bims-pideca 2024-07-21 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2024–07–21
nineteen papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

The LCLAT1/LYCAT acyltransferase is required for EGF-mediated phosphatidylinositol-3,4,5-trisphosphate generation and Akt signalling.
Phospholipase Cγ regulates lacrimal gland branching by competing with PI3K in phosphoinositide metabolism.
The Spectrum of "TFEopathies" - Flipping the mTOR Switch in Renal Tumorigenesis.
Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.
Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.
In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation.
Leveraging neighborhood representations of single-cell data to achieve sensitive DE testing with miloDE.
Single-cell sequencing of facial adipose tissue unveils FKBP5 as a therapeutic target for facial infiltrating lipomatosis.
AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.
Symbolic recording of signalling and cis-regulatory element activity to DNA.
Understanding P-Rex regulation: structural breakthroughs and emerging perspectives.
Circulating biomarkers of kidney angiomyolipoma and cysts in tuberous sclerosis complex patients.
Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.
Unilateral focal palmoplantar keratoderma associated with a postzygotic variant in PIK3CA and activation of the PI3K/AKT/mTOR pathway.
Automated cell type annotation and exploration of single-cell signaling dynamics using mass cytometry.
Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.
IGF-1 Acts through Kiss1-expressing Cells to Influence Metabolism and Reproduction.
TLNRD1 is a CCM complex component and regulates endothelial barrier integrity.
A benchmark of computational methods for correcting biases of established and unknown origin in CRISPR-Cas9 screening data.

Mol Biol Cell. 2024 Jul 18. mbcE23090361

The LCLAT1/LYCAT acyltransferase is required for EGF-mediated phosphatidylinositol-3,4,5-trisphosphate generation and Akt signalling.

Victoria Chan, Cristina Camardi, Kai Zhang, Laura A Orofiamma, Karen E Anderson, Jafarul Hoque, Leslie N Bone, Yasmin Awadeh, Daniel K C Lee, Norman J Fu, Jonathan T S Chow, Leonardo Salmena, Len R Stephens, Phillip T Hawkins, Costin N Antonescu, Roberto J Botelho.

Receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) stimulate phosphoinositide 3-kinases (PI3Ks) to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3β. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signalling promotes tumour progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signalling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signalling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signalling.

DOI: https://doi.org/10.1091/mbc.E23-09-0361
bioRxiv. 2024 Jul 02. pii: 2024.06.28.601066. [Epub ahead of print]

Phospholipase Cγ regulates lacrimal gland branching by competing with PI3K in phosphoinositide metabolism.

Qian Wang, Chenqi Tao, Yihua Wu, Karen E Anderson, Abdul Hannan, Chyuan-Sheng Lin, Phillip Hawkins, Len Stephens, Xin Zhang.

Although the regulation of branching morphogenesis by spatially distributed cues is well established, the role of intracellular signaling in determining the branching pattern remains poorly understood. In this study, we investigated the regulation and function of phospholipase C gamma (PLCγ) in Fibroblast Growth Factor (FGF) signaling in lacrimal gland development. We showed that deletion of PLCγ1 in the lacrimal gland epithelium leads to ectopic branching and acinar hyperplasia, which was phenocopied by either mutating the PLCγ1 binding site on Fgfr2 or disabling any of its SH2 domains. PLCγ1 inactivation did not change the level of Fgfr2 or affect MAPK signaling, but instead led to sustained AKT phosphorylation due to increased PIP3 production. Consistent with this, PLCγ1 mutant phenotype can be reproduced by elevation of PI3K signaling in Pten knockout and attenuated by blocking AKT signaling. This study demonstrated that PLCγ modulates PI3K signaling by shifting phosphoinositide metabolism, revealing an important role of signaling dynamics in conjunction with spatial cues in shaping branching morphogenesis.

DOI: https://doi.org/10.1101/2024.06.28.601066
Physiology (Bethesda). 2024 Jul 16.

The Spectrum of "TFEopathies" - Flipping the mTOR Switch in Renal Tumorigenesis.

