bims-pideca 2023-12-03 papers

bims-pideca

Biomed News

on Class IA PI3K signalling in development and cancer

Issue of 2023–12–03
28 papers selected by
Ralitsa Radostinova Madsen, MRC-PPU

PI3K-C2β limits mTORC1 signaling and angiogenic growth.
A guide to ERK dynamics, part 1: mechanisms and models.
A guide to ERK dynamics, part 2: downstream decoding.
Germline PTEN genotype-dependent phenotypic divergence during the early neural developmental process of forebrain organoids.
Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity.
A dynamic partitioning mechanism polarizes membrane protein distribution.
Endothelial nitric oxide synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation.
Anticancer effects of alpelisib on PIK3CA-mutated canine mammary tumor cell lines.
PRMT1 orchestrates with SAMTOR to govern mTORC1 methionine sensing via Arg-methylation of NPRL2.
An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRAS G12D inhibitors.
Measuring Drug Response with Single-Cell Growth Rate Quantification.
Temporally multiplexed imaging of dynamic signaling networks in living cells.
Fragment-sequencing unveils local tissue microenvironments at single-cell resolution.
Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36.
Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.
Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.
Insulin at the Intersection of Thermoregulation and Glucose Homeostasis.
Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput.
Environmental factor reversibly determines cellular identity through opposing Integrators that unify epigenetic and transcriptional pathways.
Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.
A Bioconductor workflow for processing, evaluating, and interpreting expression proteomics data.
PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes.
Interrogating ligand-receptor interactions using highly sensitive cellular biosensors.
System-wide analysis of RNA and protein subcellular localization dynamics.
Mitf is a Schwann cell sensor of axonal integrity that drives nerve repair.
SCALA: A complete solution for multimodal analysis of single-cell Next Generation Sequencing data.
Tiny robots made from human cells heal damaged tissue.
Acetyl-CoA is a key molecule for nephron progenitor cell pool maintenance.

Sci Signal. 2023 Nov 28. 16(813): eadg1913

PI3K-C2β limits mTORC1 signaling and angiogenic growth.

Piotr Kobialka, Judith Llena, Nerea Deleyto-Seldas, Margalida Munar-Gelabert, Jose A Dengra, Pilar Villacampa, Alba Albinyà-Pedrós, Laia Muixi, Jorge Andrade, Hielke van Splunder, Ana Angulo-Urarte, Michael Potente, Joaquim Grego-Bessa, Sandra D Castillo, Bart Vanhaesebroeck, Alejo Efeyan, Mariona Graupera.

Phosphoinositide 3-kinases (PI3Ks) phosphorylate intracellular inositol lipids to regulate signaling and intracellular vesicular trafficking. Mammals have eight PI3K isoforms, of which class I PI3Kα and class II PI3K-C2α are essential for vascular development. The class II PI3K-C2β is also abundant in endothelial cells. Using in vivo and in vitro approaches, we found that PI3K-C2β was a critical regulator of blood vessel growth by restricting endothelial mTORC1 signaling. Mice expressing a kinase-inactive form of PI3K-C2β displayed enlarged blood vessels without corresponding changes in endothelial cell proliferation or migration. Instead, inactivation of PI3K-C2β resulted in an increase in the size of endothelial cells, particularly in the sprouting zone of angiogenesis. Mechanistically, we showed that the aberrantly large size of PI3K-C2β mutant endothelial cells was caused by mTORC1 activation, which sustained growth in these cells. Consistently, pharmacological inhibition of mTORC1 with rapamycin normalized vascular morphogenesis in PI3K-C2β mutant mice. Together, these results identify PI3K-C2β as a crucial determinant of endothelial signaling and illustrate the importance of mTORC1 regulation during angiogenic growth.

DOI: https://doi.org/10.1126/scisignal.adg1913
Biochem J. 2023 Dec 13. 480(23): 1887-1907

A guide to ERK dynamics, part 1: mechanisms and models.

Abhineet Ram, Devan Murphy, Nicholaus DeCuzzi, Madhura Patankar, Jason Hu, Michael Pargett, John G Albeck.

  Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.

Keywords:  biological networks; computational models; epidermal growth factor receptor; extracellular signal-regulated kinases; mitogen-activated protein kinases; receptor tyrosine kinases

DOI:  https://doi.org/10.1042/BCJ20230276
Biochem J. 2023 Dec 13. 480(23): 1909-1928

A guide to ERK dynamics, part 2: downstream decoding.

Abhineet Ram, Devan Murphy, Nicholaus DeCuzzi, Madhura Patankar, Jason Hu, Michael Pargett, John G Albeck.

  Signaling by the extracellular signal-regulated kinase (ERK) pathway controls many cellular processes, including cell division, death, and differentiation. In this second installment of a two-part review, we address the question of how the ERK pathway exerts distinct and context-specific effects on multiple processes. We discuss how the dynamics of ERK activity induce selective changes in gene expression programs, with insights from both experiments and computational models. With a focus on single-cell biosensor-based studies, we summarize four major functional modes for ERK signaling in tissues: adjusting the size of cell populations, gradient-based patterning, wave propagation of morphological changes, and diversification of cellular gene expression states. These modes of operation are disrupted in cancer and other related diseases and represent potential targets for therapeutic intervention. By understanding the dynamic mechanisms involved in ERK signaling, there is potential for pharmacological strategies that not only simply inhibit ERK, but also restore functional activity patterns and improve disease outcomes.

