bims-cagime Biomed News
on Cancer, aging and metabolism
Issue of 2024‒06‒02
forty-two papers selected by
Kıvanç Görgülü, Technical University of Munich



  1. Contact (Thousand Oaks). 2024 Jan-Dec;7:7 25152564241255782
      One means by which cells reutilize neutral lipids stored in lipid droplets is to degrade them by autophagy. This process involves spartin, mutations of which cause the rare inherited disorder Troyer syndrome (or spastic paraplegia-20, SPG20). A recently published paper from the team led by Karin Reinsich (Yale) suggests that the molecular function of spartin and its unique highly conserved "senescence" domain is as a lipid transfer protein. Spartin binds to and transfers all lipid species found in lipid droplets, from phospholipids to triglycerides and sterol esters. This lipid transfer activity correlates with spartin's ability to sustain lipid droplet turnover. The senescence domain poses an intriguing question around the wide range of its cargoes, but intriguingly it has yet to yield up its secrets because attempts at crystallization failed and AlphaFold's prediction is unconvincing.
    Keywords:  autophagy; lipophagy; lysophagy; membrane contact sites; spastin
    DOI:  https://doi.org/10.1177/25152564241255782
  2. Cell Death Differ. 2024 May 28.
      Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is one of the deadliest forms of cancer with limited therapy options. Overexpression of the heat shock protein 70 (HSP70) is a hallmark of cancer that is strongly associated with aggressive disease and worse clinical outcomes. However, the underlying mechanisms by which HSP70 allows tumor cells to thrive under conditions of continuous stress have not been fully described. Here, we report that PDAC has the highest expression of HSP70 relative to normal tissue across all cancers analyzed. Furthermore, HSP70 expression is associated with tumor grade and is further enhanced in metastatic PDAC. We show that genetic or therapeutic ablation of HSP70 alters mitochondrial subcellular localization, impairs mitochondrial dynamics, and promotes mitochondrial swelling to induce apoptosis. Mechanistically, we find that targeting HSP70 suppresses the PTEN-induced kinase 1 (PINK1) mediated phosphorylation of dynamin-related protein 1 (DRP1). Treatment with the HSP70 inhibitor AP-4-139B was efficacious as a single agent in primary and metastatic mouse models of PDAC. In addition, we demonstrate that HSP70 inhibition promotes the AMP-activated protein kinase (AMPK) mediated phosphorylation of Beclin-1, a key regulator of autophagic flux. Accordingly, we find that the autophagy inhibitor hydroxychloroquine (HCQ) enhances the ability of AP-4-139B to mediate anti-tumor activity in vivo. Collectively, our results suggest that HSP70 is a multi-functional driver of tumorigenesis that orchestrates mitochondrial dynamics and autophagy. Moreover, these findings support the rationale for concurrent inhibition of HSP70 and autophagy as a novel therapeutic approach for HSP70-driven PDAC.
    DOI:  https://doi.org/10.1038/s41418-024-01310-9
  3. Front Immunol. 2024 ;15 1341079
      Despite the efforts, pancreatic ductal adenocarcinoma (PDAC) is still highly lethal. Therapeutic challenges reside in late diagnosis and establishment of peculiar tumor microenvironment (TME) supporting tumor outgrowth. This stromal landscape is highly heterogeneous between patients and even in the same patient. The organization of functional sub-TME with different cellular compositions provides evolutive advantages and sustains therapeutic resistance. Tumor progressively establishes a TME that can suit its own needs, including proliferation, stemness and invasion. Cancer-associated fibroblasts and immune cells, the main non-neoplastic cellular TME components, follow soluble factors-mediated neoplastic instructions and synergize to promote chemoresistance and immune surveillance destruction. Unveiling heterotypic stromal-neoplastic interactions is thus pivotal to breaking this synergism and promoting the reprogramming of the TME toward an anti-tumor milieu, improving thus the efficacy of conventional and immune-based therapies. We underscore recent advances in the characterization of immune and fibroblast stromal components supporting or dampening pancreatic cancer progression, as well as novel multi-omic technologies improving the current knowledge of PDAC biology. Finally, we put into context how the clinic will translate the acquired knowledge to design new-generation clinical trials with the final aim of improving the outcome of PDAC patients.
    Keywords:  MDSC (myeloid-derived suppressor cells); PDAC - pancreatic ductal adenocarcinoma; TILs (tumor infiltrating lymphocytes); TME (tumor microenvironment); cancer associated fibroblast (CAF); immunosuppression; immunotherapy
    DOI:  https://doi.org/10.3389/fimmu.2024.1341079
  4. Dev Cell. 2024 May 24. pii: S1534-5807(24)00266-1. [Epub ahead of print]
      Protein aggregation is a hallmark of age-related neurodegeneration. Yet, aggregation during normal aging and in tissues other than the brain is poorly understood. Here, we leverage the African turquoise killifish to systematically profile protein aggregates in seven tissues of an aging vertebrate. Age-dependent aggregation is strikingly tissue specific and not simply driven by protein expression differences. Experimental interrogation in killifish and yeast, combined with machine learning, indicates that this specificity is linked to protein-autonomous biophysical features and tissue-selective alterations in protein quality control. Co-aggregation of protein quality control machinery during aging may further reduce proteostasis capacity, exacerbating aggregate burden. A segmental progeria model with accelerated aging in specific tissues exhibits selectively increased aggregation in these same tissues. Intriguingly, many age-related protein aggregates arise in wild-type proteins that, when mutated, drive human diseases. Our data chart a comprehensive landscape of protein aggregation during vertebrate aging and identify strong, tissue-specific associations with dysfunction and disease.