Nicola Alesi, Kaushal Asrani, Tamara L Lotan, Elizabeth P Henske.

  The Mammalian Target of Rapamycin Complex 1 (mTORC1) is a serine threonine kinase that couples nutrient and growth factor signaling to the cellular control of metabolism and plays a fundamental role in aberrant proliferation in cancer. mTORC1 has previously been considered an "on/off" switch, capable of phosphorylating the entire pool of its substrates when activated. However recent studies have indicated that mTORC1 may be active towards its canonical substrates, 4EBP1 and S6K, involved in mRNA translation and protein synthesis, and inactive towards TFEB and TFE3, transcription factors involved in the regulation of lysosome biogenesis, in several pathological contexts. Among these conditions are Birt Hogg Dube (BHD) and recently, Tuberous Sclerosis Complex (TSC). Furthermore, TFEB and TFE3 hyperactivation in these syndromes, and in translocation Renal Cell Carcinomas (tRCC), drives mTORC1 activity towards the canonical substrates, through the transcriptional activation of the Rag GTPases, thereby positioning TFEB and TFE3 upstream of mTORC1 activity towards 4EBP1 and S6K. The expanding importance of TFEB and TFE3 in the pathogenesis of these renal diseases warrants a novel clinical grouping that we term "TFEopathies". Currently, there no therapeutic options directly targeting TFEB and TFE3, which represents a challenging and critically required avenue for cancer research.

Keywords:  Kidney TFEopathies; TFE3; TFEB; TSC; mTORC1

DOI:  https://doi.org/10.1152/physiol.00026.2024
Res Sq. 2024 Jul 03. pii: rs.3.rs-4595968. [Epub ahead of print]

Large-Scale Genome-Wide Optimization and Prediction of the Cre Recombinase System for Precise Genome Manipulation in Mice.

Vishnu Hosur, Valerie Erhardt, Elli Hartig, Kristian Lorenzo, Hannah Megathlin, Basile Tarchini.

The Cre-Lox recombination system is a powerful tool in mouse genetics, offering spatial-temporal control over gene expression and facilitating the large-scale generation of conditional knockout mice. Its versatility also extends to other research models, such as rats, pigs, and zebrafish. However, the Cre-Lox technology presents a set of challenges that includes high costs, a time-intensive process, and the occurrence of unpredictable recombination events, which can lead to unexpected phenotypic outcomes. To better understand factors affecting recombination, we embarked on a systematic and genome-wide analysis of Cre-mediated recombination in mice. To ensure uniformity and reproducibility, we generated 11 novel strains with conditional alleles at the ROSA26 locus, utilizing a single inbred mouse strain background, C57BL/6J. We examined several factors influencing Cre-recombination, including the inter- loxP distance, mutant loxP sites, the zygosity of the conditional alleles, chromosomal location, and the age of the breeders. We discovered that the selection of the Cre-driver strain profoundly impacts recombination efficiency. We also found that successful and complete recombination is best achieved when loxP sites are spaced between 1 to 4 kb apart, with mutant loxP sites facilitating recombination at distances of 1 to 3 kb. Furthermore, we demonstrate that complete recombination does not occur at an inter- loxP distance of ≥ 15 kb with wildtype loxP sites, nor at a distance of ≥ 7 kb with mutant lox71/66 sites. Interestingly, the age of the Cre-driver mouse at the time of breeding emerged as a critical factor in recombination efficiency, with best results observed between 8 and 20 weeks old. Moreover, crossing heterozygous floxed alleles with the Cre-driver strain resulted in more efficient recombination than using homozygous floxed alleles. Lastly, maintaining an inter- loxP distance of 4 kb or less ensures efficient recombination of the conditional allele, regardless of the chromosomal location. While CRISPR/Cas has revolutionized genome editing in mice, Cre-Lox technology remains a cornerstone for the generation of sophisticated alleles and for precise control of gene expression in mice. The knowledge gained here will enable investigators to select a Cre-Lox approach that is most efficient for their desired outcome in the generation of both germline and non-germline mouse models of human disease, thereby reducing time and cost of Cre-Lox technology-mediated genome modification.

