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1.
  • Afanasyeva, Elena A., et al. (författare)
  • Kalirin-RAC controls nucleokinetic migration in ADRN-type neuroblastoma
  • 2021
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:5
  • Tidskriftsartikel (refereegranskat)abstract
    • The migrational propensity of neuroblastoma is affected by cell identity, but the mechanisms behind the divergence remain unknown. Using RNAi and time-lapse imaging, we show that ADRN-type NB cells exhibit RAC1- and kalirin-dependent nucleokinetic (NUC) migration that relies on several integral components of neuronal migration. Inhibition of NUC migration by RAC1 and kalirin-GEF1 inhibitors occurs without hampering cell proliferation and ADRN identity. Using three clinically relevant expression dichotomies, we reveal that most of up-regulated mRNAs in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells are associated with low-risk characteristics. The computational analysis shows that, in a context of overall gene set poverty, the upregulomes in RAC1- and kalirin-GEF1-suppressed ADRN-type cells are a batch of AU-rich element-containing mRNAs, which suggests a link between NUC migration and mRNA stability. Gene set enrichment analysis-based search for vulnerabilities reveals prospective weak points in RAC1- and kalirin-GEF1-suppressed ADRN-type NB cells, including activities of H3K27- and DNA methyltransferases. Altogether, these data support the introduction of NUC inhibitors into cancer treatment research.
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2.
  • Ait-Saada, A, et al. (författare)
  • Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks
  • 2019
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 2:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.
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3.
  • Ambikan, Anoop T., et al. (författare)
  • Genome-scale metabolic models for natural and long-term drug-induced viral control in HIV infection
  • 2022
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:9
  • Tidskriftsartikel (refereegranskat)abstract
    • Genome-scale metabolic models (GSMMs) can provide novel insights into metabolic reprogramming during disease progression and therapeutic interventions. We developed a context-specific system-level GSMM of people living with HIV (PLWH) using global RNA sequencing data from PBMCs with suppressive viremia either by natural (elite controllers, PLWHEC) or drug-induced (PLWHART) control. This GSMM was compared with HIV-negative controls (HC) to provide a comprehensive systems-level metabo-transcriptomic characterization. Transcriptomic analysis identified up-regulation of oxidative phosphorylation as a characteristic of PLWHART, differentiating them from PLWHEC with dysregulated complexes I, III, and IV. The flux balance analysis identified altered flux in several intermediates of glycolysis including pyruvate, a-ketoglutarate, and glutamate, among others, in PLWHART. The in vitro pharmacological inhibition of OXPHOS complexes in a latent lymphocytic cell model (J-Lat 10.6) suggested a role for complex IV in latency reversal and immunosenescence. Furthermore, inhibition of complexes I/III/IV induced apoptosis, collectively indicating their contribution to reservoir dynamics.
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4.
  • Armenteros, Jose Juan Almagro, et al. (författare)
  • Detecting sequence signals in targeting peptides using deep learning
  • 2019
  • Ingår i: Life Science Alliance. - : LIFE SCIENCE ALLIANCE LLC. - 2575-1077. ; 2:5
  • Tidskriftsartikel (refereegranskat)abstract
    • In bioinformatics, machine learning methods have been used to predict features embedded in the sequences. In contrast to what is generally assumed, machine learning approaches can also provide new insights into the underlying biology. Here, we demonstrate this by presenting TargetP 2.0, a novel state-of-the-art method to identify N-terminal sorting signals, which direct proteins to the secretory pathway, mitochondria, and chloroplasts or other plastids. By examining the strongest signals from the attention layer in the network, we find that the second residue in the protein, that is, the one following the initial methionine, has a strong influence on the classification. We observe that two-thirds of chloroplast and thylakoid transit peptides have an alanine in position 2, compared with 20% in other plant proteins. We also note that in fungi and single-celled eukaryotes, less than 30% of the targeting peptides have an amino acid that allows the removal of the N-terminal methionine compared with 60% for the proteins without targeting peptide. The importance of this feature for predictions has not been highlighted before.
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5.
  • Asplund, Olof, et al. (författare)
  • Islet Gene View-a tool to facilitate islet research
  • 2022
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (GCG, 56%), amylin (IAPP, 52%), insulin (INS, 44%), and somatostatin (SST, 24%). Inhibition of two DEGs, UNC5D and SERPINE2, impaired glucose-stimulated insulin secretion and impacted cell survival in a human beta-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.
