bims-pideca Biomed News
on Class IA PI3K signalling in development and cancer
Issue of 2023‒10‒29
thirty-one papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

  1. Cell. 2023 Oct 18. pii: S0092-8674(23)01081-4. [Epub ahead of print]
      Lysosomes serve dual antagonistic functions in cells by mediating anabolic growth signaling and the catabolic turnover of macromolecules. How these janus-faced activities are regulated in response to cellular nutrient status is poorly understood. We show here that lysosome morphology and function are reversibly controlled by a nutrient-regulated signaling lipid switch that triggers the conversion between peripheral motile mTOR complex 1 (mTORC1) signaling-active and static mTORC1-inactive degradative lysosomes clustered at the cell center. Starvation-triggered relocalization of phosphatidylinositol 4-phosphate (PI(4)P)-metabolizing enzymes reshapes the lysosomal surface proteome to facilitate lysosomal proteolysis and to repress mTORC1 signaling. Concomitantly, lysosomal phosphatidylinositol 3-phosphate (PI(3)P), which marks motile signaling-active lysosomes in the cell periphery, is erased. Interference with this PI(3)P/PI(4)P lipid switch module impairs the adaptive response of cells to altering nutrient supply. Our data unravel a key function for lysosomal phosphoinositide metabolism in rewiring organellar membrane dynamics in response to cellular nutrient status.
    Keywords:  catabolism; functional proteomics; live correlative light and electron microscopy; lysosomes; mTOR; myotubularin; nutrient signaling; nutrients; phosphoinositides
  2. Physiol Rep. 2023 Oct;11(20): e15805
      AKT signaling plays a crucial role in muscle physiology, and is activated by stimuli, including insulin, growth factors, and exercise. Three AKT isoforms have been identified in mammals, and they possess both distinct and redundant functions. However, it is currently unknown what the predominant AKT isoform is in primary human skeletal myotubes, and very little is known regarding the effects of insulin and insulin-like growth factor-I (IGF-I) on AKT isoforms activation in human myotubes. Thus, we sought to determine the abundances of each AKT isoform in primary human skeletal myotubes and their responses to insulin or IGF-I. Analysis of protein lysates by liquid chromatography-parallel reaction monitoring/mass spectrometry revealed that AKT1 was the most abundant AKT isoform and AKT3 was the least-abundant isoform. Next, myotubes were treated with either 100 nM insulin or 10 nM IGF-I for 5, 20, 45, or 60 min. In response to insulin, there was a significant treatment effect on phosphorylation of AKT1 and AKT2, but not AKT3 (p < 0.01). In response to IGF-I, there was a significant treatment effect on phosphorylation of pan-AKT at all timepoints compared to control (p < 0.01). Next, we determined how much of the total AKT isoform pool was phosphorylated. For insulin stimulation, AKT1 was significantly higher than AKT2 at 5 min and 60 min posttreatment (p < 0.05 both) and significantly different than AKT3 at all timepoints (p < 0.05). For IGF-I stimulation, AKT1 was significantly higher than AKT2 at 45 and 60 min posttreatment (p < 0.05 both) and significantly higher than AKT3 at all timepoints (p < 0.05). Our findings reveal the differential phosphorylation patterns among the AKT isoforms and suggest a potential explanation for the regulatory role of AKT1 in skeletal muscle.
    Keywords:  PKB; anabolic; myosin; protein synthesis; skeletal muscle
  3. Cancers (Basel). 2023 Oct 12. pii: 4958. [Epub ahead of print]15(20):
      Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients.
    Keywords:  AKT; cancer; melanoma; metastasis; signaling
  4. Mol Cell. 2023 Oct 20. pii: S1097-2765(23)00800-6. [Epub ahead of print]
      Lactate has long been considered a cellular waste product. However, we found that as extracellular lactate accumulates, it also enters the mitochondrial matrix and stimulates mitochondrial electron transport chain (ETC) activity. The resulting increase in mitochondrial ATP synthesis suppresses glycolysis and increases the utilization of pyruvate and/or alternative respiratory substrates. The ability of lactate to increase oxidative phosphorylation does not depend on its metabolism. Both L- and D-lactate are effective at enhancing ETC activity and suppressing glycolysis. Furthermore, the selective induction of mitochondrial oxidative phosphorylation by unmetabolized D-lactate reversibly suppressed aerobic glycolysis in both cancer cell lines and proliferating primary cells in an ATP-dependent manner and enabled cell growth on respiratory-dependent bioenergetic substrates. In primary T cells, D-lactate enhanced cell proliferation and effector function. Together, these findings demonstrate that lactate is a critical regulator of the ability of mitochondrial oxidative phosphorylation to suppress glucose fermentation.