    Keywords:  aggregates; aging; multi-tissue; protein homeostasis; proteomics; systems biology
    DOI:  https://doi.org/10.1016/j.devcel.2024.04.014
  5. Eur J Cell Biol. 2024 May 22. pii: S0171-9335(24)00042-6. [Epub ahead of print]103(2): 151425
      The RAS-MAPK-pathway is aberrantly regulated in cancer and developmental diseases called RASopathies. While typically the impact of Ras on the proliferation of various cancer cell lines is assessed, it is poorly established how Ras affects cellular differentiation. Here we implement the C2C12 myoblast cell line to systematically study the effect of Ras mutants and Ras-pathway drugs on differentiation. We first provide evidence that a minor pool of Pax7+ progenitors replenishes a major pool of transit amplifying cells that are ready to differentiate. Our data indicate that Ras isoforms have distinct roles in the differentiating culture, where K-Ras depletion increases and H-Ras depletion decreases terminal differentiation. This assay could therefore provide significant new insights into Ras biology and Ras-driven diseases. In line with this, we found that all oncogenic Ras mutants block terminal differentiation of transit amplifying cells. By contrast, RASopathy associated K-Ras variants were less able to block differentiation. Profiling of eight targeted Ras-pathway drugs on seven oncogenic Ras mutants revealed their allele-specific activities and distinct abilities to restore normal differentiation as compared to triggering cell death. In particular, the MEK-inhibitor trametinib could broadly restore differentiation, while the mTOR-inhibitor rapamycin broadly suppressed differentiation. We expect that this quantitative assessment of the impact of Ras-pathway mutants and drugs on cellular differentiation has great potential to complement cancer cell proliferation data.
    Keywords:  Cancer; Differentiation; Flow cytometry; Inhibitors; RASopathy; Ras
    DOI:  https://doi.org/10.1016/j.ejcb.2024.151425
  6. Nat Aging. 2024 May 30.
      It has been reported that accumulation of senescent cells in various tissues contributes to pathological aging and that elimination of senescent cells (senolysis) improves age-associated pathologies. Here, we demonstrate that inhibition of sodium-glucose co-transporter 2 (SGLT2) enhances clearance of senescent cells, thereby ameliorating age-associated phenotypic changes. In a mouse model of dietary obesity, short-term treatment with the SGLT2 inhibitor canagliflozin reduced the senescence load in visceral adipose tissue and improved adipose tissue inflammation and metabolic dysfunction, but normalization of plasma glucose by insulin treatment had no effect on senescent cells. Canagliflozin extended the lifespan of mice with premature aging even when treatment was started in middle age. Metabolomic analyses revealed that short-term treatment with canagliflozin upregulated 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, enhancing immune-mediated clearance of senescent cells by downregulating expression of programmed cell death-ligand 1. These findings suggest that inhibition of SGLT2 has an indirect senolytic effect by enhancing endogenous immunosurveillance of senescent cells.
    DOI:  https://doi.org/10.1038/s43587-024-00642-y
  7. bioRxiv. 2024 May 17. pii: 2024.05.14.594197. [Epub ahead of print]
      Cancer-associated fibroblast (CAF) subpopulations in pancreatic ductal adenocarcinoma (PDAC) have been identified using single-cell RNA sequencing (scRNAseq) with divergent characteristics, but their clinical relevance remains unclear. We translate scRNAseq-derived CAF cell-subpopulation-specific marker genes to bulk RNAseq data, and develop a single- sample classifier, DeCAF, for the classification of clinically rest raining and perm issive CAF subtypes. We validate DeCAF in 19 independent bulk transcriptomic datasets across four tumor types (PDAC, mesothelioma, bladder and renal cell carcinoma). DeCAF subtypes have distinct histology features, immune landscapes, and are prognostic and predict response to therapy across cancer types. We demonstrate that DeCAF is clinically replicable and robust for the classification of CAF subtypes in patients for multiple tumor types, providing a better framework for the future development and translation of therapies against permissive CAF subtypes and preservation of restraining CAF subtypes.Significance: We introduce a replicable and robust classifier, DeCAF, that delineates the significance of the role of permissive and restraining CAF subtypes in cancer patients. DeCAF is clinically tractable, prognostic and predictive of treatment response in multiple cancer types and lays the translational groundwork for the preclinical and clinical development of CAF subtype specific therapies.
    DOI:  https://doi.org/10.1101/2024.05.14.594197
  8. Nat Commun. 2024 May 31. 15(1): 4642
      Post-translational modifications of proteins in malignant transformation and tumor maintenance of pancreatic ductal adenocarcinoma (PDAC) in the context of KRAS signaling remain poorly understood. Here, we use the KPC mouse model to examine the effect of palmitoylation on pancreatic cancer progression. ZDHHC20, upregulated by KRAS, is abnormally overexpressed and associated with poor prognosis in patients with pancreatic cancer. Dysregulation of ZDHHC20 promotes pancreatic cancer progression in a palmitoylation-dependent manner. ZDHHC20 inhibits the chaperone-mediated autophagic degradation of YTHDF3 through S-palmitoylation of Cys474, which can result in abnormal accumulation of the oncogenic product MYC and thereby promote the malignant phenotypes of cancer cells. Further, we design a biologically active YTHDF3-derived peptide to competitively inhibit YTHDF3 palmitoylation mediated by ZDHHC20, which in turn downregulates MYC expression and inhibits the progression of KRAS mutant pancreatic cancer. Thus, these findings highlight the therapeutic potential of targeting the ZDHHC20-YTHDF3-MYC signaling axis in pancreatic cancer.
    DOI:  https://doi.org/10.1038/s41467-024-49105-3
  9. Gut. 2024 May 31. pii: gutjnl-2023-331519. [Epub ahead of print]
      OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC).DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice.
    RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer.
    CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.
    Keywords:  GENE REGULATION; GENE TARGETING; IMMUNOTHERAPY; ONCOGENES; PANCREATIC CANCER
    DOI:  https://doi.org/10.1136/gutjnl-2023-331519
  10. Nat Commun. 2024 May 25. 15(1): 4469
      To facilitate inter-tissue communication and the exchange of proteins, lipoproteins, and metabolites with the circulation, hepatocytes have an intricate and efficient intracellular trafficking system regulated by small Rab GTPases. Here, we show that Rab30 is induced in the mouse liver by fasting, which is amplified in liver-specific carnitine palmitoyltransferase 2 knockout mice (Cpt2L-/-) lacking the ability to oxidize fatty acids, in a Pparα-dependent manner. Live-cell super-resolution imaging and in vivo proximity labeling demonstrates that Rab30-marked vesicles are highly dynamic and interact with proteins throughout the secretory pathway. Rab30 whole-body, liver-specific, and Rab30; Cpt2 liver-specific double knockout (DKO) mice are viable with intact Golgi ultrastructure, although Rab30 deficiency in DKO mice suppresses the serum dyslipidemia observed in Cpt2L-/- mice. Corresponding with decreased serum triglyceride and cholesterol levels, DKO mice exhibit decreased circulating but not hepatic ApoA4 protein, indicative of a trafficking defect. Together, these data suggest a role for Rab30 in the selective sorting of lipoproteins to influence hepatocyte and circulating triglyceride levels, particularly during times of excessive lipid burden.