Keywords:  cell proliferation; eukaryotic gene expression; extracellular signal-regulated kinases; gene regulatory networks; receptor tyrosine kinases

DOI:  https://doi.org/10.1042/BCJ20230277
Mol Psychiatry. 2023 Nov 29.

Germline PTEN genotype-dependent phenotypic divergence during the early neural developmental process of forebrain organoids.

Shin Chung Kang, Nicholas B Sarn, Juan Venegas, Zhibing Tan, Masahiro Hitomi, Charis Eng.

PTEN germline mutations account for ~0.2-1% of all autism spectrum disorder (ASD) cases, as well as ~17% of ASD patients with macrocephaly, making it one of the top ASD-associated risk genes. Individuals with germline PTEN mutations receive the molecular diagnosis of PTEN Hamartoma Tumor Syndrome (PHTS), an inherited cancer predisposition syndrome, about 20-23% of whom are diagnosed with ASD. We generated forebrain organoid cultures from gene-edited isogenic human induced pluripotent stem cells (hiPSCs) harboring a PTENG132D (ASD) or PTENM134R (cancer) mutant allele to model how these mutations interrupt neurodevelopmental processes. Here, we show that the PTENG132D allele disrupts early neuroectoderm formation during the first several days of organoid generation, and results in deficient electrophysiology. While organoids generated from PTENM134R hiPSCs remained morphologically similar to wild-type organoids during this early stage in development, we observed disrupted neuronal differentiation, radial glia positioning, and cortical layering in both PTEN-mutant organoids at the later stage of 72+ days of development. Perifosine, an AKT inhibitor, reduced over-activated AKT and partially corrected the abnormalities in cellular organization observed in PTENG132D organoids. Single cell RNAseq analyses on early-stage organoids revealed that genes related to neural cell fate were decreased in PTENG132D mutant organoids, and AKT inhibition was capable of upregulating gene signatures related to neuronal cell fate and CNS maturation pathways. These findings demonstrate that different PTEN missense mutations can have a profound impact on neurodevelopment at diverse stages which in turn may predispose PHTS individuals to ASD. Further study will shed light on ways to mitigate pathological impact of PTEN mutants on neurodevelopment by stage-specific manipulation of downstream PTEN signaling components.

DOI: https://doi.org/10.1038/s41380-023-02325-3
Nat Commun. 2023 Nov 29. 14(1): 7847

Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity.

Mimi Zhang, Sungsoo Kim, Hee Won Yang.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 for Rb inactivation. Here we show a non-canonical pathway for Rb inactivation and its interplay with external signals. We find that the non-phosphorylated Rb protein in quiescent cells is intrinsically unstable, offering an alternative mechanism for initiating E2F activity. Nevertheless, this pathway incompletely induces Rb-protein loss, resulting in minimal E2F activity. To trigger cell proliferation, upregulation of mitogenic signaling is required for stabilizing c-Myc, thereby augmenting E2F activity. Concurrently, stress signaling promotes Cip/Kip levels, competitively regulating cell proliferation with mitogenic signaling. In cancer, driver mutations elevate c-Myc levels, facilitating adaptation to CDK4/6 inhibitors. Differentiated cells, despite Rb-protein loss, maintain quiescence through the modulation of c-Myc and Cip/Kip levels. Our findings provide mechanistic insights into an alternative model of cell-cycle entry and the maintenance of quiescence.

DOI: https://doi.org/10.1038/s41467-023-43716-y
Nat Commun. 2023 Nov 30. 14(1): 7909

A dynamic partitioning mechanism polarizes membrane protein distribution.

Tatsat Banerjee, Satomi Matsuoka, Debojyoti Biswas, Yuchuan Miao, Dhiman Sankar Pal, Yoichiro Kamimura, Masahiro Ueda, Peter N Devreotes, Pablo A Iglesias.

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose "dynamic partitioning" as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

DOI: https://doi.org/10.1038/s41467-023-43615-2
Front Cardiovasc Med. 2023 ;10 1279868

Endothelial nitric oxide synthase (eNOS) S1176 phosphorylation status governs atherosclerotic lesion formation.

Tung D Nguyen, Nur-Taz Rahman, William C Sessa, Monica Y Lee.

   Objective: We have previously demonstrated the in vivo importance of the Akt-eNOS substrate-kinase relationship, as defective postnatal angiogenesis characteristic of global Akt1-null mice is rescued when bred to 'gain-of-function' eNOS S1176D mutant mice. While multiple studies support the vascular protective role of endothelial NO generation, the causal role of Akt1-dependent eNOS S1176 phosphorylation during atherosclerotic plaque formation is not yet clear.
Approach and results: We herein bred congenic 'loss-of-function' eNOS S1176A and 'gain-of-function' eNOS S1176D mutant mice to the exacerbated atherogenic Akt1-/-; ApoE-/- double knockout mice to definitively test the importance of Akt-mediated eNOS S1176 phosphorylation during atherogenesis. We find that a single amino acid substitution at the eNOS S1176 phosphorylation site yields divergent effects on atherosclerotic plaque formation, as an eNOS phospho-mimic aspartate (D) substitution at S1176 leads to favorable lipid profiles and decreased indices of atherosclerosis, even when on a proatherogenic Akt1 global deletion background. Conversely, mice harboring an unphosphorylatable mutation to alanine (S1176A) result in increased plasma lipids, increased lesion formation and cellular apoptosis, phenocopying the physiological consequence of eNOS deletion and/or impaired enzyme function. Furthermore, gene expression analyses of whole aortas indicate a combinatorial detriment from NO deficiency and Western Diet challenge, as 'loss-of-function' eNOS S1176A mice on a Western Diet present a unique expression pattern indicative of augmented T-cell activity when compared to eNOS S1176D mice.
Conclusions: By using genetic epistasis approaches, we conclusively demonstrate that Akt-mediated eNOS S1176 phosphorylation and subsequent eNOS activation remains to be the most physiologically relevant method of NO production to promote athero-protective effects.