    Keywords:  TCA cycle; electron transport chain; glycolysis; lactate; mitochondria; oxidative phosphorylation
  5. Cell Death Dis. 2023 Oct 25. 14(10): 700
      We here tested the potential activity and the underlying mechanisms of neuroligin-3 (NLGN3) against ischemia-reperfusion-induced neuronal cell injury. In SH-SY5Y neuronal cells and primary murine cortical neurons, NLGN3 activated Akt-mTOR and Erk signalings, and inhibited oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced cytotoxicity. Akt activation was required for NLGN3-induced neuroprotection. Gαi1/3 mediated NLGN3-induced downstream signaling activation. NLGN3-induced Akt-S6K1 activation was largely inhibited by Gαi1/3 silencing or knockout. Significantly, NLGN3-induced neuroprotection against OGD/R was almost abolished by Gαi1/3 silencing or knockout. In vivo, the middle cerebral artery occlusion (MCAO) procedure induced NLGN3 cleavage and secretion, and increased its expression and Akt activation in mouse brain tissues. ADAM10 (A Disintegrin and Metalloproteinase 10) inhibition blocked MCAO-induced NLGN3 cleavage and secretion, exacerbating ischemic brain injury in mice. Neuronal silencing of NLGN3 or Gαi1/3 in mice also inhibited Akt activation and intensified MCAO-induced ischemic brain injury. Conversely, neuronal overexpression of NLGN3 increased Akt activation and alleviated MCAO-induced ischemic brain injury. Together, NLGN3 activates Gαi1/3-Akt signaling to protect neuronal cells from ischemia-reperfusion injury.
  6. Cell Chem Biol. 2023 Oct 26. pii: S2451-9456(23)00336-7. [Epub ahead of print]
      Impaired mitochondrial dynamics causes aging-related or metabolic diseases. Yet, the molecular mechanism responsible for the impairment of mitochondrial dynamics is still not well understood. Here, we report that elevated blood insulin and/or glucagon levels downregulate mitochondrial fission through directly phosphorylating AMPKα at S496 by AKT or PKA, resulting in the impairment of AMPK-MFF-DRP1 signaling and mitochondrial dynamics and activity. Since there are significantly increased AMPKα1 phosphorylation at S496 in the liver of elderly mice, obese mice, and obese patients, we, therefore, designed AMPK-specific targeting peptides (Pa496m and Pa496h) to block AMPKα1S496 phosphorylation and found that these targeting peptides can increase AMPK kinase activity, augment mitochondrial fission and oxidation, and reduce ROS, leading to the rejuvenation of mitochondria. Furthermore, these AMPK targeting peptides robustly suppress liver glucose production in obese mice. Our data suggest these targeting peptides are promising therapeutic agents for improving mitochondrial dynamics and activity and alleviating hyperglycemia in elderly and obese patients.
    Keywords:  AKT; AMPK targeting peptides; AMPKαS496 phosphorylation; PKA; hyperglycemia; liver gluconeogenesis; mitochondrial fission
  7. Blood Adv. 2023 Oct 23. pii: bloodadvances.2022009349. [Epub ahead of print]
      Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasm characterized by the accumulation of clonal mononuclear phagocyte system cells expressing CD1a and CD207. In the past decade, molecular profiling of LCH, as well as other histiocytic neoplasms demonstrated that these diseases are driven by MAP kinase (MAPK) activating alterations, with somatic BRAFV600E mutations in >50% of LCH patients, and clinical inhibition of MAPK signaling has demonstrated remarkable clinical efficacy. At the same time, activating alterations in kinase-encoding genes such as PIK3CA, ALK, RET, and CSF1R which can activate mitogenic pathways independent from the MAPK pathway have been reported in a subset of histiocytic neoplasms with anecdotal evidence of successful targeted treatment of histiocytoses harboring driver alterations in RET, ALK, and CSF1R. However, evidence supporting the biological consequences of expression of PIK3CA mutations in hematopoietic cells has been lacking, and whether targeted inhibition of PI3K is clinically efficacious in histiocytic neoplasms is unknown. Here, we provide evidence that activating mutations in PIK3CA can drive histiocytic neoplasms in vivo using a conditional knock-in mouse expressing mutant PIK3CAH1047R in monocyte/dendritic cell progenitors. In parallel, we demonstrate successful treatment of PIK3CA-mutated, multisystemic LCH using alpelisib, an inhibitor of the alpha catalytic subunit of PI3K. Alpelisib demonstrated a tolerable safety profile at a dose of 750mg/week and clinical and metabolic complete remission in a PIK3CA-mutated LCH patient. These data demonstrate PIK3CA as a targetable non-canonical driver of LCH and underscore the importance of mutational analysis-based personalized treatment in histiocytic neoplasms.