DOI: https://doi.org/10.21203/rs.3.rs-4595968/v1
Commun Biol. 2024 Jul 19. 7(1): 884

Diversity of post-translational modifications and cell signaling revealed by single cell and single organelle mass spectrometry.

Dong-Gi Mun, Firdous A Bhat, Neha Joshi, Leticia Sandoval, Husheng Ding, Anu Jain, Jane A Peterson, Taewook Kang, Ganesh P Pujari, Jennifer L Tomlinson, Rohit Budhraja, Roman M Zenka, Nagarajan Kannan, Benjamin R Kipp, Surendra Dasari, Alexandre Gaspar-Maia, Rory L Smoot, Richard K Kandasamy, Akhilesh Pandey.

The rapid evolution of mass spectrometry-based single-cell proteomics now enables the cataloging of several thousand proteins from single cells. We investigated whether we could discover cellular heterogeneity beyond proteome, encompassing post-translational modifications (PTM), protein-protein interaction, and variants. By optimizing the mass spectrometry data interpretation strategy to enable the detection of PTMs and variants, we have generated a high-definition dataset of single-cell and nuclear proteomic-states. The data demonstrate the heterogeneity of cell-states and signaling dependencies at the single-cell level and reveal epigenetic drug-induced changes in single nuclei. This approach enables the exploration of previously uncharted single-cell and organellar proteomes revealing molecular characteristics that are inaccessible through RNA profiling.

DOI: https://doi.org/10.1038/s42003-024-06579-7
Cardiovasc Diabetol. 2024 Jul 18. 23(1): 258

In-depth phosphoproteomic profiling of the insulin signaling response in heart tissue and cardiomyocytes unveils canonical and specialized regulation.

Jonathan Samuel Achter, Estefania Torres Vega, Andrea Sorrentino, Konstantin Kahnert, Katrine Douglas Galsgaard, Pablo Hernandez-Varas, Michael Wierer, Jens Juul Holst, Jørgen Frank Pind Wojtaszewski, Robert William Mills, Rasmus Kjøbsted, Alicia Lundby.

   BACKGROUND: Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent.
METHODS: Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements.
RESULTS: We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided.
CONCLUSION: We present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.

Keywords:  Cardiac signaling; Cardiometabolic; Insulin resistance; Insulin signaling; Kinase; Metabolism; Phosphoproteomics; Phosphorylation; Proteomics; Tbc1d4

DOI:  https://doi.org/10.1186/s12933-024-02338-4
Genome Biol. 2024 Jul 18. 25(1): 189

Leveraging neighborhood representations of single-cell data to achieve sensitive DE testing with miloDE.

Alsu Missarova, Emma Dann, Leah Rosen, Rahul Satija, John Marioni.

Single-cell RNA-sequencing enables testing for differential expression (DE) between conditions at a cell type level. While powerful, one of the limitations of such approaches is that the sensitivity of DE testing is dictated by the sensitivity of clustering, which is often suboptimal. To overcome this, we present miloDE-a cluster-free framework for DE testing (available as an open-source R package). We illustrate the performance of miloDE on both simulated and real data. Using miloDE, we identify a transient hemogenic endothelia-like state in mouse embryos lacking Tal1 and detect distinct programs during macrophage activation in idiopathic pulmonary fibrosis.

DOI: https://doi.org/10.1186/s13059-024-03334-3
Stem Cell Res Ther. 2024 Jul 18. 15(1): 209

Single-cell sequencing of facial adipose tissue unveils FKBP5 as a therapeutic target for facial infiltrating lipomatosis.

Hongrui Chen, Bin Sun, Shih-Jen Chang, Zhang Yu, Yajing Qiu, Chen Hua, Xiaoxi Lin.