    DOI:  https://doi.org/10.1038/s41467-024-48959-x
  11. Nature. 2024 May 29.
      Organs have a distinctive yet often overlooked spatial arrangement in the body1-5. We propose that there is a logic to the shape of an organ and its proximity to its neighbours. Here, by using volumetric scans of many Drosophila melanogaster flies, we develop methods to quantify three-dimensional features of organ shape, position and interindividual variability. We find that both the shapes of organs and their relative arrangement are consistent yet differ between the sexes, and identify unexpected interorgan adjacencies and left-right organ asymmetries. Focusing on the intestine, which traverses the entire body, we investigate how sex differences in three-dimensional organ geometry arise. The configuration of the adult intestine is only partially determined by physical constraints imposed by adjacent organs; its sex-specific shape is actively maintained by mechanochemical crosstalk between gut muscles and vascular-like trachea. Indeed, sex-biased expression of a muscle-derived fibroblast growth factor-like ligand renders trachea sexually dimorphic. In turn, tracheal branches hold gut loops together into a male or female shape, with physiological consequences. Interorgan geometry represents a previously unrecognized level of biological complexity which might enable or confine communication across organs and could help explain sex or species differences in organ function.
    DOI:  https://doi.org/10.1038/s41586-024-07463-4
  12. Cell Rep. 2024 May 29. pii: S2211-1247(24)00622-3. [Epub ahead of print]43(6): 114294
      Ubiquitination of mitochondrial proteins provides a basis for the downstream recruitment of mitophagy machinery, yet whether ubiquitination of the machinery itself contributes to mitophagy is unknown. Here, we show that K63-linked polyubiquitination of the key mitophagy regulator TBK1 is essential for its mitophagy functions. This modification is catalyzed by the ubiquitin ligase TRIM5α and is required for TBK1 to interact with and activate a set of ubiquitin-binding autophagy adaptors including NDP52, p62/SQSTM1, and NBR1. Autophagy adaptors, along with TRIM27, enable TRIM5α to engage with TBK1 following mitochondrial damage. TRIM5α's ubiquitin ligase activity is required for the accumulation of active TBK1 on damaged mitochondria in Parkin-dependent and Parkin-independent mitophagy pathways. Our data support a model in which TRIM5α provides a mitochondria-localized, ubiquitin-based, self-amplifying assembly platform for TBK1 and mitophagy adaptors that is ultimately necessary for the recruitment of the core autophagy machinery.
    Keywords:  CP: Cell biology; NBR1; NDP52; Optineurin; TAX1BP1; TBK1; TRIM27/RFP; autophagy; p62; tripartite motif; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.celrep.2024.114294
  13. J Proteome Res. 2024 May 28.
      Quantitative proteomics has enhanced our capability to study protein dynamics and their involvement in disease using various techniques, including statistical testing, to discern the significant differences between conditions. While most focus is on what is different between conditions, exploring similarities can provide valuable insights. However, exploring similarities directly from the analyte level, such as proteins, genes, or metabolites, is not a standard practice and is not widely adopted. In this study, we propose a statistical framework called QuEStVar (Quantitative Exploration of Stability and Variability through statistical hypothesis testing), enabling the exploration of quantitative stability and variability of features with a combined statistical framework. QuEStVar utilizes differential and equivalence testing to expand statistical classifications of analytes when comparing conditions. We applied our method to an extensive data set of cancer cell lines and revealed a quantitatively stable core proteome across diverse tissues and cancer subtypes. The functional analysis of this set of proteins highlighted the molecular mechanism of cancer cells to maintain constant conditions of the tumorigenic environment via biological processes, including transcription, translation, and nucleocytoplasmic transport.
    Keywords:  bioinformatics; cancer cell lines; equivalence testing; proteomics; statistics
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00131
  14. Semin Cancer Biol. 2024 May 27. pii: S1044-579X(24)00036-1. [Epub ahead of print]
      Cancer is daunting pathology with remarkable breadth and scope, spanning genetics, epigenetics, proteomics, metalobomics and cell biology. Cellular senescence represents a stress-induced and essentially irreversible cell fate associated with aging and various age-related diseases, including malignancies. Senescent cells are characterized of morphologic alterations and metabolic reprogramming, and develop a highly active secretome termed as the senescence-associated secretory phenotype (SASP). Since the first discovery, senescence has been understood as an important barrier to tumor progression, as its induction in pre-neoplastic cells limits carcinogenesis. Paradoxically, senescent cells arising in the tumor microenvironment (TME) contribute to tumor progression, including augmented therapeutic resistance. In this article, we define typical forms of senescent cells commonly observed within the TME and how senescent cells functionally remodel their surrounding niche, affect immune responses and promote cancer evolution. Furthermore, we highlight the recently emerging pipelines of senotherapies particularly senolytics, which can selectively deplete senescent cells from affected organs in vivo and impede tumor progression by restoring therapeutic responses and securing anticancer efficacies. Together, co-targeting cancer cells and their normal but senescent counterparts in the TME holds the potential to achieve increased therapeutic benefits and restrained disease relapse in future clinical oncology.