  8. bioRxiv. 2023 Oct 10. pii: 2023.10.08.561457. [Epub ahead of print]
      The Mechanism of Action (MoA) of a drug is generally represented as a small, non-tissue-specific repertoire of high-affinity binding targets. Yet, drug activity and polypharmacology are increasingly associated with a broad range of off-target and tissue-specific effector proteins. To address this challenge, we have implemented an efficient integrative experimental and computational framework leveraging the systematic generation and analysis of drug perturbational profiles representing >700 FDA-approved and experimental oncology drugs, in cell lines selected as high-fidelity models of 23 aggressive tumor subtypes. Protein activity-based analyses revealed highly reproducible, drug-mediated modulation of tissue-specific targets, leading to generation of a proteome-wide polypharmacology map, characterization of MoA-related drug clusters and off-target effects, and identification and experimental validation of novel, tissue-specific inhibitors of undruggable oncoproteins. The proposed framework, which is easily extended to elucidating the MoA of novel small-molecule libraries, could help support more systematic and quantitative approaches to precision oncology.
  9. Nature. 2023 Oct 25.
      Identifying metabolic steps that are specifically required for the survival of cancer cells but are dispensable in normal cells remains a challenge1. Here we report a therapeutic vulnerability in a sugar nucleotide biosynthetic pathway that can be exploited in cancer cells with only a limited impact on normal cells. A systematic examination of conditionally essential metabolic enzymes revealed that UXS1, a Golgi enzyme that converts one sugar nucleotide (UDP-glucuronic acid, UDPGA) to another (UDP-xylose), is essential only in cells that express high levels of the enzyme immediately upstream of it, UGDH. This conditional relationship exists because UXS1 is required to prevent excess accumulation of UDPGA, which is produced by UGDH. UXS1 not only clears away UDPGA but also limits its production through negative feedback on UGDH. Excess UDPGA disrupts Golgi morphology and function, which impedes the trafficking of surface receptors such as EGFR to the plasma membrane and diminishes the signalling capacity of cells. UGDH expression is elevated in several cancers, including lung adenocarcinoma, and is further enhanced during chemoresistant selection. As a result, these cancer cells are selectively dependent on UXS1 for UDPGA detoxification, revealing a potential weakness in tumours with high levels of UGDH.
  10. Brief Bioinform. 2023 Sep 22. pii: bbad375. [Epub ahead of print]24(6):
      Computational reproducibility is a simple premise in theory, but is difficult to achieve in practice. Building upon past efforts and proposals to maximize reproducibility and rigor in bioinformatics, we present a framework called the five pillars of reproducible computational research. These include (1) literate programming, (2) code version control and sharing, (3) compute environment control, (4) persistent data sharing and (5) documentation. These practices will ensure that computational research work can be reproduced quickly and easily, long into the future. This guide is designed for bioinformatics data analysts and bioinformaticians in training, but should be relevant to other domains of study.
    Keywords:  bioinformatics; computational research; reproducibility; research best practices
  11. Diabetes. 2023 Oct 23. pii: db230450. [Epub ahead of print]
      Excessive insulin secretion independent of insulin resistance, defined as primary hypersecretion, is associated with obesity and an unfavorable metabolic phenotype. We examined the characteristics of the adipose tissue in youths with primary insulin hypersecretion and the longitudinal metabolic alterations influenced by the complex adipo-insular interplay. In a multiethnic cohort of non-diabetic adolescents with obesity, primary insulin hypersecretors had enhanced model-derived β-cell glucose sensitivity and rate sensitivity, but worse glucose tolerance, despite similar demographics, adiposity, and insulin resistance measured by both OGTT and euglycemic-hyperinsulinemic clamp. Hypersecretors had greater intrahepatic and visceral fat depots at abdominal MRI, hypertrophic abdominal subcutaneous adipocytes, higher FFA and leptin serum levels per fat mass, and faster in vivo lipid turnover assessed by a long-term 2H2O labeling protocol. At 2-year follow up, hypersecretors had greater fat accrual and 3-fold higher risk for abnormal glucose tolerance, while individuals with hypertrophic adipocytes or higher leptin levels showed enhanced β-cell glucose sensitivity. Primary insulin hypersecretion is associated with marked alterations in adipose tissue distribution, cellularity, and lipid dynamics, independent of whole-body adiposity and insulin resistance. Pathogenetic insight into the metabolic crosstalk between β-cell and adipocyte may help identify individuals at risk for chronic hyperinsulinemia, body weight gain, and glucose intolerance.