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6.
  • Batté, Amandine, et al. (författare)
  • Chl1 helicase controls replication fork progression by regulating dNTP pools
  • 2022
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Eukaryotic cells have evolved a replication stress response that helps to overcome stalled/collapsed replication forks and ensure proper DNA replication. The replication checkpoint protein Mrc1 plays important roles in these processes, although its functional interactions are not fully understood. Here, we show that MRC1 negatively interacts with CHL1, which encodes the helicase protein Chl1, suggesting distinct roles for these factors during the replication stress response. Indeed, whereas Mrc1 is known to facilitate the restart of stalled replication forks, we uncovered that Chl1 controls replication fork rate under replication stress conditions. Chl1 loss leads to increased RNR1 gene expression and dNTP levels at the onset of S phase likely without activating the DNA damage response. This in turn impairs the formation of RPA-coated ssDNA and subsequent checkpoint activation. Thus, the Chl1 helicase affects RPA-dependent checkpoint activation in response to replication fork arrest by ensuring proper intracellular dNTP levels, thereby controlling replication fork progression under replication stress conditions.
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7.
  • Bauer, Susanne, et al. (författare)
  • Translatome profiling in fatal familial insomnia implicates TOR signaling in somatostatin neurons
  • 2022
  • Ingår i: Life Science Alliance. - : Life Science Allience. - 2575-1077. ; 5:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Selective neuronal vulnerability is common in neurodegenerative diseases but poorly understood. In genetic prion diseases, in-cluding fatal familial insomnia (FFI) and Creutzfeldt-Jakob dis-ease (CJD), different mutations in the Prnp gene manifest as clinically and neuropathologically distinct diseases. Here we report with electroencephalography studies that theta waves are mildly increased in 21 mo old knock-in mice modeling FFI and CJD and that sleep is mildy affected in FFI mice. To define affected cell types, we analyzed cell type-specific translatomes from six neuron types of 9 mo old FFI and CJD mice. Somatostatin (SST) neurons responded the strongest in both diseases, with unex-pectedly high overlap in genes and pathways. Functional analyses revealed up-regulation of neurodegenerative disease pathways and ribosome and mitochondria biogenesis, and down-regulation of synaptic function and small GTPase-mediated signaling in FFI, implicating down-regulation of mTOR signaling as the root of these changes. In contrast, responses in glutamatergic cerebellar neurons were disease-specific. The high similarity in SST neurons of FFI and CJD mice suggests that a common therapy may be beneficial for multiple genetic prion diseases.
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8.
  • Blum, Matthias, et al. (författare)
  • A comprehensive resource for retrieving, visualizing, and integrating functional genomics data
  • 2020
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:1
  • Tidskriftsartikel (refereegranskat)abstract
    • The enormous amount of freely accessible functional genomics data is an invaluable resource for interrogating the biological function of multiple DNA-interacting players and chromatin modifications by large-scale comparative analyses. However, in practice, interrogating large collections of public data requires major efforts for (i) reprocessing available raw reads, (ii) incorporating quality assessments to exclude artefactual and low-quality data, and (iii) processing data by using high-performance computation. Here, we present qcGenomics, a user-friendly online resource for ultrafast retrieval, visualization, and comparative analysis of tens of thousands of genomics datasets to gain new functional insight from global or focused multidimensional data integration. © 2019 Blum et al.
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9.
  • Bogaert, Annelies, et al. (författare)
  • N-terminal proteoforms may engage in different protein complexes
  • 2023
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Alternative translation initiation and alternative splicing may give rise to N-terminal proteoforms, proteins that differ at their N-terminus compared with their canonical counterparts. Such proteoforms can have altered localizations, stabilities, and functions. Although proteoforms generated from splice variants can be engaged in different protein complexes, it remained to be studied to what extent this applies to N-terminal proteoforms. To address this, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. First, we generated a catalogue of N-terminal proteoforms found in the HEK293T cellular cytosol from which 22 pairs were selected for interactome profiling. In addition, we provide evidence for the expression of several N-terminal proteoforms, identified in our catalogue, across different human tissues, as well as tissue-specific expression, highlighting their biological relevance. Protein–protein interaction profiling revealed that the overlap of the interactomes for both proteoforms is generally high, showing their functional relation. We also showed that N-terminal proteoforms can be engaged in new interactions and/or lose several interactions compared with their canonical counterparts, thus further expanding the functional diversity of proteomes.
