bims-pideca 2023-04-23 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2023–04–23
27 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

PI3Kβ controls immune evasion in PTEN-deficient breast tumours.
Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.
Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells.
SynDISCO: A Mechanistic Modeling-Based Framework for Predictive Prioritization of Synergistic Drug Combinations Targeting Cell Signalling Networks.
Non-muscle myosin IIB is a driver of cellular reprogramming.
Somatic GJA4 mutation in intracranial extra-axial cavernous hemangiomas.
A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.
Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.
Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation.
Live-Cell Sender-Receiver Co-cultures for Quantitative Measurement of Paracrine Signaling Dynamics, Gene Expression, and Drug Response.
Teaching an old dog new tricks: A new tool for protein tyrosine phosphatase substrate discovery.
The IQGAP scaffolds: Critical nodes bridging receptor activation to cellular signaling.
Proteomic discovery of chemical probes that perturb protein complexes in human cells.
A hybrid single cell demultiplexing strategy that increases both cell recovery rate and calling accuracy.
The proteomic landscape of genome-wide genetic perturbations.
Integrating multiple single-cell multi-omics samples with Smmit.
Deletion of PTEN in microglia ameliorates chronic neuroinflammation following repetitive mTBI.
DeepFLR facilitates false localization rate control in phosphoproteomics.
Requirements for mammalian promoters to decode transcription factor dynamics.
Meta-Dynamic Network Modelling for Biochemical Networks.
Oncogenic CDK13 mutations impede nuclear RNA surveillance.
Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.
Primary cilia as dynamic and diverse signalling hubs in development and disease.
Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle.
PP1γ regulates neuronal insulin signaling and aggravates insulin resistance leading to AD-like phenotypes.
Multisite assessment of reproducibility in high-content cell migration imaging data.
Single-cell genomics meets human genetics.

Nature. 2023 Apr 19.

PI3Kβ controls immune evasion in PTEN-deficient breast tumours.

Johann S Bergholz, Qiwei Wang, Qi Wang, Michelle Ramseier, Sanjay Prakadan, Weihua Wang, Rong Fang, Sheheryar Kabraji, Qian Zhou, G Kenneth Gray, Kayley Abell-Hart, Shaozhen Xie, Xiaocan Guo, Hao Gu, Thanh Von, Tao Jiang, Shuang Tang, Gordon J Freeman, Hye-Jung Kim, Alex K Shalek, Thomas M Roberts, Jean J Zhao.

Loss of the PTEN tumour suppressor is one of the most common oncogenic drivers across all cancer types1. PTEN is the major negative regulator of PI3K signalling. The PI3Kβ isoform has been shown to play an important role in PTEN-deficient tumours, but the mechanisms underlying the importance of PI3Kβ activity remain elusive. Here, using a syngeneic genetically engineered mouse model of invasive breast cancer driven by ablation of both Pten and Trp53 (which encodes p53), we show that genetic inactivation of PI3Kβ led to a robust anti-tumour immune response that abrogated tumour growth in syngeneic immunocompetent mice, but not in immunodeficient mice. Mechanistically, PI3Kβ inactivation in the PTEN-null setting led to reduced STAT3 signalling and increased the expression of immune stimulatory molecules, thereby promoting anti-tumour immune responses. Pharmacological PI3Kβ inhibition also elicited anti-tumour immunity and synergized with immunotherapy to inhibit tumour growth. Mice with complete responses to the combined treatment displayed immune memory and rejected tumours upon re-challenge. Our findings demonstrate a molecular mechanism linking PTEN loss and STAT3 activation in cancer and suggest that PI3Kβ controls immune escape in PTEN-null tumours, providing a rationale for combining PI3Kβ inhibitors with immunotherapy for the treatment of PTEN-deficient breast cancer.

DOI: https://doi.org/10.1038/s41586-023-05940-w
Cell Metab. 2023 Apr 12. pii: S1550-4131(23)00126-2. [Epub ahead of print]

Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness.

Li Gu, Yahui Zhu, Kosuke Watari, Maiya Lee, Junlai Liu, Sofia Perez, Melinda Thai, Joshua E Mayfield, Bichen Zhang, Karina Cunha E Rocha, Fuming Li, Laura C Kim, Alexander C Jones, Igor H Wierzbicki, Xiao Liu, Alexandra C Newton, Tatiana Kisseleva, Jun Hee Lee, Wei Ying, David J Gonzalez, Alan R Saltiel, M Celeste Simon, Michael Karin.