  12. Cell Rep. 2023 Oct 21. pii: S2211-1247(23)01210-X. [Epub ahead of print]42(11): 113198
      Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, the emergence of drug resistance limits their long-term efficacy. Here, we show that breast cancer cells develop CDK4/6i resistance via a sequential two-step process of E2F activation. This process entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification of E2F transcriptional activity. CDK4/6i treatment halts cell proliferation in an Rb-dependent manner but dramatically reduces Rb-protein levels. However, this reduction in Rb levels insufficiently induces E2F activity. To develop CDK4/6i resistance, upregulation or activating mutations in mitogenic or hormone signaling are required to stabilize c-Myc levels, thereby augmenting E2F activity. Our analysis of pre-treatment tumor samples reveals a strong correlation between c-Myc levels, rather than Rb levels, and poor therapeutic outcomes after CDK4/6i treatment. Moreover, we propose that proteasome inhibitors can potentially reverse CDK4/6i resistance by restoring Rb levels.
    Keywords:  CDK4/6 inhibitors; CP: Cancer; E2F transcription factors; breast cancer; drug resistance; retinoblastoma protein
  13. Oncogene. 2023 Oct 24.
      PTEN loss, one of the most frequent mutations in prostate cancer (PC), is presumed to drive disease progression through AKT activation. However, two transgenic PC models with Akt activation plus Rb loss exhibited different metastatic development: Pten/RbPE:-/- mice produced systemic metastatic adenocarcinomas with high AKT2 activation, whereas RbPE:-/- mice deficient for the Src-scaffolding protein, Akap12, induced high-grade prostatic intraepithelial neoplasias and indolent lymph node dissemination, correlating with upregulated phosphotyrosyl PI3K-p85α. Using PC cells isogenic for PTEN, we show that PTEN-deficiency correlated with dependence on both p110β and AKT2 for in vitro and in vivo parameters of metastatic growth or motility, and with downregulation of SMAD4, a known PC metastasis suppressor. In contrast, PTEN expression, which dampened these oncogenic behaviors, correlated with greater dependence on p110α plus AKT1. Our data suggest that metastatic PC aggressiveness is controlled by specific PI3K/AKT isoform combinations influenced by divergent Src activation or PTEN-loss pathways.
  14. Nat Biotechnol. 2023 Oct 23.
      Pooled CRISPR screens with single-cell RNA sequencing readout (Perturb-seq) have emerged as a key technique in functional genomics, but they are limited in scale by cost and combinatorial complexity. In this study, we modified the design of Perturb-seq by incorporating algorithms applied to random, low-dimensional observations. Compressed Perturb-seq measures multiple random perturbations per cell or multiple cells per droplet and computationally decompresses these measurements by leveraging the sparse structure of regulatory circuits. Applied to 598 genes in the immune response to bacterial lipopolysaccharide, compressed Perturb-seq achieves the same accuracy as conventional Perturb-seq with an order of magnitude cost reduction and greater power to learn genetic interactions. We identified known and novel regulators of immune responses and uncovered evolutionarily constrained genes with downstream targets enriched for immune disease heritability, including many missed by existing genome-wide association studies. Our framework enables new scales of interrogation for a foundational method in functional genomics.
  15. Nat Commun. 2023 Oct 23. 14(1): 6708
      Telomeres, the ends of eukaryotic chromosomes, protect genome integrity and enable cell proliferation. Maintaining optimal telomere length in the germline and throughout life limits the risk of cancer and enables healthy aging. Telomeres in the house mouse, Mus musculus, are about five times longer than human telomeres, limiting the use of this common laboratory animal for studying the contribution of telomere biology to aging and cancer. We identified a key amino acid variation in the helicase RTEL1, naturally occurring in the short-telomere mouse species M. spretus. Introducing this variation into M. musculus is sufficient to reduce the telomere length set point in the germline and generate mice with human-length telomeres. While these mice are fertile and appear healthy, the regenerative capacity of their colonic epithelium is compromised. The engineered Telomouse reported here demonstrates a dominant role of RTEL1 in telomere length regulation and provides a unique model for aging and cancer.