Keywords:  PI3K/AKT; atherosclerosis; eNOS; endothelial; phosphorylation

DOI:  https://doi.org/10.3389/fcvm.2023.1279868
Front Vet Sci. 2023 ;10 1279535

Anticancer effects of alpelisib on PIK3CA-mutated canine mammary tumor cell lines.

Jiah Yeom, Yoonju Cho, Seoungyob Ahn, Soyoung Jeung.

  Canine mammary tumors (CMTs) are commonly observed in old and unspayed female dogs. Recently, dogs have been increasingly spaying at a young age to prevent mammary tumors. These CMTs require extensive local excision and exhibit a high probability of metastasis to the regional lymph nodes and lungs during malignancy. However, the molecular and biological mechanisms underlying CMT development have not been fully elucidated, and research in this area is limited. Therefore, in this study, we established new CMT cell lines by isolating cells from tumor tissues and investigated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), a target for human breast cancer. PIK3CA mutations were observed at a similar loci as in the human PIK3CA gene in half of all canine samples. Furthermore, we investigated whether alpelisib, a PIK3CA inhibitor approved by the U.S. Food and Drug Administration for human breast cancer treatment, along with fulvestrant, is effective for CMT treatment. Alpelisib exerted stronger anticancer effects on cell lines with PIK3CA mutations than on the wild-type cell lines. In conclusion, we established new CMT cell lines with PIK3CA mutations and confirmed the efficacy of alpelisib for CMT treatment in vitro.

Keywords:  PI3K; PIK3CA; alpelisib; canine cell line; canine mammary tumor

DOI:  https://doi.org/10.3389/fvets.2023.1279535
Cell Metab. 2023 Nov 16. pii: S1550-4131(23)00411-4. [Epub ahead of print]

PRMT1 orchestrates with SAMTOR to govern mTORC1 methionine sensing via Arg-methylation of NPRL2.

Cong Jiang, Jing Liu, Shaohui He, Wei Xu, Runzhi Huang, Weijuan Pan, Xiaolong Li, Xiaoming Dai, Jianping Guo, Tao Zhang, Hiroyuki Inuzuka, Ping Wang, John M Asara, Jianru Xiao, Wenyi Wei.

  Methionine is an essential branch of diverse nutrient inputs that dictate mTORC1 activation. In the absence of methionine, SAMTOR binds to GATOR1 and inhibits mTORC1 signaling. However, how mTORC1 is activated upon methionine stimulation remains largely elusive. Here, we report that PRMT1 senses methionine/SAM by utilizing SAM as a cofactor for an enzymatic activity-based regulation of mTORC1 signaling. Under methionine-sufficient conditions, elevated cytosolic SAM releases SAMTOR from GATOR1, which confers the association of PRMT1 with GATOR1. Subsequently, SAM-loaded PRMT1 methylates NPRL2, the catalytic subunit of GATOR1, thereby suppressing its GAP activity and leading to mTORC1 activation. Notably, genetic or pharmacological inhibition of PRMT1 impedes hepatic methionine sensing by mTORC1 and improves insulin sensitivity in aged mice, establishing the role of PRMT1-mediated methionine sensing at physiological levels. Thus, PRMT1 coordinates with SAMTOR to form the methionine-sensing apparatus of mTORC1 signaling.

Keywords:  GATOR1; NPRL2; PRMT1; arginine methylation; mTOR; methionine sensing; nutrient sensing

DOI:  https://doi.org/10.1016/j.cmet.2023.11.001
bioRxiv. 2023 Nov 18. pii: 2023.11.18.567669. [Epub ahead of print]

An integrated method for single cell proteomics with simultaneous measurements of intracellular drug concentration implicates new mechanisms for adaptation to KRAS G12D inhibitors.

Benjamin C Orsburn.

It is well established that a population of single human cells will often respond to the same drug treatment in a heterogeneous manner. In the context of chemotherapeutics, these diverse responses may lead to individual adaptation mechanisms and ultimately multiple distinct methods of resistance. The obvious question from a pharmacology perspective is how intracellular concentrations of active drug varies between individual cells, and what role does that variation play in drug response heterogeneity? To date, no integrated methods for rapidly measuring intracellular drug levels while simultaneously measuring drug responses have been described. This study describes a method for single cell preparation that allows proteins to be extracted and digested from single cells while maintaining conditions for small molecules to be simultaneously measured. The method as described allows up to 40 cells to be analyzed per instrument per day. When applied to a KRAS G12D small molecule inhibitor I observe a wide degree of intracellular levels of the drug, and that proteomic responses largely stratify based on the concentration of drug within each single cell. Further work is in progress to develop and standardize this method and - more importantly - to normalize drug measurements against direct measurements of cell volume. However, these preliminary results appear promising for the identification of single cells with unique drug response mechanisms. All data described in this study has been made publicly available through the ProteomeXchange consortium under accession PXD046002.
Abstract graphic:

DOI: https://doi.org/10.1101/2023.11.18.567669
Anal Chem. 2023 Nov 28.