   BACKGROUND: Facial infiltrating lipomatosis is characterized by excessive growth of adipose tissue. Its etiology is associated with somatic phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) variants, but the specific mechanisms are not yet fully understood.
METHODS: We collected facial adipose tissue from both FIL patients and non-FIL individuals, isolated the stromal vascular fraction (SVF) and performed single-cell transcriptome sequencing on these samples.
RESULTS: We mapped out the cellular landscape within the SVF, with a specific focus on a deeper analysis of fibro-adipogenic precursor cells (FAPs). Our analysis revealed that FAPs from FIL patients (FIL-FAPs) significantly overexpressed FK506 binding protein 51 (FKBP5) compared to FAPs from individuals without FIL. Further experiments indicated that FKBP5 is regulated by the PI3K-AKT signaling pathway. The overactivation of this pathway led to an increase in FKBP5 expression. In vitro experiments demonstrated that FKBP5 promoted adipogenic differentiation of FAPs, a process that could be hindered by FKBP5 knockdown or inhibition. Additionally, in vivo assessments confirmed FKBP5's role in adipogenesis.
CONCLUSIONS: These insights into the pathogenesis of FIL underscore FKBP5 as a promising target for developing non-surgical interventions to manage the excessive adipose tissue growth in FIL.

Keywords:  FK506 binding protein 51; Facial infiltrating lipomatosis; Fibro-adipogenic precursor cells; Phosphatidylinositol 3-kinase catalytic subunit alpha

DOI:  https://doi.org/10.1186/s13287-024-03835-9
Proc Natl Acad Sci U S A. 2024 Jul 23. 121(30): e2303642121

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells.

Isaac Zin, Arnab China, Krishnendu Khan, Jeetendra K Nag, Kommireddy Vasu, Gauravi M Deshpande, Prabar K Ghosh, Debjit Khan, Iyappan Ramachandiran, Shinjini Ganguly, Ilaria Tamagno, Belinda Willard, Valentin Gogonea, Paul L Fox.

  Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

Keywords:  ADP-ribosylation; AKT; EPRS1; PARP1; aminoacyl-tRNA synthetase

DOI:  https://doi.org/10.1073/pnas.2303642121
Nature. 2024 Jul 17.

Symbolic recording of signalling and cis-regulatory element activity to DNA.

Wei Chen, Junhong Choi, Xiaoyi Li, Jenny F Nathans, Beth Martin, Wei Yang, Nobuhiko Hamazaki, Chengxiang Qiu, Jean-Benoît Lalanne, Samuel Regalado, Haedong Kim, Vikram Agarwal, Eva Nichols, Anh Leith, Choli Lee, Jay Shendure.

Measurements of gene expression or signal transduction activity are conventionally performed using methods that require either the destruction or live imaging of a biological sample within the timeframe of interest. Here we demonstrate an alternative paradigm in which such biological activities are stably recorded to the genome. Enhancer-driven genomic recording of transcriptional activity in multiplex (ENGRAM) is based on the signal-dependent production of prime editing guide RNAs that mediate the insertion of signal-specific barcodes (symbols) into a genomically encoded recording unit. We show how this strategy can be used for multiplex recording of the cell-type-specific activities of dozens to hundreds of cis-regulatory elements with high fidelity, sensitivity and reproducibility. Leveraging signal transduction pathway-responsive cis-regulatory elements, we also demonstrate time- and concentration-dependent genomic recording of WNT, NF-κB and Tet-On activities. By coupling ENGRAM to sequential genome editing via DNA Typewriter1, we stably record information about the temporal dynamics of two orthogonal signalling pathways to genomic DNA. Finally we apply ENGRAM to integratively record the transient activity of nearly 100 transcription factor consensus motifs across daily windows spanning the differentiation of mouse embryonic stem cells into gastruloids, an in vitro model of early mammalian development. Although these are proof-of-concept experiments and much work remains to fully realize the possibilities, the symbolic recording of biological signals or states within cells, to the genome and over time, has broad potential to complement contemporary paradigms for how we make measurements in biological systems.