  16. Cardiovasc Diabetol. 2023 Oct 27. 22(1): 294
      BACKGROUND: The PI3K/AKT pathway transduces the majority of the metabolic actions of insulin. In addition to cytosolic targets, insulin-stimulated phospho-AKT also translocates to mitochondria in the myocardium. Mouse models of diabetes exhibit impaired mitochondrial AKT signaling but the implications of this on cardiac structure and function is unknown. We hypothesized that loss of mitochondrial AKT signaling is a critical step in cardiomyopathy and reduces cardiac oxidative phosphorylation.METHODS: To focus our investigation on the pathophysiological consequences of this mitochondrial signaling pathway, we generated transgenic mouse models of cardiac-specific, mitochondria-targeting, dominant negative AKT1 (CAMDAKT) and constitutively active AKT1 expression (CAMCAKT). Myocardial structure and function were examined using echocardiography, histology, and biochemical assays. We further investigated the underlying effects of mitochondrial AKT1 on mitochondrial structure and function, its interaction with ATP synthase, and explored in vivo metabolism beyond the heart.
    RESULTS: Upon induction of dominant negative mitochondrial AKT1, CAMDAKT mice developed cardiac fibrosis accompanied by left ventricular hypertrophy and dysfunction. Cardiac mitochondrial oxidative phosphorylation efficiency and ATP content were reduced, mitochondrial cristae structure was lost, and ATP synthase structure was compromised. Conversely, CAMCAKT mice were protected against development of diabetic cardiomyopathy when challenged with a high calorie diet. Activation of mitochondrial AKT1 protected cardiac function and increased fatty acid uptake in myocardium. In addition, total energy expenditure was increased in CAMCAKT mice, accompanied by reduced adiposity and reduced development of fatty liver.
    CONCLUSION: CAMDAKT mice modeled the effects of impaired mitochondrial signaling which occurs in the diabetic myocardium. Disruption of this pathway is a key step in the development of cardiomyopathy. Activation of mitochondrial AKT1 in CAMCAKT had a protective role against diabetic cardiomyopathy as well as improved metabolism beyond the heart.
    Keywords:  ATP synthase; Diabetic cardiomyopathy; Fatty liver; Heart failure; Mitochondria dysfunction; Mitochondrial AKT; Obesity
  17. bioRxiv. 2023 Oct 03. pii: 2023.10.02.560369. [Epub ahead of print]
      Oxygen (O 2 ) tension plays a key role in tissue function and pathophysiology. O 2 -controlled cell culture, in which the O 2 concentration in an incubator's gas phase is controlled, is an indispensable tool to study the role of O 2 in vivo . For this technique, it is presumed that the incubator setpoint is equal to the O 2 tension that cells experience ( i.e. , pericellular O 2 ). We discovered that physioxic (5% O 2 ) and hypoxic (1% O 2 ) setpoints regularly induce anoxic (0.0% O 2 ) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O 2 consumption is the driving factor. RNA-seq revealed that primary human hepatocytes cultured in physioxia experience ischemia-reperfusion injury due to anoxic exposure followed by rapid reoxygenation. To better understand the relationship between incubator gas phase and pericellular O 2 tensions, we developed a reaction-diffusion model that predicts pericellular O 2 tension a priori . This model revealed that the effect of cellular O 2 consumption is greatest in smaller volume culture vessels ( e.g., 96-well plate). By controlling pericellular O 2 tension in cell culture, we discovered that MCF7 cells have stronger glycolytic and glutamine metabolism responses in anoxia vs. hypoxia. MCF7 also expressed higher levels of HIF2A , CD73 , NDUFA4L2 , etc. and lower levels of HIF1A , CA9 , VEGFA, etc. in response to hypoxia vs. anoxia. Proteomics revealed that 4T1 cells had an upregulated epithelial-to-mesenchymal transition (EMT) response and downregulated reactive oxygen species (ROS) management, glycolysis, and fatty acid metabolism pathways in hypoxia vs. anoxia. Collectively, these results reveal that breast cancer cells respond non-monotonically to low O 2 , suggesting that anoxic cell culture is not suitable to model hypoxia. We demonstrate that controlling atmospheric O 2 tension in cell culture incubators is insufficient to control O 2 in cell culture and introduce the concept of pericellular O 2 -controlled cell culture .
