan C Materna, Stephanie Woo.
      The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.
    Keywords:  CRISPR-Cas; Protein tagging; Split fluorescent protein; Zebrafish
    DOI:  https://doi.org/10.1016/j.ydbio.2024.06.011
  19. Nat Commun. 2024 Jun 25. 15(1): 5279
    Automated neuronal reconstruction with super-multicolour Tetbow labelling and threshold-based clustering of colour hues.
    Marcus N Leiwe, Satoshi Fujimoto, Toshikazu Baba, Daichi Moriyasu, Biswanath Saha, Richi Sakaguchi, Shigenori Inagaki, Takeshi Imai.
      Fluorescence imaging is widely used for the mesoscopic mapping of neuronal connectivity. However, neurite reconstruction is challenging, especially when neurons are densely labelled. Here, we report a strategy for the fully automated reconstruction of densely labelled neuronal circuits. Firstly, we establish stochastic super-multicolour labelling with up to seven different fluorescent proteins using the Tetbow method. With this method, each neuron is labelled with a unique combination of fluorescent proteins, which are then imaged and separated by linear unmixing. We also establish an automated neurite reconstruction pipeline based on the quantitative analysis of multiple dyes (QDyeFinder), which identifies neurite fragments with similar colour combinations. To classify colour combinations, we develop unsupervised clustering algorithm, dCrawler, in which data points in multi-dimensional space are clustered based on a given threshold distance. Our strategy allows the reconstruction of neurites for up to hundreds of neurons at the millimetre scale without using their physical continuity.
    DOI:  https://doi.org/10.1038/s41467-024-49455-y
  20. bioRxiv. 2024 Jun 10. pii: 2024.06.10.598255. [Epub ahead of print]
    Cholesterol inhibits assembly and activation of the EphA2 receptor.
    Ryan J Schuck, Alyssa E Ward, Amita R Sahoo, Jennifer A Rybak, Robert J Pyron, Thomas N Trybala, Timothy B Simmons, Joshua A Baccile, Ioannis Sgouralis, Matthias Buck, Rajan Lamichhane, Francisco N Barrera.
      The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.
    DOI:  https://doi.org/10.1101/2024.06.10.598255
  21. J Cell Biol. 2024 Aug 05. pii: e202401074. [Epub ahead of print]223(8):
    Determining how many cells to average for statistical testing of microscopy experiments.
    Adam Zweifach.
      From a statistical standpoint, individual cells are typically not independent experimental replicates. To test for differences in mean, cells from each experimental sample can be averaged and each sample's average treated as an n of 1. Here, I outline how to determine how many cells to average per sample.
    DOI:  https://doi.org/10.1083/jcb.202401074
  22. Cell. 2024 Jun 20. pii: S0092-8674(24)00645-7. [Epub ahead of print]
    Multiplexed single-cell characterization of alternative polyadenylation regulators.
    Madeline H Kowalski, Hans-Hermann Wessels, Johannes Linder, Carol Dalgarno, Isabella Mascio, Saket Choudhary, Austin Hartman, Yuhan Hao, Anshul Kundaje, Rahul Satija.
      Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.
    Keywords:  Perturb-seq; RNA processing; alternative polyadenylation; cleavage and polyadenylation; post-transcriptional regulation
    DOI:  https://doi.org/10.1016/j.cell.2024.06.005
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