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10.
  • Bonekamp, NA, et al. (författare)
  • High levels of TFAM repress mammalian mitochondrial DNA transcription in vivo
  • 2021
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Mitochondrial transcription factor A (TFAM) is compacting mitochondrial DNA (dmtDNA) into nucleoids and directly controls mtDNA copy number. Here, we show that the TFAM-to-mtDNA ratio is critical for maintaining normal mtDNA expression in different mouse tissues. Moderately increased TFAM protein levels increase mtDNA copy number but a normal TFAM-to-mtDNA ratio is maintained resulting in unaltered mtDNA expression and normal whole animal metabolism. Mice ubiquitously expressing very high TFAM levels develop pathology leading to deficient oxidative phosphorylation (OXPHOS) and early postnatal lethality. The TFAM-to-mtDNA ratio varies widely between tissues in these mice and is very high in skeletal muscle leading to strong repression of mtDNA expression and OXPHOS deficiency. In the heart, increased mtDNA copy number results in a near normal TFAM-to-mtDNA ratio and maintained OXPHOS capacity. In liver, induction of LONP1 protease and mitochondrial RNA polymerase expression counteracts the silencing effect of high TFAM levels. TFAM thus acts as a general repressor of mtDNA expression and this effect can be counterbalanced by tissue-specific expression of regulatory factors.
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11.
  • Breidenstein, Annika, et al. (författare)
  • PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs
  • 2024
  • Ingår i: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 7:8
  • Tidskriftsartikel (refereegranskat)abstract
    • A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded type IV secretion systems (T4SSs). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many, their function remains unclear. Here, we characterize PrgE, a proposed SSB from the Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very unusual DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string supported by its N-terminal tail. In vitro studies highlight the plasticity of PrgE oligomerization and confirm the importance of the N-terminus. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from eukaryotic SSBs.
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12.
  • Breidenstein, Annika, et al. (författare)
  • Structural and functional characterization of TraI from pKM101 reveals basis for DNA processing
  • 2023
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Type 4 secretion systems are large and versatile protein machineries that facilitate the spread of antibiotic resistance and other virulence factors via horizontal gene transfer. Conjugative type 4 secretion systems depend on relaxases to process the DNA in preparation for transport. TraI from the well-studied conjugative plasmid pKM101 is one such relaxase. Here, we report the crystal structure of the trans-esterase domain of TraI in complex with its substrate oriT DNA, highlighting the conserved DNA-binding mechanism of conjugative relaxases. In addition, we present an apo structure of the trans-esterase domain of TraI that includes most of the flexible thumb region. This allows us for the first time to visualize the large conformational change of the thumb subdomain upon DNA binding. We also characterize the DNA binding, nicking, and religation activity of the trans-esterase domain, helicase domain, and full-length TraI. Unlike previous indications in the literature, our results reveal that the TraI trans-esterase domain from pKM101 behaves in a conserved manner with its homologs from the R388 and F plasmids.
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13.
  • Cardiello, Joseph F, et al. (författare)
  • Evaluation of genetic demultiplexing of single-cell sequencing data from model species
  • 2023
  • Ingår i: Life Science Alliance. - 2575-1077. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Single-cell sequencing (sc-seq) provides a species agnostic tool to study cellular processes. However, these technologies are expensive and require sufficient cell quantities and biological replicates to avoid artifactual results. An option to address these problems is pooling cells from multiple individuals into one sc-seq library. In humans, genotype-based computational separation (i.e., demultiplexing) of pooled sc-seq samples is common. This approach would be instrumental for studying non-isogenic model organisms. We set out to determine whether genotype-based demultiplexing could be more broadly applied among species ranging from zebrafish to non-human primates. Using such non-isogenic species, we benchmark genotype-based demultiplexing of pooled sc-seq datasets against various ground truths. We demonstrate that genotype-based demultiplexing of pooled sc-seq samples can be used with confidence in several non-isogenic model organisms and uncover limitations of this method. Importantly, the only genomic resource required for this approach is sc-seq data and a de novo transcriptome. The incorporation of pooling into sc-seq study designs will decrease cost while simultaneously increasing the reproducibility and experimental options in non-isogenic model organisms.
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14.
  • Cascales, HS, et al. (författare)
  • Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1
  • 2021
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.
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15.