  Insulin inhibits gluconeogenesis and stimulates glucose conversion to glycogen and lipids. How these activities are coordinated to prevent hypoglycemia and hepatosteatosis is unclear. Fructose-1,6-bisphosphatase (FBP1) is rate controlling for gluconeogenesis. However, inborn human FBP1 deficiency does not cause hypoglycemia unless accompanied by fasting or starvation, which also trigger paradoxical hepatomegaly, hepatosteatosis, and hyperlipidemia. Hepatocyte FBP1-ablated mice exhibit identical fasting-conditional pathologies along with AKT hyperactivation, whose inhibition reversed hepatomegaly, hepatosteatosis, and hyperlipidemia but not hypoglycemia. Surprisingly, fasting-mediated AKT hyperactivation is insulin dependent. Independently of its catalytic activity, FBP1 prevents insulin hyperresponsiveness by forming a stable complex with AKT, PP2A-C, and aldolase B (ALDOB), which specifically accelerates AKT dephosphorylation. Enhanced by fasting and weakened by elevated insulin, FBP1:PP2A-C:ALDOB:AKT complex formation, which is disrupted by human FBP1 deficiency mutations or a C-terminal FBP1 truncation, prevents insulin-triggered liver pathologies and maintains lipid and glucose homeostasis. Conversely, an FBP1-derived complex disrupting peptide reverses diet-induced insulin resistance.

Keywords:  AKT; FBP1; hepatomegaly; hepatosteatosis

DOI:  https://doi.org/10.1016/j.cmet.2023.03.021
Elife. 2023 Apr 20. pii: e85542. [Epub ahead of print]12

Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells.

Baolong Xia, Raghuvir Viswanatha, Yanhui Hu, Stephanie E Mohr, Norbert Perrimon.

  Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells.

Keywords:  CRISPR activation; D. melanogaster; genetic screening; genetics; genomics; rapamycin resistance gene

DOI:  https://doi.org/10.7554/eLife.85542
Methods Mol Biol. 2023 ;2634 357-381

SynDISCO: A Mechanistic Modeling-Based Framework for Predictive Prioritization of Synergistic Drug Combinations Targeting Cell Signalling Networks.

Sung-Young Shin, Lan K Nguyen.

  The widespread development of resistance to cancer monotherapies has prompted the need to identify combinatorial treatment approaches that circumvent drug resistance and achieve more durable clinical benefit. However, given the vast space of possible combinations of existing drugs, the inaccessibility of drug screens to candidate targets with no available drugs, and the significant heterogeneity of cancers, exhaustive experimental testing of combination treatments remains highly impractical. There is thus an urgent need to develop computational approaches that complement experimental efforts and aid the identification and prioritization of effective drug combinations. Here, we provide a practical guide to SynDISCO, a computational framework that leverages mechanistic ODE modeling to predict and prioritize synergistic combination treatments directed at signaling networks. We demonstrate the key steps of SynDISCO and its application to the EGFR-MET signaling network in triple negative breast cancer as an illustrative example. SynDISCO is, however, a network- and cancer-independent framework, and given a suitable ODE model of the network of interest, it could be leveraged to discover cancer-specific combination treatments.

Keywords:  Computational simulation; Drug combination; Drug synergy; Mechanistic modeling; ODE model; Signaling network; SynDISCO

DOI:  https://doi.org/10.1007/978-1-0716-3008-2_17
Mol Biol Cell. 2023 Apr 19. mbcE21080386

Non-muscle myosin IIB is a driver of cellular reprogramming.

Amanda E Balaban, Ly T S Nguyen, Eleana Parajón, Douglas N Robinson.

Nonmuscle myosin IIB (NMIIB) is considered a primary force generator during cell motility. Yet, many cell types, including motile cells, do not necessarily express NMIIB. Given the potential of cell engineering for the next wave of technologies, adding back NMIIB could be a strategy for creating super-cells with strategically altered cell morphology and motility. However, we wondered what unforeseen consequences could arise from such an approach. Here, we leveraged pancreatic cancer cells, which do not express NMIIB. We generated a series of cells where we added back NMIIB and strategic mutants that increase the ADP-bound time or alter the phosphorylation control of bipolar filament assembly. We characterized the cellular phenotypes and conducted RNA-seq analysis. The addition of NMIIB and the different mutants each has specific consequences for cell morphology, metabolism, cortical tension, mechanoresponsiveness, and gene expression. Major modes of ATP production are shifted, including alterations in spare respiratory capacity and the dependence upon glycolysis or oxidative phosphorylation. Several metabolic and growth pathways undergo significant changes in gene expression. This work demonstrates that NMIIB is highly integrated with many cellular systems and simple cell engineering has profound impact that extends beyond the primary contractile activity presumably being added to the cells.

DOI: https://doi.org/10.1091/mbc.E21-08-0386
Stroke Vasc Neurol. 2023 Apr 18. pii: svn-2022-002227. [Epub ahead of print]

Somatic GJA4 mutation in intracranial extra-axial cavernous hemangiomas.

Ran Huo, Yingxi Yang, Hongyuan Xu, Shaozhi Zhao, Dong Song, Jiancong Weng, Ruochen Ma, Yingfan Sun, Jie Wang, Yuming Jiao, Junze Zhang, Qiheng He, Ruolei Wu, Shuo Wang, Ji-Zong Zhao, Junting Zhang, Jiguang Wang, Yong Cao.