    Keywords:  senescence-associated secretory phenotype; senescent cell; senolytics; senotherapy; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.semcancer.2024.05.002
  15. Sci Adv. 2024 May 31. 10(22): eadj4370
      Tumor heterogeneity is a primary factor that contributes to treatment failure. Predictive tools, capable of classifying cancer cells based on their functions, may substantially enhance therapy and extend patient life span. The connection between cell biomechanics and cancer cell functions is used here to classify cells through mechanical measurements, via particle uptake. Machine learning (ML) was used to classify cells based on single-cell patterns of uptake of particles with diverse sizes. Three pairs of human cancer cell subpopulations, varied in their level of drug resistance or malignancy, were studied. Cells were allowed to interact with fluorescently labeled polystyrene particles ranging in size from 0.04 to 3.36 μm and analyzed for their uptake patterns using flow cytometry. ML algorithms accurately classified cancer cell subtypes with accuracy rates exceeding 95%. The uptake data were especially advantageous for morphologically similar cell subpopulations. Moreover, the uptake data were found to serve as a form of "normalization" that could reduce variation in repeated experiments.
    DOI:  https://doi.org/10.1126/sciadv.adj4370
  16. J Surg Oncol. 2024 May 27.
      BACKGROUND: The benefit of adjuvant therapy (AT) remains unclear in pancreatic ductal adenocarcinoma (PDAC) after neoadjuvant therapy (NAT) and surgical resection.METHODS: The 2019 National Cancer Database was queried for patients with non-metastatic PDAC who received NAT followed by pancreaticoduodenectomy. Only patients with data regarding receipt of AT were included. Patients were classified if they had nodal down-staging specifically, or any downstaging (Tumor, Nodal, or overall). Propensity score matching (PSM) adjusted for pretreatment covariate imbalance between groups. The weighted Kaplan-Meier method and log-rank test were used to estimate the cumulative survival.
    RESULTS: After exclusion criteria and PSM, a total of 2784 patients remained; 1689 (60.7%) received AT and 1095 (39.3%) did not receive AT. Among all, those with additional AT had a significantly improved overall survival (OS) (p < 0.001). Upon evaluation of patients without downstaging after NAT, those who received AT had improved OS (no nodal downstaging or any downstaging; p = 0.002; p = 0.001). When evaluating patients with downstaging after NAT, those receiving AT did not have improved OS (nodal downstaging or any downstaging: p = 0.352; p = 0.99).
    CONCLUSION: Response to NAT appears to correlate with the benefit of AT following pancreaticoduodenectomy; patients who have a favorable response to NAT may not benefit from AT.
    Keywords:  adjuvant therapy; chemotherapy; neoadjuvant therapy; pancreatic cancer
    DOI:  https://doi.org/10.1002/jso.27689
  17. Nat Methods. 2024 May 27.
      The dynamics of cellular membrane tension and its role in mechanosensing, which is the ability of cells to respond to physical stimuli, remain incompletely understood, mainly due to the lack of appropriate tools. Here, we report a force-controlled nanopipette-based method that combines fluidic force microscopy with fluorescence imaging for precise manipulation of the cellular membrane tension while monitoring the impact on single-cell mechanosensitivity. The force-controlled nanopipette enables control of the indentation force imposed on the cell cortex as well as of the aspiration pressure applied to the plasma membrane. We show that this setup can be used to concurrently monitor the activation of Piezo1 mechanosensitive ion channels via calcium imaging. Moreover, the spatiotemporal behavior of the tension propagation is assessed with the fluorescent membrane tension probe Flipper-TR, and further dissected using molecular dynamics modeling. Finally, we demonstrate that aspiration and indentation act independently on the cellular mechanobiological machinery, that indentation induces a local pre-tension in the membrane, and that membrane tension stays confined by links to the cytoskeleton.
    DOI:  https://doi.org/10.1038/s41592-024-02277-8
  18. bioRxiv. 2024 May 19. pii: 2024.05.14.594162. [Epub ahead of print]
      Three-dimensional (3D) optical microscopy, combined with advanced tissue clearing, permits in situ interrogation of the tumor microenvironment (TME) in large volumetric tumors for preclinical cancer research. Light sheet (also known as ultramicroscopy) and confocal fluorescence microscopy are often used to achieve macroscopic and microscopic 3D images of optically cleared tumor tissues, respectively. Although each technique offers distinct fields of view (FOVs) and spatial resolution, the combination of these two optical microscopy techniques to obtain correlative multiscale 3D images from the same tumor tissues has not yet been explored. To establish correlative multiscale 3D optical microscopy, we developed a method for optically marking defined regions of interest (ROIs) within a cleared mouse tumor by employing a UV light-activated visible dye and Z-axis position-selective UV irradiation in a light sheet microscope system. By integrating this method with subsequent tissue processing, including physical ROI marking, reversal of tissue clearing, tissue macrosectioning, and multiplex immunofluorescence, we established a workflow that enables the tracking and 3D imaging of ROIs within tumor tissues through sequential light sheet and confocal fluorescence microscopy. This approach allowed for quantitative 3D spatial analysis of the immune response in the TME of a mouse mammary tumor following cancer immunotherapy at multiple spatial scales. The workflow also facilitated the direct localization of a metastatic lesion within a whole mouse brain. These results demonstrate that our ROI tracking method and its associated workflow offer a novel approach for correlative multiscale 3D optical microscopy, with the potential to provide new insights into tumor heterogeneity, metastasis, and response to therapy at various spatial levels.
    DOI:  https://doi.org/10.1101/2024.05.14.594162
  19. Trends Biochem Sci. 2024 May 24. pii: S0968-0004(24)00112-9. [Epub ahead of print]
      Autophagy sequesters cytoplasmic portions into autophagosomes. While selective cargo is engulfed by elongation of cup-shaped isolation membranes (IMs), the morphogenesis of non-selective IMs remains elusive. Based on recent observations, we propose a novel model for autophagosome morphogenesis wherein active regulation of the IM rim serves the physiological roles of autophagy.