DOI: https://doi.org/10.1038/s41586-024-07706-4
Biochem Soc Trans. 2024 Jul 18. pii: BST20231546. [Epub ahead of print]

Understanding P-Rex regulation: structural breakthroughs and emerging perspectives.

Gareth D Jones, Andrew M Ellisdon.

  Rho GTPases are a family of highly conserved G proteins that regulate numerous cellular processes, including cytoskeleton organisation, migration, and proliferation. The 20 canonical Rho GTPases are regulated by ∼85 guanine nucleotide exchange factors (GEFs), with the largest family being the 71 Diffuse B-cell Lymphoma (Dbl) GEFs. Dbl GEFs promote GTPase activity through the highly conserved Dbl homology domain. The specificity of GEF activity, and consequently GTPase activity, lies in the regulation and structures of the GEFs themselves. Dbl GEFs contain various accessory domains that regulate GEF activity by controlling subcellular localisation, protein interactions, and often autoinhibition. This review focuses on the two phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3)-dependent Rac exchangers (P-Rex), particularly the structural basis of P-Rex1 autoinhibition and synergistic activation. First, we discuss structures that highlight the conservation of P-Rex catalytic and phosphoinositide binding activities. We then explore recent breakthroughs in uncovering the structural basis for P-Rex1 autoinhibition and detail the proposed minimal two-step model of how PI(3,4,5)P3 and Gβγ synergistically activate P-Rex1 at the membrane. Additionally, we discuss the further layers of P-Rex regulation provided by phosphorylation and P-Rex2-PTEN coinhibitory complex formation, although these mechanisms remain incompletely understood. Finally, we leverage the available data to infer how cancer-associated mutations in P-Rex2 destabilise autoinhibition and evade PTEN coinhibitory complex formation, leading to increased P-Rex2 GEF activity and driving cancer progression and metastasis.

Keywords:  G-proteins; GTPases; Rac; guanine nucleotide exchange factor; phosphatidylinositol; structural biology

DOI:  https://doi.org/10.1042/BST20231546
iScience. 2024 Jul 19. 27(7): 110265

Circulating biomarkers of kidney angiomyolipoma and cysts in tuberous sclerosis complex patients.

Varvara I Rubtsova, Yujin Chun, Joohwan Kim, Cuauhtemoc B Ramirez, Sunhee Jung, Wonsuk Choi, Miranda E Kelly, Miranda L Lopez, Elizabeth Cassidy, Gabrielle Rushing, Dean J Aguiar, Wei Ling Lau, Rebecca S Ahdoot, Moyra Smith, Aimee L Edinger, Sang-Guk Lee, Cholsoon Jang, Gina Lee.

  Patients with tuberous sclerosis complex (TSC) develop multi-organ disease manifestations, with kidney angiomyolipomas (AML) and cysts being one of the most common and deadly. Early and regular AML/cyst detection and monitoring are vital to lower TSC patient morbidity and mortality. However, the current standard of care involves imaging-based methods that are not designed for rapid screening, posing challenges for early detection. To identify potential diagnostic screening biomarkers of AML/cysts, we performed global untargeted metabolomics in blood samples from 283 kidney AML/cyst-positive or -negative TSC patients using mass spectrometry. We identified 7 highly sensitive chemical features, including octanoic acid, that predict kidney AML/cysts in TSC patients. Patients with elevated octanoic acid have lower levels of very long-chain fatty acids (VLCFAs), suggesting that dysregulated peroxisome activity leads to overproduction of octanoic acid via VLCFA oxidation. These data highlight AML/cysts blood biomarkers for TSC patients and offers valuable metabolic insights into the disease.

Keywords:  Clinical genetics; Endocrinology; Pathophysiology

DOI:  https://doi.org/10.1016/j.isci.2024.110265
J Biol Chem. 2024 Jul 14. pii: S0021-9258(24)02076-3. [Epub ahead of print] 107575

Phosphorylation of GCN2 by mTOR confers adaptation to conditions of hyper-mTOR activation under stress.

Odai Darawshi, Olaya Yassin, Miri Shmuel, Ronald C Wek, S Jalil Mahdizadeh, Leif A Eriksson, Maria Hatzoglou, Boaz Tirosh.