   OBJECTIVE: Extra-axial cavernous hemangiomas (ECHs) are sporadic and rare intracranial occupational lesions that usually occur within the cavernous sinus. The aetiology of ECHs remains unknown.
METHODS: Whole-exome sequencing was performed on ECH lesions from 12 patients (discovery cohort) and droplet digital polymerase-chain-reaction (ddPCR) was used to confirm the identified mutation in 46 additional cases (validation cohort). Laser capture microdissection (LCM) was carried out to capture and characterise subgroups of tissue cells. Mechanistic and functional investigations were carried out in human umbilical vein endothelial cells and a newly established mouse model.
RESULTS: We detected somatic GJA4 mutation (c.121G>T, p.G41C) in 5/12 patients with ECH in the discovery cohort and confirmed the finding in the validation cohort (16/46). LCM followed by ddPCR revealed that the mutation was enriched in lesional endothelium. In vitro experiments in endothelial cells demonstrated that the GJA4 mutation activated SGK-1 signalling that in turn upregulated key genes involved in cell hyperproliferation and the loss of arterial specification. Compared with wild-type littermates, mice overexpressing the GJA4 mutation developed ECH-like pathological morphological characteristics (dilated venous lumen and elevated vascular density) in the retinal superficial vascular plexus at the postnatal 3 weeks, which were reversed by an SGK1 inhibitor, EMD638683.
CONCLUSIONS: We identified a somatic GJA4 mutation that presents in over one-third of ECH lesions and proposed that ECHs are vascular malformations due to GJA4-induced activation of the SGK1 signalling pathway in brain endothelial cells.

Keywords:  Vascular Malformations

DOI:  https://doi.org/10.1136/svn-2022-002227
Nucleic Acids Res. 2023 Apr 18. pii: gkad255. [Epub ahead of print]

A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells.

Antonio Carusillo, Sibtain Haider, Raul Schäfer, Manuel Rhiel, Daniel Türk, Kay O Chmielewski, Julia Klermund, Laura Mosti, Geoffroy Andrieux, Richard Schäfer, Tatjana I Cornu, Toni Cathomen, Claudio Mussolino.

Precise genome editing requires the resolution of nuclease-induced DNA double strand breaks (DSBs) via the homology-directed repair (HDR) pathway. In mammals, this is typically outcompeted by non-homologous end-joining (NHEJ) that can generate potentially genotoxic insertion/deletion mutations at DSB sites. Because of higher efficacy, clinical genome editing has been restricted to imperfect but efficient NHEJ-based approaches. Hence, strategies that promote DSB resolution via HDR are essential to facilitate clinical transition of HDR-based editing strategies and increase safety. Here we describe a novel platform that consists of a Cas9 fused to DNA repair factors to synergistically inhibit NHEJ and favor HDR for precise repairing of Cas-induced DSBs. Compared to canonical CRISPR/Cas9, the increase in error-free editing ranges from 1.5-fold to 7-fold in multiple cell lines and in primary human cells. This novel CRISPR/Cas9 platform accepts clinically relevant repair templates, such as oligodeoxynucleotides (ODNs) and adeno-associated virus (AAV)-based vectors, and has a lower propensity to induce chromosomal translocations as compared to benchmark CRISPR/Cas9. The observed reduced mutational burden, resulting from diminished indel formation at on- and off-target sites, provides a remarkable gain in safety and advocates this novel CRISPR system as an attractive tool for therapeutic applications depending on precision genome editing.

DOI: https://doi.org/10.1093/nar/gkad255
Cell Syst. 2023 Apr 19. pii: S2405-4712(23)00078-9. [Epub ahead of print]14(4): 252-257

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Frederick R Adler, Alexander R A Anderson, Abhinav Bhushan, Paul Bogdan, Jose Javier Bravo-Cordero, Amy Brock, Yun Chen, Edna Cukierman, Kathleen E DelGiorno, Gerald V Denis, Meghan C Ferrall-Fairbanks, Zev Jordan Gartner, Ronald N Germain, Deborah M Gordon, Ginger Hunter, Mohit Kumar Jolly, Loukia Georgiou Karacosta, Karthikeyan Mythreye, Parag Katira, Rajan P Kulkarni, Matthew L Kutys, Arthur D Lander, Ashley M Laughney, Herbert Levine, Emil Lou, Pedro R Lowenstein, Kristyn S Masters, Dana Pe'er, Shelly R Peyton, Manu O Platt, Jeremy E Purvis, Gerald Quon, Jennifer K Richer, Nicole C Riddle, Analiz Rodriguez, Joshua C Snyder, Gregory Lee Szeto, Claire J Tomlin, Itai Yanai, Ioannis K Zervantonakis, Hannah Dueck.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

DOI: https://doi.org/10.1016/j.cels.2023.03.002
Nat Chem. 2023 Apr 17.

Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation.