    Keywords:  Atg2; Atg24; DFCP1; PI3P; autophagy; phagophore
    DOI:  https://doi.org/10.1016/j.tibs.2024.05.002
  20. J Biol Chem. 2024 May 23. pii: S0021-9258(24)01910-0. [Epub ahead of print] 107409
      About 18% of all human cancers carry a mutation in the KRAS gene making it among the most sought-after anti-cancer targets. However, mutant KRas protein has proved remarkably undruggable. The recent approval of the first generation of RAS inhibitors therefore marks a seminal milestone in the history of cancer research. Inevitably though, it also raises the predictable challenges of limited drug efficacies and acquired resistance. Hence, new approaches that improve our understanding of the tumorigenic mechanisms of oncogenic RAS within more physiological settings continue to be essential. Here, we have employed the near-diploid human hTERT RPE-1 cells to generate isogenic cell lines in which one of the endogenous KRAS alleles carries an oncogenic KRAS mutation at glycine 12. Cells with a KRASG12V/+, KRASG12C/+, or KRASG12D/+ genotype, together with wild-type KRASG12G(WT)/+ cells, reveal that oncogenic KRAS.G12X mutations increase cell proliferation rate and cell motility and reduced focal adhesions in KRASG12V/+ cells. EGF-induced phosphorylation of ERK and AKT was comparable between KRASG12V/+, KRASG12C/+, KRASG12D/+, and KRASG12G(WT)/+ cells. Interestingly, KRASG12X/+ cells showed varying responses to distinct inhibitors with the KRASG12V/+ and KRASG12D/+ cells more sensitive to hydroxyurea and MEK inhibitors, U0126 and trametinib, but more resistant to PI3K inhibitor, PIK-90, than the KRASG12G(WT)/+ cells. A combination of low doses of hydroxyurea and U0126 showed an additive inhibition on growth rate that was greater in KRASG12V/+ than wild-type cells. Collectively, these cell lines will be a valuable resource for studying oncogenic RAS signalling and developing effective anti-KRAS reagents with minimum cytotoxicity on wild-type cells.
    Keywords:  Small GTPase; cell cycle; cell motility; cell proliferation; cell signalling; focal adhesion
    DOI:  https://doi.org/10.1016/j.jbc.2024.107409
  21. Nucleic Acids Res. 2024 May 25. pii: gkae423. [Epub ahead of print]
      Protein-protein interaction experiments still yield many false positive interactions. The socioaffinity metric can distinguish true protein-protein interactions from noise based on available data. Here, we present WeSA (Weighted SocioAffinity), which considers large datasets of interaction proteomics data (IntAct, BioGRID, the BioPlex) to score human protein interactions and, in a statistically robust way, flag those (even from a single experiment) that are likely to be false positives. ROC analysis (using CORUM-PDB positives and Negatome negatives) shows that WeSA improves over other measures of interaction confidence. WeSA shows consistently good results over all datasets (up to: AUC = 0.93 and at best threshold: TPR = 0.84, FPR = 0.11, Precision = 0.98). WeSA is freely available without login (wesa.russelllab.org). Users can submit their own data or look for organized information on human protein interactions using the web server. Users can either retrieve available information for a list of proteins of interest or calculate scores for new experiments. The server outputs either pre-computed or updated WeSA scores for the input enriched with information from databases. The summary is presented as a table and a network-based visualization allowing the user to remove those nodes/edges that the method considers spurious.
    DOI:  https://doi.org/10.1093/nar/gkae423
  22. JCI Insight. 2024 May 30. pii: e174329. [Epub ahead of print]
      The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
    Keywords:  Cancer; Cell biology; Cytoskeleton; Drug screens; Oncology
    DOI:  https://doi.org/10.1172/jci.insight.174329
  23. Cell Stress. 2024 ;8 56-58
      Anoikis is a common programmed death for most of detached cells, but cancer cells can obtain anoikis resistance to facilitate their distant metastasis through the circulation system. Researches have indicated that enhanced autophagic flux accounts for the survival of many cancer cells under detached conditions. Targeting ATG4B, the key factor of autophagy progress, can inhibit cancer metastasis in vitro, but ATG4B-deficient mice are susceptible to many serious diseases, which indicates the potential uncontrolled side effects of direct targeting of ATG4B. In our recent research, we confirmed that ATG4B is a novel RNA binding protein in the gastric cancer (GC) cell. It interacts with circSPECC1 which consequently facilitates the liquid-liquid phase separation and ubiquitination of ATG4B. Additionally, the m6A reader ELAVL1 inhibits the expression of circSPECC1 to enhance the expression of ATG4B and anoikis resistance of GC cells. Further, we screened out an FDA-approved compound, lopinavir, to restore circSPECC1 abundance and suppress GC metastasis. In conclusion, our research identified a novel signal pathway (ELAVL1-circSPECC1-ATG4B-autophagy) to facilitate anoikis resistance and metastasis of GC cells and screened out a compound with clinical application potential to block this pathway, providing a novel strategy for the prevention of GC metastasis.
    Keywords:  ATG4B; anoikis; autophagy; circSPECC1; liquid-liquid phase separation; lopinavir
    DOI:  https://doi.org/10.15698/cst2024.05.296
  24. J Biol Chem. 2024 May 28. pii: S0021-9258(24)01919-7. [Epub ahead of print] 107418
      ATP-citrate lyase (ACLY) links carbohydrate and lipid metabolism and provides nucleocytosolic acetyl-CoA necessary for protein acetylation. ACLY has two major splice isoforms: the full-length canonical "long" isoform and an uncharacterized "short" isoform in which exon 14 is spliced out. Exon 14 encodes 10 amino acids within a disordered region of the protein and includes at least 1 site that is dynamically phosphorylated. Both isoforms are expressed in healthy tissues to varying degrees. Analysis of human transcriptomic data revealed that the Percent Spliced In (PSI) of exon 14, i.e., the proportion of long isoform, is increased in several cancers and correlated with poorer overall survival in a pan-cancer analysis, though not in individual tumor types, which prompted us to explore potential biochemical and functional differences between ACLY isoforms. Here, we show that there are no discernible differences in enzymatic activity or stability between isoforms or phosphomutants of ACLY in vitro. Similarly, both isoforms and phosphomutants were able to rescue ACLY functions, including fatty acid synthesis and bulk histone acetylation, when re-expressed in Acly knockout cells. Deletion of Acly exon 14 in mice did not overtly impact development or metabolic physiology, nor did it attenuate tumor burden in a genetic model of intestinal cancer. Notably, expression of epithelial splicing regulatory protein 1 (ESRP1) is highly correlated with ACLY PSI. We report that ACLY splicing is regulated by ESRP1. In turn, both ESRP1 expression and ACLY PSI are correlated with specific immune signatures in tumors. Despite these intriguing patterns of ACLY splicing in healthy and cancer tissues, functional differences between the isoforms remain elusive.