  18. Front Bioeng Biotechnol. 2023 ;11 1218957
      After the development of 3D cell culture methods in the middle of the last century and the plethora of data generated with this culture configuration up to date, it could be shown that a three-dimensional arrangement of cells in most of the cases leads to a more physiological behavior of the generated tissue. However, a major determinant for an organotypic function, namely, the dissolved oxygen concentration in the used in vitro-system, has been neglected in most of the studies. This is due to the fact that the oxygen measurement in the beginning was simply not feasible and, if so, disturbed the measurement and/or the in vitro-system itself. This is especially true for the meanwhile more widespread use of 3D culture systems. Therefore, the tissues analyzed by these techniques can be considered as the Schrödinger's cat in 3D cell biology. In this perspective paper we will outline how the measurement and, moreover, the regulation of the dissolved oxygen concentration in vitro-3D culture systems could be established at all and how it may be possible to determine the oxygen concentration in organoid cultures and the respiratory capacity via mito stress tests, especially in spheroids in the size range of a few hundred micrometers, under physiological culture conditions, without disturbances or stress induction in the system and in a high-throughput fashion. By this, such systems will help to more efficiently translate tissue engineering approaches into new in vitro-platforms for fundamental and applied research as well as preclinical safety testing and clinical applications.
    Keywords:  3D cell culture; in vitro; microcavity sensor arrays; optical O2 measurement; organoid; physiological oxygen concentration
  19. Dev Cell. 2023 Oct 18. pii: S1534-5807(23)00521-X. [Epub ahead of print]
      Mutations in the degradative ubiquitin ligase anaphase-promoting complex (APC) alter neurodevelopment by impairing proteasomal protein clearance, but our understanding of their molecular and cellular pathogenesis remains limited. Here, we employ the proteomic-based discovery of APC substrates in APC mutant mouse brain and human cell lines and identify the chromosome-passenger complex (CPC), topoisomerase 2a (Top2a), and Ki-67 as major chromatin factors targeted by the APC during neuronal differentiation. These substrates accumulate in phosphorylated form, suggesting that they fail to be eliminated after mitosis during terminal differentiation. The accumulation of the CPC kinase Aurora B within constitutive heterochromatin and hyperphosphorylation of its target histone 3 are corrected in the mutant brain by pharmacologic Aurora B inhibition. Surprisingly, the reduction of Ki-67, but not H3S10ph, rescued the function of constitutive heterochromatin in APC mutant neurons. These results expand our understanding of how ubiquitin signaling regulates chromatin during neurodevelopment and identify potential therapeutic targets in APC-related disorders.
    Keywords:  H3S10ph; Ki-67; anaphase-promoting complex; chromatin; chromosome-passenger complex; heterochromatin; neurodevelopment; proteomics/phosphoproteomics; topoisomerase; ubiquitin ligase
  20. Nat Commun. 2023 Oct 27. 14(1): 6774
      Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.
  21. PLoS Biol. 2023 Oct;21(10): e3002354
      The N-terminal tails of eukaryotic histones are frequently posttranslationally modified. The role of these modifications in transcriptional regulation is well-documented. However, the extent to which the enzymatic processes of histone posttranslational modification might affect metabolic regulation is less clear. Here, we investigated how histone methylation might affect metabolism using metabolomics, proteomics, and RNA-seq data from cancer cell lines, primary tumour samples and healthy tissue samples. In cancer, the expression of histone methyltransferases (HMTs) was inversely correlated to the activity of NNMT, an enzyme previously characterised as a methyl sink that disposes of excess methyl groups carried by the universal methyl donor S-adenosyl methionine (SAM or AdoMet). In healthy tissues, histone methylation was inversely correlated to the levels of an alternative methyl sink, PEMT. These associations affected the levels of multiple histone marks on chromatin genome-wide but had no detectable impact on transcriptional regulation. We show that HMTs with a variety of different associations to transcription are co-regulated by the Retinoblastoma (Rb) tumour suppressor in human cells. Rb-mutant cancers show increased total HMT activity and down-regulation of NNMT. Together, our results suggest that the total activity of HMTs affects SAM metabolism, independent of transcriptional regulation.