Emily R Ruskowitz, Brizzia G Munoz-Robles, Alder C Strange, Carson H Butcher, Sebastian Kurniawan, Jeremy R Filteau, Cole A DeForest.

Proteins provide essential functional regulation of many bioprocesses across all scales of life; however, new techniques to specifically modulate protein activity within living systems and in engineered biomaterials are needed to better interrogate fundamental cell signalling and guide advanced decisions of biological fate. Here we establish a generalizable strategy to rapidly and irreversibly activate protein function with full spatiotemporal control. Through the development of a genetically encoded and light-activated SpyLigation (LASL), bioactive proteins can be stably reassembled from non-functional split fragment pairs following brief exposure (typically minutes) to cytocompatible light. Employing readily accessible photolithographic processing techniques to specify when, where and how much photoligation occurs, we demonstrate precise protein activation of UnaG, NanoLuc and Cre recombinase using LASL in solution, biomaterials and living mammalian cells, as well as optical control over protein subcellular localization. Looking forward, we expect that these photoclick-based optogenetic approaches will find tremendous utility in probing and directing complex cellular fates in both time and three-dimensional space.

DOI: https://doi.org/10.1038/s41557-023-01152-x
Methods Mol Biol. 2023 ;2634 285-314

Live-Cell Sender-Receiver Co-cultures for Quantitative Measurement of Paracrine Signaling Dynamics, Gene Expression, and Drug Response.

Michael Pargett, Abhineet R Ram, Vaibhav Murthy, Alexander E Davies.

  Paracrine signaling is a fundamental process regulating tissue development, repair, and pathogenesis of diseases such as cancer. Herein we describe a method for quantitatively measuring paracrine signaling dynamics, and resultant gene expression changes, in living cells using genetically encoded signaling reporters and fluorescently tagged gene loci. We discuss considerations for selecting paracrine "sender-receiver" cell pairs, appropriate reporters, the use of this system to ask diverse experimental questions and screen drugs blocking intracellular communication, data collection, and the use of computational approaches to model and interpret these experiments.

Keywords:  Cancer; Drug screening; ERK; Intercellular communication; Live-cell microscopy; MAPK; Single cell

DOI:  https://doi.org/10.1007/978-1-0716-3008-2_13
J Biol Chem. 2023 Apr 18. pii: S0021-9258(23)01759-3. [Epub ahead of print] 104731

Teaching an old dog new tricks: A new tool for protein tyrosine phosphatase substrate discovery.

Anton M Bennett.

The identification of substrates for protein tyrosine phosphatases (PTPs) is critical for a complete understanding of how these enzymes function. In a recent study in the JBC, Bonham et al. developed a modified method combining substrate-trapping mutations with proximity-labeling mass spectrometry to identify the protein substrates and interactors of PTP1B. This method revealed interaction networks in breast cancer cell models and discovered novel targets of PTP1B that regulate HER2 signaling pathways. This strategy represents a versatile new tool for identifying the functional interactions between PTPs and their substrates.

DOI: https://doi.org/10.1016/j.jbc.2023.104731
J Cell Biol. 2023 Jun 05. pii: e202205062. [Epub ahead of print]222(6):

The IQGAP scaffolds: Critical nodes bridging receptor activation to cellular signaling.

Louise Thines, Francis J Roushar, Andrew C Hedman, David B Sacks.

The scaffold protein IQGAP1 assembles multiprotein signaling complexes to influence biological functions. Cell surface receptors, particularly receptor tyrosine kinases and G-protein coupled receptors, are common IQGAP1 binding partners. Interactions with IQGAP1 modulate receptor expression, activation, and/or trafficking. Moreover, IQGAP1 couples extracellular stimuli to intracellular outcomes via scaffolding of signaling proteins downstream of activated receptors, including mitogen-activated protein kinases, constituents of the phosphatidylinositol 3-kinase pathway, small GTPases, and β-arrestins. Reciprocally, some receptors influence IQGAP1 expression, subcellular localization, binding properties, and post-translational modifications. Importantly, the receptor:IQGAP1 crosstalk has pathological implications ranging from diabetes and macular degeneration to carcinogenesis. Here, we describe the interactions of IQGAP1 with receptors, summarize how they modulate signaling, and discuss their contribution to pathology. We also address the emerging functions in receptor signaling of IQGAP2 and IQGAP3, the other human IQGAP proteins. Overall, this review emphasizes the fundamental roles of IQGAPs in coupling activated receptors to cellular homeostasis.

DOI: https://doi.org/10.1083/jcb.202205062
Mol Cell. 2023 Apr 18. pii: S1097-2765(23)00239-3. [Epub ahead of print]

Proteomic discovery of chemical probes that perturb protein complexes in human cells.