    DOI:  https://doi.org/10.1016/j.jbc.2024.107418
  25. Cell Chem Biol. 2024 May 20. pii: S2451-9456(24)00179-X. [Epub ahead of print]
      Aspartate is crucial for nucleotide synthesis, ammonia detoxification, and maintaining redox balance via the malate-aspartate-shuttle (MAS). To disentangle these multiple roles of aspartate metabolism, tools are required that measure aspartate concentrations in real time and in live cells. We introduce AspSnFR, a genetically encoded green fluorescent biosensor for intracellular aspartate, engineered through displaying and screening biosensor libraries on mammalian cells. In live cells, AspSnFR is able to precisely and quantitatively measure cytosolic aspartate concentrations and dissect its production from glutamine. Combining high-content imaging of AspSnFR with pharmacological perturbations exposes differences in metabolic vulnerabilities of aspartate levels based on nutrient availability. Further, AspSnFR facilitates tracking of aspartate export from mitochondria through SLC25A12, the MAS' key transporter. We show that SLC25A12 is a rapidly responding and direct route to couple Ca2+ signaling with mitochondrial aspartate export. This establishes SLC25A12 as a crucial link between cellular signaling, mitochondrial respiration, and metabolism.
    DOI:  https://doi.org/10.1016/j.chembiol.2024.05.002
  26. Cancer Sci. 2024 May 27.
      Senescent cells promote cancer development and progression through chronic inflammation caused by a senescence-associated secretory phenotype (SASP). Although various senotherapeutic strategies targeting senescent cells have been developed for the prevention and treatment of cancers, technology for the in vivo detection and evaluation of senescent cell accumulation has not yet been established. Here, we identified activatable fluorescent probes targeting dipeptidylpeptidase-4 (DPP4) as an effective probe for detecting senescent cells through an enzymatic activity-based screening of fluorescent probes. We also determined that these probes were highly, selectively, and rapidly activated in senescent cells during live cell imaging. Furthermore, we successfully visualized senescent cells in the organs of mice using DPP4-targeted probes. These results are expected to lead to the development of a diagnostic technology for noninvasively detecting senescent cells in vivo and could play a role in the application of DPP4 prodrugs for senotherapy.
    Keywords:  DPP4 and senotherapy; fluorescent probe; imaging; senescence
    DOI:  https://doi.org/10.1111/cas.16229
  27. Mol Cell Proteomics. 2024 May 24. pii: S1535-9476(24)00081-1. [Epub ahead of print] 100791
      Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed "proteome birthdating" that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways.
    Keywords:  Aging; Mass spectrometry (MS); Protein turnover; Proteomics; Proteostasis; Ubiquitin Proteasome System (UPS)
    DOI:  https://doi.org/10.1016/j.mcpro.2024.100791
  28. Sci Adv. 2024 May 31. 10(22): eade7753
      Lipid droplets (LDs) comprise a triglyceride core surrounded by a lipid monolayer enriched with proteins, many of which function in LD homeostasis. How proteins are targeted to the growing LD is still unclear. Rab1b, a GTPase regulating secretory transport, was recently associated with targeting proteins to LDs in a Drosophila RNAi screen. LD formation was prevented in human hepatoma cells overexpressing dominant-negative Rab1b. We thus hypothesized that Rab1b recruits lipid-synthesizing enzymes, facilitating LD growth. Here, FRET between diacylglycerol acyltransferase 2 (DGAT2) and Rab1b and activity mutants of the latter demonstrated that Rab1b promotes DGAT2 ER to the LD surface redistribution. Last, alterations in LD metabolism and DGAT2 redistribution, consistent with Rab1b activity, were caused by mutations in the Rab1b-GTPase activating protein TBC1D20 in Warburg Micro syndrome (WARBM) model mice fibroblasts. These data contribute to our understanding of the mechanism of Rab1b in LD homeostasis and WARBM, a devastating autosomal-recessive disorder caused by mutations in TBC1D20.
    DOI:  https://doi.org/10.1126/sciadv.ade7753
  29. bioRxiv. 2024 May 16. pii: 2024.05.06.592777. [Epub ahead of print]
      Chimeras have played a foundational role in biology, for example by enabling the classification of developmental processes into those driven intrinsically by individual cells versus those driven extrinsically by their extracellular environment. Here, we extend this framework to decompose evolutionary divergence in gene expression and other quantitative traits into cell-intrinsic, extrinsic, and intrinsic-extrinsic interaction components. Applying this framework to reciprocal rat-mouse chimeras, we found that the majority of gene expression divergence is attributable to cell-intrinsic factors, though extrinsic factors also play an integral role. For example, a rat-like extracellular environment extrinsically up-regulates the expression of a key transcriptional regulator of the endoplasmic reticulum (ER) stress response in some but not all cell types, which in turn strongly predicts extrinsic up-regulation of its target genes and of the ER stress response pathway as a whole. This effect is also seen at the protein level, suggesting propagation through multiple regulatory levels. We also demonstrate that our framework is applicable to a cellular trait, neuronal differentiation, and estimated the intrinsic and extrinsic contributions to its divergence. Finally, we show that imprinted genes are dramatically mis-expressed in species-mismatched environments, suggesting that mismatch between rapidly evolving intrinsic and extrinsic mechanisms controlling gene imprinting may contribute to barriers to interspecies chimerism. Overall, our conceptual framework opens new avenues to investigate the mechanistic basis of evolutionary divergence in gene expression and other quantitative traits in any multicellular organism.
    DOI:  https://doi.org/10.1101/2024.05.06.592777
  30. PLoS Comput Biol. 2024 May 31. 20(5): e1012169
      On any given day, we make countless reaching movements to objects around us. While such ubiquity may suggest uniformity, each movement's speed is unique-why is this? Reach speed is known to be influenced by accuracy; we slow down to sustain high accuracy. However, in other forms of movement like walking or running, metabolic cost is often the primary determinant of movement speed. Here we bridge this gap and ask: how do metabolic cost and accuracy interact to determine speed of reaching movements? First, we systematically measure the effect of increasing mass on the metabolic cost of reaching across a range of movement speeds. Next, in a sequence of three experiments, we examine how added mass affects preferred reaching speed across changing accuracy requirements. We find that, while added mass consistently increases metabolic cost thereby leading to slower metabolically optimal movement speeds, self-selected reach speeds are slower than those predicted by an optimization of metabolic cost alone. We then demonstrate how a single model that considers both accuracy and metabolic costs can explain preferred movement speeds. Together, our findings provide a unifying framework to illuminate the combined effects of metabolic cost and accuracy on movement speed and highlight the integral role metabolic cost plays in determining reach speed.