Adaptation to shortage in free amino acids (AA) is mediated by two pathways, the integrated stress response (ISR) and the mechanistic target of rapamycin (mTOR). In response to reduced levels, primarily of leucine or arginine, mTOR in its complex 1 configuration (mTORC1) is suppressed leading to a decrease in translation initiation and elongation. The eIF2α kinase general control nonderepressible 2 (GCN2) is activated by uncharged tRNAs, leading to induction of the ISR in response to a broader range of AA shortage. ISR confers a reduced translation initiation, while promoting the selective synthesis of stress proteins, such as ATF4. To efficiently adapt to AA starvation, the two pathways are cross-regulated at multiple levels. Here we identified a new mechanism of ISR/mTORC1 crosstalk that optimizes survival under AA starvation, when mTORC1 is forced to remain active. mTORC1 activation during acute AA shortage, augmented ATF4 expression in a GCN2-dependent manner. Under these conditions, enhanced GCN2 activity was not dependent on tRNA sensing, inferring a different activation mechanism. We identified a labile physical interaction between GCN2 and mTOR that results in a phosphorylation of GCN2 on serine 230 by mTOR, which promotes GCN2 activity. When examined under prolonged AA starvation, GCN2 phosphorylation by mTOR promoted survival. Our data unveils an adaptive mechanism to AA starvation, when mTORC1 evades inhibition.

DOI: https://doi.org/10.1016/j.jbc.2024.107575
Eur J Dermatol. 2024 Jun 01. 34(3): 287-293

Unilateral focal palmoplantar keratoderma associated with a postzygotic variant in PIK3CA and activation of the PI3K/AKT/mTOR pathway.

Zhuoqing Gong, Sha Peng, Huijun Wang, Xingyuan Jiang, Xiaoping Ke, Zhimiao Lin.

  Palmoplantar keratoderma (PPK) is a group of -disorders with genetic and phenotypic heterogeneity featuring skin thickening of the palms and soles. More than 60 genes involved in various biological processes are implicated in PPK. PIK3CA is an oncogene encoding p110α, and its somatic variants contribute to a spectrum of congenital overgrowth disorders, including epidermal nevi (EN). To identify the genetic basis and elucidate the pathogenesis of a patient with unilateral focal PPK. Whole-exome sequencing and Sanger sequencing combined with laser capture microdissection (LCM) were performed on genomic DNA extracted from the patient's peripheral blood and skin lesion. Skin biopsies were taken from the lesion of the patient and normal controls for immunofluorescence. Molecular docking was performed using Alphafold2-multimer. A three-year-old girl presented with unilateral focal PPK with an identified missense -variant (c.3140A>G, p.His1047Arg) in PIK3CA from affected tissue. This variant only existed in the lesional epidermis. Elevated PI3K/AKT/mTOR signalling in the affected epidermis and an increased number of Ki67-positive keratinocytes were demonstrated. Molecular docking indicated instability of the p110α-p85α dimer caused by the PIK3CA His1047Arg variant. We describe the first PPK case associated with a variant in PIK3CA, which expands the spectrum of PIK3CA-related disorders. Our study further underscores the importance of the PI3K/AKT/mTOR pathway in the homeostasis of skin keratinization.

Keywords:  PI3K/AKT/mTOR pathway; PIK3CA; mosaicism; palmoplantar keratoderma; postzygotic variant

DOI:  https://doi.org/10.1684/ejd.2024.4704
iScience. 2024 Jul 19. 27(7): 110261

Automated cell type annotation and exploration of single-cell signaling dynamics using mass cytometry.

Dimitrios Kleftogiannnis, Sonia Gavasso, Benedicte Sjo Tislevoll, Nisha van der Meer, Inga K F Motzfeldt, Monica Hellesøy, Stein-Erik Gullaksen, Emmanuel Griessinger, Oda Fagerholt, Andrea Lenartova, Yngvar Fløisand, Jan Jacob Schuringa, Bjørn Tore Gjertsen, Inge Jonassen.