Michael R Lazear, Jarrett R Remsberg, Martin G Jaeger, Katherine Rothamel, Hsuan-Lin Her, Kristen E DeMeester, Evert Njomen, Simon J Hogg, Jahan Rahman, Landon R Whitby, Sang Joon Won, Michael A Schafroth, Daisuke Ogasawara, Minoru Yokoyama, Garrett L Lindsey, Haoxin Li, Jason Germain, Sabrina Barbas, Joan Vaughan, Thomas W Hanigan, Vincent F Vartabedian, Christopher J Reinhardt, Melissa M Dix, Seong Joo Koo, Inha Heo, John R Teijaro, Gabriel M Simon, Brahma Ghosh, Omar Abdel-Wahab, Kay Ahn, Alan Saghatelian, Bruno Melillo, Stuart L Schreiber, Gene W Yeo, Benjamin F Cravatt.

  Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

Keywords:  activity-based protein profiling; chemical probe; covalent; cysteine; proteasome; protein complexes; proteomics; size-exclusion chromatography; spliceosome

DOI:  https://doi.org/10.1016/j.molcel.2023.03.026
bioRxiv. 2023 Apr 04. pii: 2023.04.02.535299. [Epub ahead of print]

A hybrid single cell demultiplexing strategy that increases both cell recovery rate and calling accuracy.

Lei Li, Jiayi Sun, Yanbin Fu, Siriruk Changrob, Joshua J C McGrath, Patrick C Wilson.

Recent advances in single cell RNA sequencing allow users to pool multiple samples into one run and demultiplex in downstream analysis, greatly increasing the experimental efficiency and cost-effectiveness. However, the expensive reagents for cell labeling, limited pooling capacity, non-ideal cell recovery rate and calling accuracy remain great challenges for this approach. To date, there are two major demultiplexing methods, antibody-based cell hashing and Single Nucleotide Polymorphism (SNP)-based genomic signature profiling, and each method has advantages and limitations. Here, we propose a hybrid demultiplexing strategy that increases calling accuracy and cell recovery at the same time. We first develop a computational algorithm that significantly increases calling accuracy of cell hashing. Next, we cluster all single cells based on their SNP profiles. Finally, we integrate results from both methods to make corrections and retrieve cells that are only identifiable in one method but not the other. By testing on several real-world datasets, we demonstrate that this hybrid strategy combines advantages of both methods, resulting in increased cell recovery and calling accuracy at lower cost.
Highlights: An improved algorithm for cell hashing that distinguishes true positive from background for each individual hashtag at higher accuracyThis hybrid strategy increases cell recovery and calling accuracy while lowering experimental costThis hybrid demultiplexing strategy is applicable for single-cell RNA sequencing with different donor species, subjects, and cell populationsDoublet rate is a major determinant of the performance of SNP-based demultiplexing method.

DOI: https://doi.org/10.1101/2023.04.02.535299
Cell. 2023 Apr 17. pii: S0092-8674(23)00300-8. [Epub ahead of print]

The proteomic landscape of genome-wide genetic perturbations.

Christoph B Messner, Vadim Demichev, Julia Muenzner, Simran K Aulakh, Natalie Barthel, Annika Röhl, Lucía Herrera-Domínguez, Anna-Sophia Egger, Stephan Kamrad, Jing Hou, Guihong Tan, Oliver Lemke, Enrica Calvani, Lukasz Szyrwiel, Michael Mülleder, Kathryn S Lilley, Charles Boone, Georg Kustatscher, Markus Ralser.

  Functional genomic strategies have become fundamental for annotating gene function and regulatory networks. Here, we combined functional genomics with proteomics by quantifying protein abundances in a genome-scale knockout library in Saccharomyces cerevisiae, using data-independent acquisition mass spectrometry. We find that global protein expression is driven by a complex interplay of (1) general biological properties, including translation rate, protein turnover, the formation of protein complexes, growth rate, and genome architecture, followed by (2) functional properties, such as the connectivity of a protein in genetic, metabolic, and physical interaction networks. Moreover, we show that functional proteomics complements current gene annotation strategies through the assessment of proteome profile similarity, protein covariation, and reverse proteome profiling. Thus, our study reveals principles that govern protein expression and provides a genome-spanning resource for functional annotation.

Keywords:  Saccharomyces cerevisiae; data-independent acquisition; deletion; functional genomics; functional proteomics; gene annotation; high throughput; knockout; quantitative proteomics; systems biology

DOI:  https://doi.org/10.1016/j.cell.2023.03.026
bioRxiv. 2023 Apr 06. pii: 2023.04.06.535857. [Epub ahead of print]

Integrating multiple single-cell multi-omics samples with Smmit.

Changxin Wan, Zhicheng Ji.

Multi-sample single-cell multi-omics datasets, which simultaneously measure multiple data modalities in the same cells and in multiple samples, facilitate the study of gene expression and gene regulatory activities on a population scale. Existing integration methods can integrate either multiple samples or multiple modalities, but not both simultaneously. To address this limitation, we developed Smmit, a computational pipeline that leverages existing integration methods to simultaneously integrate both samples and modalities and produces a unified representation of reduced dimensions. We demonstrate Smmitâ€™s capability to integrate information across samples and modalities while preserving cell type differences in two real datasets. Smmit is an R software package that is freely available at Github: https://github.com/zji90/Smmit.