  • Cinato, Mathieu, et al. (författare)
  • Cardiac Plin5 interacts with SERCA2 and promotes calcium handling and cardiomyocyte contractility
  • 2023
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:4
  • Tidskriftsartikel (refereegranskat)abstract
    • The adult heart develops hypertrophy to reduce ventricular wall stress and maintain cardiac function in response to an increased workload. Although pathological hypertrophy generally prog-resses to heart failure, physiological hypertrophy may be car-dioprotective. Cardiac-specific overexpression of the lipid-droplet protein perilipin 5 (Plin5) promotes cardiac hypertrophy, but it is unclear whether this response is beneficial. We analyzed RNA -sequencing data from human left ventricle and showed that car-diac PLIN5 expression correlates with up-regulation of cardiac contraction-related processes. To investigate how elevated cardiac Plin5 levels affect cardiac contractility, we generated mice with cardiac-specific overexpression of Plin5 (MHC-Plin5 mice). These mice displayed increased left ventricular mass and cardiomyocyte size but preserved heart function. Quantitative proteomics identified sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) as a Plin5-interacting protein. In situ proximity ligation assay further confirmed the Plin5/SERCA2 interaction. Live imaging showed in-creases in intracellular Ca2+ release during contraction, Ca2+ removal during relaxation, and SERCA2 function in MHC-Plin5 versus WT cardiomyocytes. These results identify a role of Plin5 in improving cardiac contractility through enhanced Ca2+ signaling.
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17.
  • Dudenhöffer-Pfeifer, Monika, et al. (författare)
  • Immunoediting is not a primary transformation event in a murine model of MLL-ENL AML
  • 2018
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 1:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Although it is firmly established that endogenous immunity can prevent cancer outgrowth, with a range of immunomodulatory strategies reaching clinical use, most studies on the topic have been restricted to solid cancers. This applies in particular to cancer initiation, where model constraints have precluded investigations of immunosurveillance and immunoediting during the multistep progression into acute myeloid leukemia (AML). Here, we used a mouse model where the chimeric transcription factor MLL-ENL can be conditionally activated in vivo as a leukemic “first-hit,” which is followed by spontaneous transformation into AML. We observed similar disease kinetics regardless of whether AML developed in WT or immunocompromised hosts, despite more permissive preleukemic environments in the latter. When assessing transformed AML cells from either primary immunocompetent or immunocompromised hosts, AML cells from all sources could be targets of endogenous immunity. Our data argue against immunoediting in response to selective pressure from endogenous immunity as a universal primary transformation event in AML.
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18.
  • Fabrik, Ivo, et al. (författare)
  • Lung macrophages utilize unique cathepsin K-dependent phagosomal machinery to degrade intracellular collagen
  • 2023
  • Ingår i: Life Science Alliance (LSA). - : Life Science Alliance, LLC. - 2575-1077. ; 6:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Resident tissue macrophages are organ-specialized phagocytes responsible for the maintenance and protection of tissue ho-meostasis. It is well established that tissue diversity is reflected by the heterogeneity of resident tissue macrophage origin and phenotype. However, much less is known about tissue-specific phagocytic and proteolytic macrophage functions. Here, using a quantitative proteomics approach, we identify cathepsins as key determinants of phagosome maturation in primary peritoneum-, lung-, and brain-resident macrophages. The data further uncover cathepsin K (CtsK) as a molecular marker for lung phagosomes required for intracellular protein and collagen degradation. Pharmacological blockade of CtsK activity diminished phag-osomal proteolysis and collagenolysis in lung-resident mac-rophages. Furthermore, profibrotic TGF-beta negatively regulated CtsK-mediated phagosomal collagen degradation indepen-dently from classical endocytic-proteolytic pathways. In humans, phagosomal CtsK activity was reduced in COPD lung macrophages and non-COPD lung macrophages exposed to cig-arette smoke extract. Taken together, this study provides a comprehensive map of how peritoneal, lung, and brain tissue environment shapes phagosomal composition, revealing CtsK as a key molecular determinant of lung phagosomes contributing to phagocytic collagen clearance in lungs.
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20.