Measuring Drug Response with Single-Cell Growth Rate Quantification.

Fiona Xi Xu, Ruibing Wu, Kailun Hu, Dan Fu.

Intratumoral heterogeneity is a substantial cause of drug resistance development during chemotherapy or other drug treatments for cancer. Therefore, monitoring and measuring cell exposure and response to drugs at the single-cell level are crucial. Previous research suggested that the single-cell growth rate can be used to investigate drug-cell interactions. However, currently established methods for quantifying single-cell growth are limited to isolated or monolayer cells. Here, we introduce a technique that accurately measures both 2D and 3D cell growth rates using label-free ratiometric stimulated Raman scattering (SRS) microscopy. We use deuterated amino acids, leucine, isoleucine, and valine, as tracers and measure the C-D SRS signal from deuterium-labeled proteins and the C-H SRS signal from unlabeled proteins simultaneously to determine the cell growth rate at the single-cell level. The technique offers single-cell level drug sensitivity measurement with a shorter turnaround time (within 12 h) than most traditional assays. The submicrometer resolution of the imaging technique allows us to examine the effects of chemotherapeutic drugs, including kinase inhibitors, mitotic inhibitors, and topoisomerase II inhibitors, on both the cell growth rate and morphology. The capability of quantifying 3D cell growth rates provides insight into a deeper understanding of the cell-drug interaction in the actual tumor environment.

DOI: https://doi.org/10.1021/acs.analchem.3c03434
Cell. 2023 Nov 21. pii: S0092-8674(23)01227-8. [Epub ahead of print]

Temporally multiplexed imaging of dynamic signaling networks in living cells.

Yong Qian, Orhan T Celiker, Zeguan Wang, Burcu Guner-Ataman, Edward S Boyden.

  Molecular signals interact in networks to mediate biological processes. To analyze these networks, it would be useful to image many signals at once, in the same living cell, using standard microscopes and genetically encoded fluorescent reporters. Here, we report temporally multiplexed imaging (TMI), which uses genetically encoded fluorescent proteins with different clocklike properties-such as reversibly photoswitchable fluorescent proteins with different switching kinetics-to represent different cellular signals. We linearly decompose a brief (few-second-long) trace of the fluorescence fluctuations, at each point in a cell, into a weighted sum of the traces exhibited by each fluorophore expressed in the cell. The weights then represent the signal amplitudes. We use TMI to analyze relationships between different kinase activities in individual cells, as well as between different cell-cycle signals, pointing toward broad utility throughout biology in the analysis of signal transduction cascades in living systems.

Keywords:  TMI; cell cycle; cytoskeleton; fluorescent indicators; fluorescent proteins; live-cell imaging; microscopy; organelle; protein kinase; signal transduction; temporally multiplexed imaging

DOI:  https://doi.org/10.1016/j.cell.2023.11.010
Nat Commun. 2023 Nov 27. 14(1): 7775

Fragment-sequencing unveils local tissue microenvironments at single-cell resolution.

Kristina Handler, Karsten Bach, Costanza Borrelli, Salvatore Piscuoglio, Xenia Ficht, Ilhan E Acar, Andreas E Moor.

Cells collectively determine biological functions by communicating with each other-both through direct physical contact and secreted factors. Consequently, the local microenvironment of a cell influences its behavior, gene expression, and cellular crosstalk. Disruption of this microenvironment causes reciprocal changes in those features, which can lead to the development and progression of diseases. Hence, assessing the cellular transcriptome while simultaneously capturing the spatial relationships of cells within a tissue provides highly valuable insights into how cells communicate in health and disease. Yet, methods to probe the transcriptome often fail to preserve native spatial relationships, lack single-cell resolution, or are highly limited in throughput, i.e. lack the capacity to assess multiple environments simultaneously. Here, we introduce fragment-sequencing (fragment-seq), a method that enables the characterization of single-cell transcriptomes within multiple spatially distinct tissue microenvironments. We apply fragment-seq to a murine model of the metastatic liver to study liver zonation and the metastatic niche. This analysis reveals zonated genes and ligand-receptor interactions enriched in specific hepatic microenvironments. Finally, we apply fragment-seq to other tissues and species, demonstrating the adaptability of our method.

DOI: https://doi.org/10.1038/s41467-023-43005-8
Nat Commun. 2023 Nov 29. 14(1): 7859

Endosomal Arl4A attenuates EGFR degradation by binding to the ESCRT-II component VPS36.

Shin-Jin Lin, Ming-Chieh Lin, Tsai-Jung Liu, Yueh-Tso Tsai, Ming-Ting Tsai, Fang-Jen S Lee.