DOI: https://doi.org/10.1101/2023.04.06.535857
Mol Cell Neurosci. 2023 Apr 19. pii: S1044-7431(23)00049-0. [Epub ahead of print] 103855

Deletion of PTEN in microglia ameliorates chronic neuroinflammation following repetitive mTBI.

Andrew Pearson, Camila Ortiz, Max Eisenbaum, Clara Arrate, Mackenzie Browning, Michael Mullan, Corbin Bachmeier, Fiona Crawford, Joseph O Ojo.

  Traumatic brain injury is a leading cause of morbidity and mortality in adults and children in developed nations. Following the primary injury, microglia, the resident innate immune cells of the CNS, initiate several inflammatory signaling cascades and pathophysiological responses that may persist chronically; chronic neuroinflammation following TBI has been closely linked to the development of neurodegeneration and neurological dysfunction. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that have been shown to regulate several key mechanisms in the inflammatory response to TBI. Increasing evidence has shown that the modulation of the PI3K/AKT signaling pathway has the potential to influence the cellular response to inflammatory stimuli. However, directly targeting PI3K signaling poses several challenges due to its regulatory role in several cell survival pathways. We have previously identified that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN), the major negative regulator of PI3K/AKT signaling, is dysregulated following exposure to repetitive mild traumatic brain injury (r-mTBI). Moreover, this dysregulated PI3K/AKT signaling was correlated with chronic microglial-mediated neuroinflammation. Therefore, we interrogated microglial-specific PTEN as a therapeutic target in TBI by generating a microglial-specific, Tamoxifen inducible conditional PTEN knockout model using a CX3CR1 Cre recombinase mouse line PTENfl/fl/CX3CR1+/CreERT2 (mcg-PTENcKO), and exposed them to our 20-hit r-mTBI paradigm. Animals were treated with tamoxifen at 76 days post-last injury, and the effects of microglia PTEN deletion on immune-inflammatory responses were assessed at 90-days post last injury. We observed that the deletion of microglial PTEN ameliorated the proinflammatory response to repetitive brain trauma, not only reducing chronic microglial activation and proinflammatory cytokine production but also rescuing TBI-induced reactive astrogliosis, demonstrating that these effects extended beyond microglia alone. Additionally, we observed that the pharmacological inhibition of PTEN with BpV(HOpic) ameliorated the LPS-induced activation of microglial NFκB signaling in vitro. Together, these data provide support for the role of PTEN as a regulator of chronic neuroinflammation following repetitive mild TBI.

Keywords:  Microglia; Microglial activation; Neuroinflammation; PTEN; TBI; Traumatic brain injury

DOI:  https://doi.org/10.1016/j.mcn.2023.103855
Nat Commun. 2023 Apr 20. 14(1): 2269

DeepFLR facilitates false localization rate control in phosphoproteomics.

Yu Zong, Yuxin Wang, Yi Yang, Dan Zhao, Xiaoqing Wang, Chengpin Shen, Liang Qiao.

Protein phosphorylation is a post-translational modification crucial for many cellular processes and protein functions. Accurate identification and quantification of protein phosphosites at the proteome-wide level are challenging, not least because efficient tools for protein phosphosite false localization rate (FLR) control are lacking. Here, we propose DeepFLR, a deep learning-based framework for controlling the FLR in phosphoproteomics. DeepFLR includes a phosphopeptide tandem mass spectrum (MS/MS) prediction module based on deep learning and an FLR assessment module based on a target-decoy approach. DeepFLR improves the accuracy of phosphopeptide MS/MS prediction compared to existing tools. Furthermore, DeepFLR estimates FLR accurately for both synthetic and biological datasets, and localizes more phosphosites than probability-based methods. DeepFLR is compatible with data from different organisms, instruments types, and both data-dependent and data-independent acquisition approaches, thus enabling FLR estimation for a broad range of phosphoproteomics experiments.

DOI: https://doi.org/10.1038/s41467-023-38035-1
Nucleic Acids Res. 2023 Apr 18. pii: gkad273. [Epub ahead of print]

Requirements for mammalian promoters to decode transcription factor dynamics.

Enoch B Antwi, Yassine Marrakchi, Özgün Çiçek, Thomas Brox, Barbara Di Ventura.

In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.

DOI: https://doi.org/10.1093/nar/gkad273
Methods Mol Biol. 2023 ;2634 167-189

Meta-Dynamic Network Modelling for Biochemical Networks.

Anthony Hart, Lan K Nguyen.