    DOI:  https://doi.org/10.1371/journal.pcbi.1012169
  31. Cell Oncol (Dordr). 2024 May 28.
      PURPOSE: Pancreatic Ductal Adenocarcinoma (PDAC) remains a challenging disease due to its complex biology and aggressive behavior with an urgent need for efficient therapeutic strategies. To assess therapy response, pre-clinical PDAC organoid-based models in combination with accurate real-time monitoring are required.METHODS: We established stable live-imaging organoid/peripheral blood mononuclear cells (PBMCs) co-cultures and introduced OrganoIDNet, a deep-learning-based algorithm, capable of analyzing bright-field images of murine and human patient-derived PDAC organoids acquired with live-cell imaging. We investigated the response to the chemotherapy gemcitabine in PDAC organoids and the PD-L1 inhibitor Atezolizumab, cultured with or without HLA-matched PBMCs over time. Results obtained with OrganoIDNet were validated with the endpoint proliferation assay CellTiter-Glo.
    RESULTS: Live cell imaging in combination with OrganoIDNet accurately detected size-specific drug responses of organoids to gemcitabine over time, showing that large organoids were more prone to cytotoxic effects. This approach also allowed distinguishing between healthy and unhealthy status and measuring eccentricity as organoids' reaction to therapy. Furthermore, imaging of a new organoids/PBMCs sandwich-based co-culture enabled longitudinal analysis of organoid responses to Atezolizumab, showing an increased potency of PBMCs tumor-killing in an organoid-individual manner when Atezolizumab was added.
    CONCLUSION: Optimized PDAC organoid imaging analyzed by OrganoIDNet represents a platform capable of accurately detecting organoid responses to standard PDAC chemotherapy over time. Moreover, organoid/immune cell co-cultures allow monitoring of organoid responses to immunotherapy, offering dynamic insights into treatment behavior within a co-culture setting with PBMCs. This setup holds promise for real-time assessment of immunotherapeutic effects in individual patient-derived PDAC organoids.
    Keywords:  Artificial intelligence; Co-cultures; Gemcitabine; Immunotherapy; Organoids; PDAC
    DOI:  https://doi.org/10.1007/s13402-024-00958-2
  32. bioRxiv. 2024 May 18. pii: 2024.05.15.594354. [Epub ahead of print]
      Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-β-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-β pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer.Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-β, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.
    DOI:  https://doi.org/10.1101/2024.05.15.594354
  33. Sci Adv. 2024 May 31. 10(22): eadn0235
      The ability of cells to organize into tissues with proper structure and function requires the effective coordination of proliferation, migration, polarization, and differentiation across length scales. Skeletal muscle is innately anisotropic; however, few biomaterials can emulate mechanical anisotropy to determine its influence on tissue patterning without introducing confounding topography. Here, we demonstrate that substrate stiffness anisotropy coordinates contractility-driven collective cellular dynamics resulting in C2C12 myotube alignment over millimeter-scale distances. When cultured on mechanically anisotropic liquid crystalline polymer networks (LCNs) lacking topography, C2C12 myoblasts collectively polarize in the stiffest direction. Cellular coordination is amplified through reciprocal cell-ECM dynamics that emerge during fusion, driving global myotube-ECM ordering. Conversely, myotube alignment was restricted to small local domains with no directional preference on mechanically isotropic LCNs of the same chemical formulation. These findings provide valuable insights for designing biomaterials that mimic anisotropic microenvironments and underscore the importance of stiffness anisotropy in orchestrating tissue morphogenesis.
    DOI:  https://doi.org/10.1126/sciadv.adn0235
  34. J Biomed Opt. 2024 Jun;29(6): 065003
      Significance: Hematogenous metastasis is mediated by circulating tumor cells (CTCs) and CTC clusters (CTCCs). We recently developed "diffuse in vivo flow cytometry" (DiFC) to detect fluorescent protein (FP) expressing CTCs in small animals. Extending DiFC to allow detection of two FPs simultaneously would allow concurrent study of different CTC sub-populations or heterogeneous CTCCs in the same animal.Aim: The goal of this work was to develop and validate a two-color DiFC system capable of non-invasively detecting circulating cells expressing two distinct FPs.
    Approach: A DiFC instrument was designed and built to detect cells expressing either green FP (GFP) or tdTomato. We tested the instrument in tissue-mimicking flow phantoms in vitro and in multiple myeloma bearing mice in vivo.
    Results: In phantoms, we could accurately differentiate GFP+ and tdTomato+ CTCs and CTCCs. In tumor-bearing mice, CTC numbers expressing both FPs increased during disease. Most CTCCs (86.5%) expressed single FPs with the remainder both FPs. These data were supported by whole-body hyperspectral fluorescence cryo-imaging of the mice.
    Conclusions: We showed that two-color DiFC can detect two populations of CTCs and CTCCs concurrently. This instrument could allow study of tumor development and response to therapies for different sub-populations in the same animal.
    Keywords:  circulating tumor cell clusters; circulating tumor cells; fluorescence; optical devices
    DOI:  https://doi.org/10.1117/1.JBO.29.6.065003
  35. bioRxiv. 2024 May 15. pii: 2024.05.13.594009. [Epub ahead of print]
      Coarse-grained (CG) models have been developed for studying membrane proteins at physiologically relevant scales. Such methods, including popular CG lipid models, exhibit stability and efficiency at moderate scales, but they can become impractical or even unusable beyond a critical size due to various technical issues. Here, we report that these scale-dependent issues can arise from progressively slower relaxation dynamics and become confounded by unforeseen instabilities observed only at larger scales. To address these issues, we systemically optimized a 4-site solvent-free CG lipid model that is suitable for conducting micron-scale molecular dynamics simulations of membrane proteins under various membrane properties. We applied this lipid model to explore the long-range membrane deformation induced by a large mechanosensitive ion channel, PIEZO. We show that the optimized CG models are powerful in elucidating the structural and dynamic interplay between PIEZO and the membrane. Furthermore, we anticipate that our methodological insights can prove useful for resolving issues stemming from scale-dependent limitations of similar CG methodologies.