  22. Development. 2023 Oct 15. pii: dev201610. [Epub ahead of print]150(20):
      Metabolism is crucial for development through supporting cell growth, energy production, establishing cell identity, developmental signaling and pattern formation. In many model systems, development occurs alongside metabolic transitions as cells differentiate and specialize in metabolism that supports new functions. Some cells exhibit metabolic flexibility to circumvent mutations or aberrant signaling, whereas other cell types require specific nutrients for developmental progress. Metabolic gradients and protein modifications enable pattern formation and cell communication. On an organism level, inadequate nutrients or stress can limit germ cell maturation, implantation and maturity through diapause, which slows metabolic activities until embryonic activation under improved environmental conditions.
    Keywords:  Diapause; Embryogenesis; Metabolism; Signaling
  23. Proteomes. 2023 Sep 27. pii: 28. [Epub ahead of print]11(4):
      The budding yeast Saccharomyces cerevisiae is a powerful model system that is widely used to investigate many cellular processes. The harvesting of yeast cells is the first step in almost every experimental procedure. Here, yeast cells are isolated from their growth medium, collected, and used for successive experiments or analysis. The two most common methods to harvest S. cerevisiae are centrifugation and filtration. Understanding if and how centrifugation and filtration affect yeast physiology is essential with respect to downstream data interpretation. Here, we profile and compare the proteomes and the phosphoproteomes, using isobaric label-based quantitative mass spectrometry, of three common methods used to harvest S. cerevisiae cells: low-speed centrifugation, high-speed centrifugation, and filtration. Our data suggest that, while the proteome was stable across the tested conditions, hundreds of phosphorylation events were different between centrifugation and filtration. Our analysis shows that, under our experimental conditions, filtration may cause both cell wall and osmotic stress at higher levels compared to centrifugation, implying harvesting-method-specific stresses. Thus, considering that the basal activation levels of specific stresses may differ under certain harvesting conditions is an important, but often overlooked, aspect of experimental design.
    Keywords:  TMTpro; phosphoproteome analysis; proteome analysis; stress; yeast
  24. Science. 2023 Oct 27. 382(6669): 360
      How rodents survive on summits is a mystery.
  25. bioRxiv. 2023 Oct 10. pii: 2023.10.08.561452. [Epub ahead of print]
      During development and disease progression, cells are subject to osmotic and mechanical stresses that modulate cell volume, which fundamentally influences cell homeostasis and has been linked to a variety of cellular functions. It is not well understood how the mechanobiological state of cells is programmed by the interplay of intracellular organization and complex extracellular mechanics when stimulated by cell volume modulation. Here, by controlling cell volume via osmotic pressure, we evaluate physical phenotypes (including cell shape, morphodynamics, traction force, and extracellular matrix (ECM) remodeling) and molecular signaling (YAP), and we uncover fundamental transitions in active biophysical states. We demonstrate that volumetric compression shifts the ratiometric balance of Rho GTPase activities, thereby altering mechanosensing and cytoskeletal organization in a reversible manner. Specifically, volumetric compression controls cell spreading, adhesion formation, and YAP nuclear translocation, while maintaining cell contractile activity. Furthermore, we show that on physiologically relevant fibrillar collagen I matrices, which are highly non-elastic, cells exhibit additional modes of cell volume-dependent mechanosensing that are not observable on elastic substrates. Notably, volumetric compression regulates the dynamics of cell-ECM interactions and irreversible ECM remodeling via Rac-directed protrusion dynamics, at both the single-cell level and the multicellular level. Our findings support that cell volume is a master biophysical regulator and reveal its roles in cell mechanical state transition, cell-ECM interactions, and biophysical tissue programming.
  26. Biophys J. 2023 Oct 23. pii: S0006-3495(23)00655-0. [Epub ahead of print]
      Phenotypic adaptation is a universal feature of biological systems navigating highly variable environments. Recent empirical data support the role of memory-driven decision-making in cellular systems navigating uncertain future nutrient landscapes, wherein a distinct growth phenotype emerges in fluctuating conditions. We develop a simple stochastic mathematical model to describe memory-driven cellular adaptation required for systems to optimally navigate such uncertainty. In this framework, adaptive populations traverse dynamic environments by inferring future variation from a memory of prior states, and memory capacity imposes a fundamental trade-off between the speed and accuracy of adaptation to new fluctuating environments. Our results suggest that observed growth reductions that occur in fluctuating environments are a direct consequence of optimal decision-making and result from bet-hedging and occasional phenotypic-environmental mismatch. We anticipate that this modeling framework will be useful for studying the role of memory in phenotypic adaptation, including in the design of temporally varying therapies against adaptive systems.