Ligand-induced epidermal growth factor receptor (EGFR) endocytosis followed by endosomal EGFR signaling and lysosomal degradation plays important roles in controlling multiple biological processes. ADP-ribosylation factor (Arf)-like protein 4 A (Arl4A) functions at the plasma membrane to mediate cytoskeletal remodeling and cell migration, whereas its localization at endosomal compartments remains functionally unknown. Here, we report that Arl4A attenuates EGFR degradation by binding to the endosomal sorting complex required for transport (ESCRT)-II component VPS36. Arl4A plays a role in prolonging the duration of EGFR ubiquitinylation and deterring endocytosed EGFR transport from endosomes to lysosomes under EGF stimulation. Mechanistically, the Arl4A-VPS36 direct interaction stabilizes VPS36 and ESCRT-III association, affecting subsequent recruitment of deubiquitinating-enzyme USP8 by CHMP2A. Impaired Arl4A-VPS36 interaction enhances EGFR degradation and clearance of EGFR ubiquitinylation. Together, we discover that Arl4A negatively regulates EGFR degradation by binding to VPS36 and attenuating ESCRT-mediated late endosomal EGFR sorting.

DOI: https://doi.org/10.1038/s41467-023-42979-9
Sci Transl Med. 2023 Nov 29. 15(724): eadd0499

Enhanced mTORC1 signaling and protein synthesis in pathologic α-synuclein cellular and animal models of Parkinson's disease.

Mohammed Repon Khan, Xiling Yin, Sung-Ung Kang, Jaba Mitra, Hu Wang, Taekyung Ryu, Saurav Brahmachari, Senthilkumar S Karuppagounder, Yasuyoshi Kimura, Aanishaa Jhaldiyal, Hyun Hee Kim, Hao Gu, Rong Chen, Javier Redding-Ochoa, Juan Troncoso, Chan Hyun Na, Taekjip Ha, Valina L Dawson, Ted M Dawson.

Pathologic α-synuclein plays an important role in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD). Disruption of proteostasis is thought to be central to pathologic α-synuclein toxicity; however, the molecular mechanism of this deregulation is poorly understood. Complementary proteomic approaches in cellular and animal models of PD were used to identify and characterize the pathologic α-synuclein interactome. We report that the highest biological processes that interacted with pathologic α-synuclein in mice included RNA processing and translation initiation. Regulation of catabolic processes that include autophagy were also identified. Pathologic α-synuclein was found to bind with the tuberous sclerosis protein 2 (TSC2) and to trigger the activation of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), which augmented mRNA translation and protein synthesis, leading to neurodegeneration. Genetic and pharmacologic inhibition of mTOR and protein synthesis rescued the dopamine neuron loss, behavioral deficits, and aberrant biochemical signaling in the α-synuclein preformed fibril mouse model and Drosophila transgenic models of pathologic α-synuclein-induced degeneration. Pathologic α-synuclein furthermore led to a destabilization of the TSC1-TSC2 complex, which plays an important role in mTORC1 activity. Constitutive overexpression of TSC2 rescued motor deficits and neuropathology in α-synuclein flies. Biochemical examination of PD postmortem brain tissues also suggested deregulated mTORC1 signaling. These findings establish a connection between mRNA translation deregulation and mTORC1 pathway activation that is induced by pathologic α-synuclein in cellular and animal models of PD.

DOI: https://doi.org/10.1126/scitranslmed.add0499
Nucleic Acids Res. 2023 Nov 30. pii: gkad1125. [Epub ahead of print]

Self-delivering, chemically modified CRISPR RNAs for AAV co-delivery and genome editing in vivo.

Han Zhang, Karen Kelly, Jonathan Lee, Dimas Echeverria, David Cooper, Rebecca Panwala, Nadia Amrani, Zexiang Chen, Nicholas Gaston, Atish Wagh, Gregory A Newby, Jun Xie, David R Liu, Guangping Gao, Scot A Wolfe, Anastasia Khvorova, Jonathan K Watts, Erik J Sontheimer.

Guide RNAs offer programmability for CRISPR-Cas9 genome editing but also add challenges for delivery. Chemical modification, which has been key to the success of oligonucleotide therapeutics, can enhance the stability, distribution, cellular uptake, and safety of nucleic acids. Previously, we engineered heavily and fully modified SpyCas9 crRNA and tracrRNA, which showed enhanced stability and retained activity when delivered to cultured cells in the form of the ribonucleoprotein complex. In this study, we report that a short, fully stabilized oligonucleotide (a 'protecting oligo'), which can be displaced by tracrRNA annealing, can significantly enhance the potency and stability of a heavily modified crRNA. Furthermore, protecting oligos allow various bioconjugates to be appended, thereby improving cellular uptake and biodistribution of crRNA in vivo. Finally, we achieved in vivo genome editing in adult mouse liver and central nervous system via co-delivery of unformulated, chemically modified crRNAs with protecting oligos and AAV vectors that express tracrRNA and either SpyCas9 or a base editor derivative. Our proof-of-concept establishment of AAV/crRNA co-delivery offers a route towards transient editing activity, target multiplexing, guide redosing, and vector inactivation.

DOI: https://doi.org/10.1093/nar/gkad1125
bioRxiv. 2023 Nov 17. pii: 2023.11.17.566254. [Epub ahead of print]

Insulin at the Intersection of Thermoregulation and Glucose Homeostasis.