  Mass cytometry by time-of-flight (CyTOF) is an emerging technology allowing for in-depth characterization of cellular heterogeneity in cancer and other diseases. Unfortunately, high-dimensional analyses of CyTOF data remain quite demanding. Here, we deploy a bioinformatics framework that tackles two fundamental problems in CyTOF analyses namely (1) automated annotation of cell populations guided by a reference dataset and (2) systematic utilization of single-cell data for effective patient stratification. By applying this framework on several publicly available datasets, we demonstrate that the Scaffold approach achieves good trade-off between sensitivity and specificity for automated cell type annotation. Additionally, a case study focusing on a cohort of 43 leukemia patients reported salient interactions between signaling proteins that are sufficient to predict short-term survival at time of diagnosis using the XGBoost algorithm. Our work introduces an automated and versatile analysis framework for CyTOF data with many applications in future precision medicine projects.

Keywords:  Bioinformatics; Cancer; Machine learning

DOI:  https://doi.org/10.1016/j.isci.2024.110261
Proc Natl Acad Sci U S A. 2024 Jul 23. 121(30): e2319782121

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Daniel Maxim Iascone, Xue Zhang, Patricia Brafford, Clementina Mesaros, Yogev Sela, Samuel Hofbauer, Shirley L Zhang, Sukanya Madhwal, Kieona Cook, Pavel Pivarshev, Ben Z Stanger, Stewart Anderson, Chi V Dang, Amita Sehgal.

  Crosstalk between metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to disease. Here, we investigated whether maintenance of circadian rhythms depends on specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to signal from a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function across a series of pancreatic adenocarcinoma cell lines. Metabolic profiling of congenic tumor cell clones revealed substantial diversity among these lines that we used to identify clones to generate circadian reporter lines. We observed diverse circadian profiles among these lines that varied with their metabolic phenotype: The most hypometabolic line [exhibiting low levels of oxidative phosphorylation (OxPhos) and glycolysis] had the strongest rhythms, while the most hypermetabolic line had the weakest rhythms. Pharmacological enhancement of OxPhos decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, inhibition of OxPhos enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

Keywords:  adenocarcinoma; cancer; circadian rhythms; luciferase; metabolism

DOI:  https://doi.org/10.1073/pnas.2319782121
bioRxiv. 2024 Jul 04. pii: 2024.07.02.601722. [Epub ahead of print]

IGF-1 Acts through Kiss1-expressing Cells to Influence Metabolism and Reproduction.

Mengjie Wang, Seamus M Pugh, Judy Daboul, David Miller, Yong Xu, Jennifer W Hill.

   Objective: Kisspeptin, encoded by the Kiss1 gene, ties puberty and fertility to energy status; however, the metabolic factors that control Kiss1-expressing cells need to be clarified.
Methods: To evaluate the impact of IGF-1 on the metabolic and reproductive functions of kisspeptin producing cells, we created mice with IGF-1 receptor deletion driven by the Kiss1 promoter (IGF1RKiss1 mice). Previous studies have shown IGF-1 and insulin can bind to each other's receptor, permitting IGF-1 signaling in the absence of IGF1R. Therefore, we also generated mice with simultaneous deletion of the IGF1R and insulin receptor (IR) in Kiss1-expressing cells (IGF1R/IRKiss1 mice).
Results: Loss of IGF1R in Kiss1 cells caused stunted body length. In addition, female IGF1RKiss1 mice displayed lower body weight and food intake plus higher energy expenditure and physical activity. This phenotype was linked to higher proopiomelanocortin (POMC) expression and heightened brown adipose tissue (BAT) thermogenesis. Male IGF1RKiss1 mice had mild changes in metabolic functions. Moreover, IGF1RKiss1 mice of both sexes experienced delayed puberty. Notably, male IGF1RKiss1 mice had impaired adulthood fertility accompanied by lower gonadotropin and testosterone levels. Thus, IGF1R in Kiss1-expressing cells impacts metabolism and reproduction in a sex-specific manner. IGF1R/IRKiss1 mice had higher fat mass and glucose intolerance, suggesting IGF1R and IR in Kiss1-expressing cells together regulate body composition and glucose homeostasis.
Conclusions: Overall, our study shows that IGF1R and IR in Kiss1 have cooperative roles in body length, metabolism, and reproduction.