  ODE modelling requires accurate knowledge of parameter and state variable values to deliver accurate and robust predictions. Parameters and state variables, however, are rarely static and immutable entities, especially in a biological context. This observation undermines the predictions made by ODE models that rely on specific parameter and state variable values and limits the contexts in which their predictions remain accurate and useful. Meta-dynamic network (MDN) modelling is a technique that can be synergistically integrated into an ODE modelling pipeline to assist in overcoming these limitations. The core mechanic of MDN modelling is the generation of a large number of model instances, each with a unique set of parameters and/or state variable values, followed by the simulation of each to determine how parameter and state variable variation affects protein dynamics. This process reveals the range of possible protein dynamics for a given network topology. Since MDN modelling is integrated with traditional ODE modelling, it can also be used to investigate the underlying causal mechanics. This technique is particularly suited to the investigation of network behaviors in systems that are highly heterogenous or systems wherein the network properties can change over time. MDN is a collection of principles rather than a strict protocol, so in this chapter, we have introduced the core principles using an example, the Hippo-ERK crosstalk signalling network.

Keywords:  ERK pathway; Heterogeneity; Hippo pathway; Meta-dynamic network modelling; ODE modelling; Protein dynamics; Signalling networks

DOI:  https://doi.org/10.1007/978-1-0716-3008-2_8
Science. 2023 Apr 21. 380(6642): eabn7625

Oncogenic CDK13 mutations impede nuclear RNA surveillance.

Megan L Insco, Brian J Abraham, Sara J Dubbury, Ines H Kaltheuner, Sofia Dust, Constance Wu, Kevin Y Chen, David Liu, Stanislav Bellaousov, Anna M Cox, Benjamin J E Martin, Tongwu Zhang, Calvin G Ludwig, Tania Fabo, Rodsy Modhurima, Dakarai E Esgdaille, Telmo Henriques, Kevin M Brown, Stephen J Chanock, Matthias Geyer, Karen Adelman, Phillip A Sharp, Richard A Young, Paul L Boutz, Leonard I Zon.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

DOI: https://doi.org/10.1126/science.abn7625
Mol Cell. 2023 Apr 20. pii: S1097-2765(23)00213-7. [Epub ahead of print]83(8): 1340-1349.e7

Uncoupled glycerol-3-phosphate shuttle in kidney cancer reveals that cytosolic GPD is essential to support lipid synthesis.

Cong-Hui Yao, Joon Seok Park, Kiran Kurmi, Song-Hua Hu, Giulia Notarangelo, Joseph Crowley, Heidi Jacobson, Sheng Hui, Arlene H Sharpe, Marcia C Haigis.

  The glycerol-3-phosphate shuttle (G3PS) is a major NADH shuttle that regenerates reducing equivalents in the cytosol and produces energy in the mitochondria. Here, we demonstrate that G3PS is uncoupled in kidney cancer cells where the cytosolic reaction is ∼4.5 times faster than the mitochondrial reaction. The high flux through cytosolic glycerol-3-phosphate dehydrogenase (GPD) is required to maintain redox balance and support lipid synthesis. Interestingly, inhibition of G3PS by knocking down mitochondrial GPD (GPD2) has no effect on mitochondrial respiration. Instead, loss of GPD2 upregulates cytosolic GPD on a transcriptional level and promotes cancer cell proliferation by increasing glycerol-3-phosphate supply. The proliferative advantage of GPD2 knockdown tumor can be abolished by pharmacologic inhibition of lipid synthesis. Taken together, our results suggest that G3PS is not required to run as an intact NADH shuttle but is instead truncated to support complex lipid synthesis in kidney cancer.

Keywords:  GPD; NAD; glycerol; glycerol-3-phosphate dehydrogenase; glycerol-3-phosphate shuttle; kidney cancer; lipids; metabolism; mitochondria

DOI:  https://doi.org/10.1016/j.molcel.2023.03.023
Nat Rev Genet. 2023 Apr 18.

Primary cilia as dynamic and diverse signalling hubs in development and disease.

Pleasantine Mill, Søren T Christensen, Lotte B Pedersen.

Primary cilia, antenna-like sensory organelles protruding from the surface of most vertebrate cell types, are essential for regulating signalling pathways during development and adult homeostasis. Mutations in genes affecting cilia cause an overlapping spectrum of >30 human diseases and syndromes, the ciliopathies. Given the immense structural and functional diversity of the mammalian cilia repertoire, there is a growing disconnect between patient genotype and associated phenotypes, with variable severity and expressivity characteristic of the ciliopathies as a group. Recent technological developments are rapidly advancing our understanding of the complex mechanisms that control biogenesis and function of primary cilia across a range of cell types and are starting to tackle this diversity. Here, we examine the structural and functional diversity of primary cilia, their dynamic regulation in different cellular and developmental contexts and their disruption in disease.

DOI: https://doi.org/10.1038/s41576-023-00587-9
Elife. 2023 Apr 19. pii: e83338. [Epub ahead of print]12

Microtubule-mediated GLUT4 trafficking is disrupted in insulin resistant skeletal muscle.