  • Gorospe, Choco Michael, et al. (författare)
  • Mitochondrial membrane potential acts as a retrograde signal to regulate cell cycle progression
  • 2023
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Mitochondria are central to numerous metabolic pathways whereby mitochondrial dysfunction has a profound impact and can manifest in disease. The consequences of mitochondrial dysfunction can be ameliorated by adaptive responses that rely on crosstalk from the mitochondria to the rest of the cell. Such mito-cellular signalling slows cell cycle progression in mitochondrial DNA-deficient (ρ0) Saccharomyces cerevisiae cells, but the initial trigger of the response has not been thoroughly studied. Here, we show that decreased mitochondrial membrane potential (ΔΨm) acts as the initial signal of mitochondrial stress that delays G1-to-S phase transition in both ρ0 and control cells containing mtDNA. Accordingly, experimentally increasing ΔΨm was sufficient to restore timely cell cycle progression in ρ0 cells. In contrast, cellular levels of oxidative stress did not correlate with the G1-to-S delay. Restored G1-to-S transition in ρ0 cells with a recovered ΔΨm is likely attributable to larger cell size, whereas the timing of G1/S transcription remained delayed. The identification of ΔΨm as a regulator of cell cycle progression may have implications for disease states involving mitochondrial dysfunction.
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21.
  • Gresle, MM, et al. (författare)
  • Multiple sclerosis risk variants regulate gene expression in innate and adaptive immune cells
  • 2020
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:7
  • Tidskriftsartikel (refereegranskat)abstract
    • At least 200 single-nucleotide polymorphisms (SNPs) are associated with multiple sclerosis (MS) risk. A key function that could mediate SNP-encoded MS risk is their regulatory effects on gene expression. We performed microarrays using RNA extracted from purified immune cell types from 73 untreated MS cases and 97 healthy controls and then performed Cis expression quantitative trait loci mapping studies using additive linear models. We describe MS risk expression quantitative trait loci associations for 129 distinct genes. By extending these models to include an interaction term between genotype and phenotype, we identify MS risk SNPs with opposing effects on gene expression in cases compared with controls, namely, rs2256814 MYT1 in CD4 cells (q = 0.05) and rs12087340 RF00136 in monocyte cells (q = 0.04). The rs703842 SNP was also associated with a differential effect size on the expression of the METTL21B gene in CD8 cells of MS cases relative to controls (q = 0.03). Our study provides a detailed map of MS risk loci that function by regulating gene expression in cell types relevant to MS.
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22.
  • Grudtsyna, Valeriia, et al. (författare)
  • Extracellular matrix sensing via modulation of orientational order of integrins and F-actin in focal adhesions
  • 2023
  • Ingår i: Life Science Alliance. - 2575-1077. ; 6:10
  • Tidskriftsartikel (refereegranskat)abstract
    • Specificity of cellular responses to distinct cues from the ECM requires precise and sensitive decoding of physical information. However, how known mechanisms of mechanosensing like force-dependent catch bonds and conformational changes in FA proteins can confer that this sensitivity is not known. Using polarization microscopy and computational modeling, we identify dynamic changes in an orientational order of FA proteins as a molecular organizational mechanism that can fine-tune cell sensitivity to the ECM. We find that αV integrins and F-actin show precise changes in the orientational order in an ECM-mediated integrin activation-dependent manner. These changes are sensitive to ECM density and are regulated independent of myosin-II activity though contractility can enhance this sensitivity. A molecular-clutch model demonstrates that the orientational order of integrin-ECM binding coupled to directional catch bonds can capture cellular responses to changes in ECM density. This mechanism also captures decoupling of ECM density sensing from stiffness sensing thus elucidating specificity. Taken together, our results suggest relative geometric organization of FA molecules as an important molecular architectural feature and regulator of mechanotransduction.
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25.
  • Hong, Mun-Gwan, et al. (författare)
  • Profiles of histidine-rich glycoprotein associate with age and risk of all-cause mortality
  • 2020
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:10, s. e202000817-
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite recognizing aging as a common risk factor of many human diseases, little is known about its molecular traits. To identify age-associated proteins circulating in human blood, we screened 156 individuals aged 50–92 using exploratory and multiplexed affinity proteomics assays. Profiling eight additional study sets (N = 3,987), performing antibody validation, and conducting a meta-analysis revealed a consistent age association (P = 6.61 × 10−6) for circulating histidine-rich glycoprotein (HRG). Sequence variants of HRG influenced how the protein was recognized in the immunoassays. Indeed, only the HRG profiles affected by rs9898 were associated with age and predicted the risk of mortality (HR = 1.25 per SD; 95% CI = 1.12–1.39; P = 6.45 × 10−5) during a follow-up period of 8.5 yr after blood sampling (IQR = 7.7–9.3 yr). Our affinity proteomics analysis found associations between the particular molecular traits of circulating HRG with age and all-cause mortality. The distinct profiles of this multipurpose protein could serve as an accessible and informative indicator of the physiological processes related to biological aging.