    Keywords:  Cellular adaptation; Optimal cellular decision-making; Phenotypic switching; Stochastic dynamic programming
  27. bioRxiv. 2023 Oct 18. pii: 2023.10.13.562064. [Epub ahead of print]
      Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.
  28. Nat Rev Cancer. 2023 Oct 26.
      Cancers undergo sequential changes to proton (H+) concentration and sensing that are consequences of the disease and facilitate its further progression. The impact of protonation state on protein activity can arise from alterations to amino acids or their titration. Indeed, many cancer-initiating mutations influence pH balance, regulation or sensing in a manner that enables growth and invasion outside normal constraints as part of oncogenic transformation. These cancer-supporting effects become more prominent when tumours develop an acidic microenvironment owing to metabolic reprogramming and disordered perfusion. The ensuing intracellular and extracellular pH disturbances affect multiple aspects of tumour biology, ranging from proliferation to immune surveillance, and can even facilitate further mutagenesis. As a selection pressure, extracellular acidosis accelerates disease progression by favouring acid-resistant cancer cells, which are typically associated with aggressive phenotypes. Although acid-base disturbances in tumours often occur alongside hypoxia and lactate accumulation, there is now ample evidence for a distinct role of H+-operated responses in key events underpinning cancer. The breadth of these actions presents therapeutic opportunities to change the trajectory of disease.
  29. PLoS Biol. 2023 Oct;21(10): e3002341
      There is a growing appreciation that the direct interaction between bacteriophages and the mammalian host can facilitate diverse and unexplored symbioses. Yet the impact these bacteriophages may have on mammalian cellular and immunological processes is poorly understood. Here, we applied highly purified phage T4, free from bacterial by-products and endotoxins to mammalian cells and analyzed the cellular responses using luciferase reporter and antibody microarray assays. Phage preparations were applied in vitro to either A549 lung epithelial cells, MDCK-I kidney cells, or primary mouse bone marrow derived macrophages with the phage-free supernatant serving as a comparative control. Highly purified T4 phages were rapidly internalized by mammalian cells and accumulated within macropinosomes but did not activate the inflammatory DNA response TLR9 or cGAS-STING pathways. Following 8 hours of incubation with T4 phage, whole cell lysates were analyzed via antibody microarray that detected expression and phosphorylation levels of human signaling proteins. T4 phage application led to the activation of AKT-dependent pathways, resulting in an increase in cell metabolism, survival, and actin reorganization, the last being critical for macropinocytosis and potentially regulating a positive feedback loop to drive further phage internalization. T4 phages additionally down-regulated CDK1 and its downstream effectors, leading to an inhibition of cell cycle progression and an increase in cellular growth through a prolonged G1 phase. These interactions demonstrate that highly purified T4 phages do not activate DNA-mediated inflammatory pathways but do trigger protein phosphorylation cascades that promote cellular growth and survival. We conclude that mammalian cells are internalizing bacteriophages as a resource to promote cellular growth and metabolism.
  30. Nat Commun. 2023 Oct 27. 14(1): 6598
      L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.
  31. Gigascience. 2022 Dec 28. pii: giad082. [Epub ahead of print]12
      BACKGROUND: In recent years, 3-dimensional (3D) spheroid models have become increasingly popular in scientific research as they provide a more physiologically relevant microenvironment that mimics in vivo conditions. The use of 3D spheroid assays has proven to be advantageous as it offers a better understanding of the cellular behavior, drug efficacy, and toxicity as compared to traditional 2-dimensional cell culture methods. However, the use of 3D spheroid assays is impeded by the absence of automated and user-friendly tools for spheroid image analysis, which adversely affects the reproducibility and throughput of these assays.RESULTS: To address these issues, we have developed a fully automated, web-based tool called SpheroScan, which uses the deep learning framework called Mask Regions with Convolutional Neural Networks (R-CNN) for image detection and segmentation. To develop a deep learning model that could be applied to spheroid images from a range of experimental conditions, we trained the model using spheroid images captured using IncuCyte Live-Cell Analysis System and a conventional microscope. Performance evaluation of the trained model using validation and test datasets shows promising results.
    CONCLUSION: SpheroScan allows for easy analysis of large numbers of images and provides interactive visualization features for a more in-depth understanding of the data. Our tool represents a significant advancement in the analysis of spheroid images and will facilitate the widespread adoption of 3D spheroid models in scientific research. The source code and a detailed tutorial for SpheroScan are available at
    Keywords:  3D spheroids; Image analysis; Mask R-CNN; deep learning; high-throughput screening; image segmentation