Nathan C Winn, Michael W Schleh, Jamie N Garcia, Louise Lantier, Owen P McGuinness, Joslin A Blair, Alyssa H Hasty, David H Wasserman.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28°C) and room (laboratory) temperature (RT, ∼22°C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue specific glucose uptake were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.
Highlights: Energy expenditure and insulin-mediated glucose fluxes are reduced in thermoneutral (TN)-adapted mice versus room 'laboratory' temperature (RT)-adapted mice.Reduced insulin sensitivity manifests in TN mice regardless of whether they are TN-adapted or short-term transitioned from RT-adapted to TN.TN-adapted mice are resistant to the RT-induced increase in whole-body insulin sensitivity even though metabolic rate is increased.TN-adapted mice switched to RT meets increased thermogenic needs, not by increasing glucose uptake, but by partitioning a greater fraction of glucose from glycogen storage to glycolysis.Brown fat glucose uptake sensitively increases with RT and decreases with TN by an insulin-independent mechanism.

DOI: https://doi.org/10.1101/2023.11.17.566254
bioRxiv. 2023 Nov 14. pii: 2023.11.11.566649. [Epub ahead of print]

Proteome-wide base editor screens to assess phosphorylation site functionality in high-throughput.

Patrick H Kennedy, Amin Alborzian Deh Sheikh, Matthew Balakar, Alexander C Jones, Meagan E Olive, Mudra Hegde, Maria I Matias, Natan Pirete, Rajan Burt, Jonathan Levy, Tamia Little, Patrick G Hogan, David R Liu, John G Doench, Alexandra C Newton, Rachel A Gottschalk, Carl de Boer, Suzie Alarcón, Gregory Newby, Samuel A Myers.

Signaling pathways that drive gene expression are typically depicted as having a dozen or so landmark phosphorylation and transcriptional events. In reality, thousands of dynamic post-translational modifications (PTMs) orchestrate nearly every cellular function, and we lack technologies to find causal links between these vast biochemical pathways and genetic circuits at scale. Here, we describe "signaling-to-transcription network" mapping through the development of PTM-centric base editing coupled to phenotypic screens, directed by temporally-resolved phosphoproteomics. Using T cell activation as a model, we observe hundreds of unstudied phosphorylation sites that modulate NFAT transcriptional activity. We identify the phosphorylation-mediated nuclear localization of the phosphatase PHLPP1 which promotes NFAT but inhibits NFκB activity. We also find that specific phosphosite mutants can alter gene expression in subtle yet distinct patterns, demonstrating the potential for fine-tuning transcriptional responses. Overall, base editor screening of PTM sites provides a powerful platform to dissect PTM function within signaling pathways.

DOI: https://doi.org/10.1101/2023.11.11.566649
Bioessays. 2023 Nov 27. e2300084

Environmental factor reversibly determines cellular identity through opposing Integrators that unify epigenetic and transcriptional pathways.

Hiroki Takahashi, Ryo Ito, Yoshihiro Matsumura, Juro Sakai.

  Organisms must adapt to environmental stresses to ensure their survival and prosperity. Different types of stresses, including thermal, mechanical, and hypoxic stresses, can alter the cellular state that accompanies changes in gene expression but not the cellular identity determined by a chromatin state that remains stable throughout life. Some tissues, such as adipose tissue, demonstrate remarkable plasticity and adaptability in response to environmental cues, enabling reversible cellular identity changes; however, the mechanisms underlying these changes are not well understood. We hypothesized that positive and/or negative "Integrators" sense environmental cues and coordinate the epigenetic and transcriptional pathways required for changes in cellular identity. Adverse environmental factors such as pollution disrupt the coordinated control contributing to disease development. Further research based on this hypothesis will reveal how organisms adapt to fluctuating environmental conditions, such as temperature, extracellular matrix stiffness, oxygen, cytokines, and hormonal cues by changing their cellular identities.

Keywords:  beige adipocytes; cellular identity; chromatin state; pollution; protein kinase; protein phosphatase; transdifferentiation

DOI:  https://doi.org/10.1002/bies.202300084
J Clin Invest. 2023 Nov 30. pii: e173280. [Epub ahead of print]

Sustained hyperglycemia specifically targets translation of mRNAs for insulin secretion.

Abigael Cheruiyot, Jennifer Hollister-Lock, Brooke A Sullivan, Hui Pan, Jonathan M Dreyfuss, Susan Bonner-Weir, Jean E Schaffer.

  Pancreatic beta-cells are specialized for coupling glucose metabolism to insulin peptide production and secretion. Acute glucose exposure robustly and coordinately increases translation of proinsulin and proteins required for secretion of mature insulin peptide. By contrast, chronically elevated glucose levels that occur during diabetes impair beta-cell insulin secretion and have been shown experimentally to suppress insulin translation. Whether translation of other genes critical for insulin secretion are similarly downregulated by chronic high glucose is unknown. Here, we used high-throughput ribosome profiling and nascent proteomics in MIN6 insulinoma cells to elucidate the genome-wide impact of sustained high glucose on beta-cell mRNA translation. Prior to induction of ER stress or suppression of global translation, sustained high glucose suppressed glucose-stimulated insulin secretion and downregulated translation of not only insulin, but also of mRNAs related to insulin secretory granule formation, exocytosis, and metabolism-coupled insulin secretion. Translation of these mRNAs was also downregulated in primary rat and human islets following ex-vivo incubation with sustained high glucose and in an in vivo model of chronic mild hyperglycemia. Furthermore, translational downregulation decreased cellular abundance of these proteins. Our study uncovered a translational regulatory circuit during beta-cell glucose toxicity that impairs expression of proteins with critical roles in beta-cell function.