Jonas R Knudsen, Kaspar W Persson, Carlos Henriquez-Olguin, Zhencheng Li, Nicolas Di Leo, Sofie A Hesselager, Steffen H Raun, Janne R Hingst, Raphaël Trouillon, Martin Wohlwend, Jørgen F P Wojtaszewski, Martin A M Gijs, Thomas Elbenhardt Jensen.

  Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2h Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell-surface recruitable GLUT4 pool via kinesin-1 mediated trafficking.

Keywords:  cell biology; human; mouse; rat

DOI:  https://doi.org/10.7554/eLife.83338
Cell Commun Signal. 2023 Apr 21. 21(1): 82

PP1γ regulates neuronal insulin signaling and aggravates insulin resistance leading to AD-like phenotypes.

Yamini Yadav, Medha Sharma, Chinmoy Sankar Dey.

   BACKGROUND: PP1γ is one of the isoforms of catalytic subunit of a Ser/Thr phosphatase PP1. The role of PP1γ in cellular regulation is largely unknown. The present study investigated the role of PP1γ in regulating neuronal insulin signaling and insulin resistance in neuronal cells. PP1 was inhibited in mouse neuroblastoma cells (N2a) and human neuroblastoma cells (SH-SY5Y). The expression of PP1α and PP1γ was determined in insulin resistant N2a, SH-SY5Y cells and in high-fat-diet-fed-diabetic mice whole-brain-lysates. PP1α and PP1γ were silenced by siRNA in N2a and SH-SY5Y cells and effect was tested on AKT isoforms, AS160 and GSK3 isoforms using western immunoblot, GLUT4 translocation by confocal microscopy and glucose uptake by fluorescence-based assay.
RESULTS: Results showed that, in one hand PP1γ, and not PP1α, regulates neuronal insulin signaling and insulin resistance by regulating phosphorylation of AKT2 via AKT2-AS160-GLUT4 axis. On the other hand, PP1γ regulates phosphorylation of GSK3β via AKT2 while phosphorylation of GSK3α via MLK3. Imbalance in this regulation results into AD-like phenotype.
CONCLUSION: PP1γ acts as a linker, regulating two pathophysiological conditions, neuronal insulin resistance and AD. Video Abstract.

Keywords:  AKT isoforms; Alzheimer disease; Insulin resistance; MLK3; Neuronal insulin signaling; PP1γ

DOI:  https://doi.org/10.1186/s12964-023-01071-x
Mol Syst Biol. 2023 Apr 17. e11490

Multisite assessment of reproducibility in high-content cell migration imaging data.

Jianjiang Hu, Xavier Serra-Picamal, Gert-Jan Bakker, Marleen Van Troys, Sabina Winograd-Katz, Nil Ege, Xiaowei Gong, Yuliia Didan, Inna Grosheva, Omer Polansky, Karima Bakkali, Evelien Van Hamme, Merijn van Erp, Manon Vullings, Felix Weiss, Jarama Clucas, Anna M Dowbaj, Erik Sahai, Christophe Ampe, Benjamin Geiger, Peter Friedl, Matteo Bottai, Staffan Strömblad.

  High-content image-based cell phenotyping provides fundamental insights into a broad variety of life science disciplines. Striving for accurate conclusions and meaningful impact demands high reproducibility standards, with particular relevance for high-quality open-access data sharing and meta-analysis. However, the sources and degree of biological and technical variability, and thus the reproducibility and usefulness of meta-analysis of results from live-cell microscopy, have not been systematically investigated. Here, using high-content data describing features of cell migration and morphology, we determine the sources of variability across different scales, including between laboratories, persons, experiments, technical repeats, cells, and time points. Significant technical variability occurred between laboratories and, to lesser extent, between persons, providing low value to direct meta-analysis on the data from different laboratories. However, batch effect removal markedly improved the possibility to combine image-based datasets of perturbation experiments. Thus, reproducible quantitative high-content cell image analysis of perturbation effects and meta-analysis depend on standardized procedures combined with batch correction.

Keywords:  batch effect removal; cell migration; high-content imaging; reproducibility; variability

DOI:  https://doi.org/10.15252/msb.202211490
Nat Rev Genet. 2023 Apr 21.

Single-cell genomics meets human genetics.

Anna S E Cuomo, Aparna Nathan, Soumya Raychaudhuri, Daniel G MacArthur, Joseph E Powell.

Single-cell genomic technologies are revealing the cellular composition, identities and states in tissues at unprecedented resolution. They have now scaled to the point that it is possible to query samples at the population level, across thousands of individuals. Combining single-cell information with genotype data at this scale provides opportunities to link genetic variation to the cellular processes underpinning key aspects of human biology and disease. This strategy has potential implications for disease diagnosis, risk prediction and development of therapeutic solutions. But, effectively integrating large-scale single-cell genomic data, genetic variation and additional phenotypic data will require advances in data generation and analysis methods. As single-cell genetics begins to emerge as a field in its own right, we review its current state and the challenges and opportunities ahead.

DOI: https://doi.org/10.1038/s41576-023-00599-5