    DOI:  https://doi.org/10.1101/2024.05.13.594009
  36. Sci Adv. 2024 May 31. 10(22): eadk9681
      In response to energy and nutrient shortage, the liver triggers several catabolic processes to promote survival. Despite recent progress, the precise molecular mechanisms regulating the hepatic adaptation to fasting remain incompletely characterized. Here, we report the identification of hydroxysteroid dehydrogenase-like 2 (HSDL2) as a mitochondrial protein highly induced by fasting. We show that the activation of PGC1α-PPARα and the inhibition of the PI3K-mTORC1 axis stimulate HSDL2 expression in hepatocytes. We found that HSDL2 depletion decreases cholesterol conversion to bile acids (BAs) and impairs FXR activity. HSDL2 knockdown also reduces mitochondrial respiration, fatty acid oxidation, and TCA cycle activity. Bioinformatics analyses revealed that hepatic Hsdl2 expression positively associates with the postprandial excursion of various BA species in mice. We show that liver-specific HSDL2 depletion affects BA metabolism and decreases circulating cholesterol levels upon refeeding. Overall, our report identifies HSDL2 as a fasting-induced mitochondrial protein that links nutritional signals to BAs and cholesterol homeostasis.
    DOI:  https://doi.org/10.1126/sciadv.adk9681
  37. JACS Au. 2024 May 27. 4(5): 1841-1853
      Cell-like materials that sense environmental cues can serve as next-generation biosensors and help advance the understanding of intercellular communication. Currently, bottom-up engineering of protocell models from molecular building blocks remains a grand challenge chemists face. Herein, we describe giant unilamellar vesicles (GUVs) with biomimetic lipid membranes capable of sensing environmental redox cues. The GUVs employ activity-based sensing through designer phospholipids that are fluorescently activated in response to specific reductive (hydrogen sulfide) or oxidative (hydrogen peroxide) conditions. These synthetic phospholipids are derived from 1,2-dipalmitoyl-rac-glycero-3-phosphocholine and they possess a headgroup with heterocyclic aromatic motifs. Despite their structural deviation from the phosphocholine headgroup, the designer phospholipids (0.5-1.0 mol %) mixed with natural lipids can vesiculate, and the resulting GUVs (7-20 μm in diameter) remain intact over the course of redox sensing. All-atom molecular dynamics simulations gave insight into how these lipids are positioned within the hydrophobic core of the membrane bilayer and at the membrane-water interface. This work provides a purely chemical method to investigate potential redox signaling and opens up new design opportunities for soft materials that mimic protocells.
    DOI:  https://doi.org/10.1021/jacsau.4c00041
  38. Nat Rev Gastroenterol Hepatol. 2024 May 28.
      Despite extensive research and improvements in understanding colorectal cancer (CRC), its metastatic form continues to pose a substantial challenge, primarily owing to limited therapeutic options and a poor prognosis. This Review addresses the emerging focus on metastatic CRC (mCRC), which has historically been under-studied compared with primary CRC despite its lethality. We delve into two crucial aspects: the molecular and cellular determinants facilitating CRC metastasis and the principles guiding the evolution of metastatic disease. Initially, we examine the genetic alterations integral to CRC metastasis, connecting them to clinically marked characteristics of advanced CRC. Subsequently, we scrutinize the role of cellular heterogeneity and plasticity in metastatic spread and therapy resistance. Finally, we explore how the tumour microenvironment influences metastatic disease, emphasizing the effect of stromal gene programmes and the immune context. The ongoing research in these fields holds immense importance, as its future implications are projected to revolutionize the treatment of patients with mCRC, hopefully offering a promising outlook for their survival.
    DOI:  https://doi.org/10.1038/s41575-024-00934-z
  39. Nat Struct Mol Biol. 2024 May 29.
      α-Helical integral membrane proteins comprise approximately 25% of the proteome in all organisms. The membrane proteome is highly diverse, varying in the number, topology, spacing and properties of transmembrane domains. This diversity imposes different constraints on the insertion of different regions of a membrane protein into the lipid bilayer. Here, we present a cohesive framework to explain membrane protein biogenesis, in which different parts of a nascent substrate are triaged between Oxa1 and SecY family members for insertion. In this model, Oxa1 family proteins insert transmembrane domains flanked by short translocated segments, whereas the SecY channel is required for insertion of transmembrane domains flanked by long translocated segments. Our unifying model rationalizes evolutionary, genetic, biochemical and structural data across organisms and provides a foundation for future mechanistic studies of membrane protein biogenesis.
    DOI:  https://doi.org/10.1038/s41594-024-01296-5
  40. Soft Matter. 2024 May 28.
      The nucleus of eukaryotic cells typically makes up around 30% of the cell volume and has significantly different mechanics, which can make it effectively up to ten times stiffer than the surrounding cytoplasm. Therefore it is an important element for cell mechanics, but a quantitative understanding of its mechanical role during whole cell dynamics is largely missing. Here we demonstrate that elastic phase fields can be used to describe dynamical cell processes in adhesive or confining environments in which the nucleus acts as a stiff inclusion in an elastic cytoplasm. We first introduce and verify our computational method and then study several prevalent cell-mechanical measurement methods. For cells on adhesive patterns, we find that nuclear stress is shielded by the adhesive pattern. For cell compression between two parallel plates, we obtain force-compression curves that allow us to extract an effective modulus for the cell-nucleus composite. For micropipette aspiration, the effect of the nucleus on the effective modulus is found to be much weaker, highlighting the complicated interplay between extracellular geometry and cell mechanics that is captured by our approach. We also show that our phase field approach can be used to investigate the effects of Kelvin-Voigt-type viscoelasticity and cortical tension.
    DOI:  https://doi.org/10.1039/d4sm00345d