Keywords:  Body weight; IGF-1 receptor; Insulin receptor; Kiss1-expressing cells; Reproduction

DOI:  https://doi.org/10.1101/2024.07.02.601722
J Cell Biol. 2024 Sep 02. pii: e202310030. [Epub ahead of print]223(9):

TLNRD1 is a CCM complex component and regulates endothelial barrier integrity.

Neil J Ball, Sujan Ghimire, Gautier Follain, Ada O Pajari, Diana Wurzinger, Monika Vaitkevičiūtė, Alana R Cowell, Bence Berki, Johanna Ivaska, Ilkka Paatero, Benjamin T Goult, Guillaume Jacquemet.

We previously identified talin rod domain-containing protein 1 (TLNRD1) as a potent actin-bundling protein in vitro. Here, we report that TLNRD1 is expressed in the vasculature in vivo. Its depletion leads to vascular abnormalities in vivo and modulation of endothelial cell monolayer integrity in vitro. We demonstrate that TLNRD1 is a component of the cerebral cavernous malformations (CCM) complex through its direct interaction with CCM2, which is mediated by a hydrophobic C-terminal helix in CCM2 that attaches to a hydrophobic groove on the four-helix domain of TLNRD1. Disruption of this binding interface leads to CCM2 and TLNRD1 accumulation in the nucleus and actin fibers. Our findings indicate that CCM2 controls TLNRD1 localization to the cytoplasm and inhibits its actin-bundling activity and that the CCM2-TLNRD1 interaction impacts endothelial actin stress fiber and focal adhesion formation. Based on these results, we propose a new pathway by which the CCM complex modulates the actin cytoskeleton and vascular integrity.

DOI: https://doi.org/10.1083/jcb.202310030
Genome Biol. 2024 Jul 19. 25(1): 192

A benchmark of computational methods for correcting biases of established and unknown origin in CRISPR-Cas9 screening data.

Alessandro Vinceti, Raffaele M Iannuzzi, Isabella Boyle, Lucia Trastulla, Catarina D Campbell, Francisca Vazquez, Joshua M Dempster, Francesco Iorio.

BACKGROUND: CRISPR-Cas9 dropout screens are formidable tools for investigating biology with unprecedented precision and scale. However, biases in data lead to potential confounding effects on interpretation and compromise overall quality. The activity of Cas9 is influenced by structural features of the target site, including copy number amplifications (CN bias). More worryingly, proximal targeted loci tend to generate similar gene-independent responses to CRISPR-Cas9 targeting (proximity bias), possibly due to Cas9-induced whole chromosome-arm truncations or other genomic structural features and different chromatin accessibility levels.
RESULTS: We benchmarked eight computational methods, rigorously evaluating their ability to reduce both CN and proximity bias in the two largest publicly available cell-line-based CRISPR-Cas9 screens to date. We also evaluated the capability of each method to preserve data quality and heterogeneity by assessing the extent to which the processed data allows accurate detection of true positive essential genes, established oncogenetic addictions, and known/novel biomarkers of cancer dependency. Our analysis sheds light on the ability of each method to correct biases under different scenarios. AC-Chronos outperforms other methods in correcting both CN and proximity biases when jointly processing multiple screens of models with available CN information, whereas CRISPRcleanR is the top performing method for individual screens or when CN information is not available. In addition, Chronos and AC-Chronos yield a final dataset better able to recapitulate known sets of essential and non-essential genes.
CONCLUSIONS: Overall, our investigation provides guidance for the selection of the most appropriate bias-correction method, based on its strengths, weaknesses and experimental settings.

DOI: https://doi.org/10.1186/s13059-024-03336-1