Keywords:  Beta cells; Endocrinology; Islet cells; Metabolism; Translation

DOI:  https://doi.org/10.1172/JCI173280
F1000Res. 2023 ;12 1402

A Bioconductor workflow for processing, evaluating, and interpreting expression proteomics data.

Charlotte Hutchings, Charlotte S Dawson, Thomas Krueger, Kathryn S Lilley, Lisa M Breckels.

  Background: Expression proteomics involves the global evaluation of protein abundances within a system. In turn, differential expression analysis can be used to investigate changes in protein abundance upon perturbation to such a system. Methods: Here, we provide a workflow for the processing, analysis and interpretation of quantitative mass spectrometry-based expression proteomics data. This workflow utilizes open-source R software packages from the Bioconductor project and guides users end-to-end and step-by-step through every stage of the analyses. As a use-case we generated expression proteomics data from HEK293 cells with and without a treatment. Of note, the experiment included cellular proteins labelled using tandem mass tag (TMT) technology and secreted proteins quantified using label-free quantitation (LFQ). Results: The workflow explains the software infrastructure before focusing on data import, pre-processing and quality control. This is done individually for TMT and LFQ datasets. The application of statistical differential expression analysis is demonstrated, followed by interpretation via gene ontology enrichment analysis. Conclusions: A comprehensive workflow for the processing, analysis and interpretation of expression proteomics is presented. The workflow is a valuable resource for the proteomics community and specifically beginners who are at least familiar with R who wish to understand and make data-driven decisions with regards to their analyses.

Keywords:  Bioconductor; QFeatures; bottom-up proteomics; data processing; differential expression; limma; mass spectrometry; proteomics; quality control; shotgun proteomics

DOI:  https://doi.org/10.12688/f1000research.139116.1
Elife. 2023 Nov 29. pii: RP89331. [Epub ahead of print]12

PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes.

Abdoulie O Touray, Rishi Rajesh, Tony Isebe, Tamara Sternlieb, Mira Loock, Oksana Kutova, Igor Cestari.

  African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

Keywords:  allosteric regulation; antigenic variation; chromosomes; gene expression; infectious disease; microbiology; phosphoinositides; telomere; telomere silencing; trypanosoma

DOI:  https://doi.org/10.7554/eLife.89331
Nat Commun. 2023 Nov 28. 14(1): 7804

Interrogating ligand-receptor interactions using highly sensitive cellular biosensors.

Maximilian A Funk, Judith Leitner, Marlene C Gerner, Jasmin M Hammerler, Benjamin Salzer, Manfred Lehner, Claire Battin, Simon Gumpelmair, Karin Stiasny, Katharina Grabmeier-Pfistershammer, Peter Steinberger.

Interactions of membrane-resident proteins are important targets for therapeutic interventions but most methods to study them are either costly, laborious or fail to reflect the physiologic interaction of membrane resident proteins in trans. Here we describe highly sensitive cellular biosensors as a tool to study receptor-ligand pairs. They consist of fluorescent reporter cells that express chimeric receptors harboring ectodomains of cell surface molecules and intracellular signaling domains. We show that a broad range of molecules can be integrated into this platform and we demonstrate its applicability to highly relevant research areas, including the characterization of immune checkpoints and the probing of cells for the presence of receptors or ligands. The platform is suitable to evaluate the interactions of viral proteins with host receptors and to test for neutralization capability of drugs or biological samples. Our results indicate that cellular biosensors have broad utility as a tool to study protein-interactions.

DOI: https://doi.org/10.1038/s41467-023-43589-1
Nat Methods. 2023 Nov 30.

System-wide analysis of RNA and protein subcellular localization dynamics.

Eneko Villanueva, Tom Smith, Mariavittoria Pizzinga, Mohamed Elzek, Rayner M L Queiroz, Robert F Harvey, Lisa M Breckels, Oliver M Crook, Mie Monti, Veronica Dezi, Anne E Willis, Kathryn S Lilley.

Although the subcellular dynamics of RNA and proteins are key determinants of cell homeostasis, their characterization is still challenging. Here we present an integrative framework to simultaneously interrogate the dynamics of the transcriptome and proteome at subcellular resolution by combining two methods: localization of RNA (LoRNA) and a streamlined density-based localization of proteins by isotope tagging (dLOPIT) to map RNA and protein to organelles (nucleus, endoplasmic reticulum and mitochondria) and membraneless compartments (cytosol, nucleolus and cytosolic granules). Interrogating all RNA subcellular locations at once enables system-wide quantification of the proportional distribution of RNA. We obtain a cell-wide overview of localization dynamics for 31,839 transcripts and 5,314 proteins during the unfolded protein response, revealing that endoplasmic reticulum-localized transcripts are more efficiently recruited to cytosolic granules than cytosolic RNAs, and that the translation initiation factor eIF3d is key to sustaining cytoskeletal function. Overall, we provide the most comprehensive overview so far of RNA and protein subcellular localization dynamics.

DOI: https://doi.org/10.1038/s41592-023-02101-9
Cell Rep. 2023 Nov 28. pii: S2211-1247(23)01294-9. [Epub ahead of print]42(11): 113282

Mitf is a Schwann cell sensor of axonal integrity that drives nerve repair.

Lydia Daboussi, Giancarlo Costaguta, Miriam Gullo, Nicole Jasinski, Veronica Pessino, Brendan O'Leary, Karen Lettieri, Shawn Driscoll, Samuel L Pfaff.

  Schwann cells respond to acute axon damage by transiently transdifferentiating into specialized repair cells that restore sensorimotor function. However, the molecular systems controlling repair cell formation and function are not well defined, and consequently, it is unclear whether this form of cellular plasticity has a role in peripheral neuropathies. Here, we identify Mitf as a transcriptional sensor of axon damage under the control of Nrg-ErbB-PI3K-PI5K-mTorc2 signaling. Mitf regulates a core transcriptional program for generating functional repair Schwann cells following injury and during peripheral neuropathies caused by CMT4J and CMT4D. In the absence of Mitf, core genes for epithelial-to-mesenchymal transition, metabolism, and dedifferentiation are misexpressed, and nerve repair is disrupted. Our findings demonstrate that Schwann cells monitor axonal health using a phosphoinositide signaling system that controls Mitf nuclear localization, which is critical for activating cellular plasticity and counteracting neural disease.

Keywords:  CP: Neuroscience; Mitf; Schwann cell; injury; neurodegeneration; peripheral nerve; phosphoinositide; regeneration; transdifferentiation

DOI:  https://doi.org/10.1016/j.celrep.2023.113282
Comput Struct Biotechnol J. 2023 ;21 5382-5393

SCALA: A complete solution for multimodal analysis of single-cell Next Generation Sequencing data.

Christos Tzaferis, Evangelos Karatzas, Fotis A Baltoumas, Georgios A Pavlopoulos, George Kollias, Dimitris Konstantopoulos.

  Analysis and interpretation of high-throughput transcriptional and chromatin accessibility data at single-cell (sc) resolution are still open challenges in the biomedical field. The existence of countless bioinformatics tools, for the different analytical steps, increases the complexity of data interpretation and the difficulty to derive biological insights. In this article, we present SCALA, a bioinformatics tool for analysis and visualization of single-cell RNA sequencing (scRNA-seq) and Assay for Transposase-Accessible Chromatin using sequencing (scATAC-seq) datasets, enabling either independent or integrative analysis of the two modalities. SCALA combines standard types of analysis by integrating multiple software packages varying from quality control to the identification of distinct cell populations and cell states. Additional analysis options enable functional enrichment, cellular trajectory inference, ligand-receptor analysis, and regulatory network reconstruction. SCALA is fully parameterizable, presenting data in tabular format and producing publication-ready visualizations. The different available analysis modules can aid biomedical researchers in exploring, analyzing, and visualizing their data without any prior experience in coding. We demonstrate the functionality of SCALA through two use-cases related to TNF-driven arthritic mice, handling both scRNA-seq and scATAC-seq datasets. SCALA is developed in R, Shiny and JavaScript and is mainly available as a standalone version, while an online service of more limited capacity can be found at http://scala.pavlopouloslab.info or https://scala.fleming.gr.

Keywords:  Automated analysis of single-cell Next Generation Sequencing data; Integrative analysis of single-cell Next Generation Sequencing data; Single-cell ATAC-seq analysis; Single-cell RNA sequencing analysis

DOI:  https://doi.org/10.1016/j.csbj.2023.10.032
Nature. 2023 Nov 30.

Tiny robots made from human cells heal damaged tissue.

Matthew Hutson.



Keywords:  Engineering; Personalized medicine

DOI:  https://doi.org/10.1038/d41586-023-03777-x
Nat Commun. 2023 Nov 25. 14(1): 7733

Acetyl-CoA is a key molecule for nephron progenitor cell pool maintenance.

Fabiola Diniz, Nguyen Yen Nhi Ngo, Mariel Colon-Leyva, Francesca Edgington-Giordano, Sylvia Hilliard, Kevin Zwezdaryk, Jiao Liu, Samir S El-Dahr, Giovane G Tortelote.

Nephron endowment at birth impacts long-term renal and cardiovascular health, and it is contingent on the nephron progenitor cell (NPC) pool. Glycolysis modulation is essential for determining NPC fate, but the underlying mechanism is unclear. Combining RNA sequencing and quantitative proteomics we identify 267 genes commonly targeted by Wnt activation or glycolysis inhibition in NPCs. Several of the impacted pathways converge at Acetyl-CoA, a co-product of glucose metabolism. Notably, glycolysis inhibition downregulates key genes of the Mevalonate/cholesterol pathway and stimulates NPC differentiation. Sodium acetate supplementation rescues glycolysis inhibition effects and favors NPC maintenance without hindering nephrogenesis. Six2Cre-mediated removal of ATP-citrate lyase (Acly), an enzyme that converts citrate to acetyl-CoA, leads to NPC pool depletion, glomeruli count reduction, and increases Wnt4 expression at birth. Sodium acetate supplementation counters the effects of Acly deletion on cap-mesenchyme. Our findings show a pivotal role of acetyl-CoA metabolism in kidney development and uncover new avenues for manipulating nephrogenesis and preventing adult kidney disease.

DOI: https://doi.org/10.1038/s41467-023-43513-7