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26.
  • Javadi, Ala, et al. (författare)
  • Assembly mechanisms of the bacterial cytoskeletal protein FilP
  • 2019
  • Ingår i: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 2:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite low-sequence homology, the intermediate filament (IF)–like protein FilP from Streptomyces coelicolor displays structural and biochemical similarities to the metazoan nuclear IF lamin. FilP, like IF proteins, is composed of central coiled-coil domains interrupted by short linkers and flanked by head and tail domains. FilP polymerizes into repetitive filament bundles with paracrystalline properties. However, the cations Na+ and K+ are found to induce the formation of a FilP hexagonal meshwork with the same 60-nm repetitive unit as the filaments. Studies of polymerization kinetics, in combination with EM techniques, enabled visualization of the basic building block — a transiently soluble rod-shaped FilP molecule—and its assembly into protofilaments and filament bundles. Cryoelectron tomography provided a 3D view of the FilP bundle structure and an original assembly model of an IF-like protein of prokaryotic origin, thereby enabling a comparison with the assembly of metazoan IF.
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27.
  • Jespersen, Nathan, et al. (författare)
  • Systematic identification of recognition motifs for the hub protein LC8
  • 2019
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 2:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Hub proteins participate in cellular regulation by dynamic binding of multiple proteins within interaction networks. The hub protein LC8 reversibly interacts with more than 100 partners through a flexible pocket at its dimer interface. To explore the diversity of the LC8 partner pool, we screened for LC8 binding partners using a proteomic phage display library composed of peptides from the human proteome, which had no bias toward a known LC8 motif. Of the identified hits, we validated binding of 29 peptides using isothermal titration calorimetry. Of the 29 peptides, 19 were entirely novel, and all had the canonical TQT motif anchor. A striking observation is that numerous peptides containing the TQT anchor do not bind LC8, indicating that residues outside of the anchor facilitate LC8 interactions. Using both LC8-binding and nonbinding peptides containing the motif anchor, we developed the "LC8Pred" algorithm that identifies critical residues flanking the anchor and parses random sequences to predict LC8-binding motifs with similar to 78% accuracy. Our findings significantly expand the scope of the LC8 hub interactome.
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28.
  • Karlina, Ruth, et al. (författare)
  • Identification and characterization of distinct brown adipocyte subtypes in C57BL/6J mice
  • 2021
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Brown adipose tissue (BAT) plays an important role in the regulation of body weight and glucose homeostasis. Although increasing evidence supports white adipose tissue heterogeneity, little is known about heterogeneity within murine BAT. Recently, UCP1 high and low expressing brown adipocytes were identified, but a developmental origin of these subtypes has not been studied. To obtain more insights into brown preadipocyte heterogeneity, we use single-cell RNA sequencing of the BAT stromal vascular fraction of C57/BL6 mice and characterize brown preadipocyte and adipocyte clonal cell lines. Statistical analysis of gene expression profiles from brown preadipocyte and adipocyte clones identify markers distinguishing brown adipocyte subtypes. We confirm the presence of distinct brown adipocyte populations in vivo using the markers EIF5, TCF25, and BIN1. We also demonstrate that loss of Bin1 enhances UCP1 expression and mitochondrial respiration, suggesting that BIN1 marks dormant brown adipocytes. The existence of multiple brown adipocyte subtypes suggests distinct functional properties of BAT depending on its cellular composition, with potentially distinct functions in thermogenesis and the regulation of whole body energy homeostasis.
  •  
29.
  • Konings, Sabine C, et al. (författare)
  • Apolipoprotein E intersects with amyloid-β within neurons
  • 2023
  • Ingår i: Life Science Alliance. - 2575-1077. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Apolipoprotein E4 (ApoE4) is the most important genetic risk factor for Alzheimer's disease (AD). Among the earliest changes in AD is endosomal enlargement in neurons, which was reported as enhanced in ApoE4 carriers. ApoE is thought to be internalized into endosomes of neurons, whereas β-amyloid (Aβ) accumulates within neuronal endosomes early in AD. However, it remains unknown whether ApoE and Aβ intersect intracellularly. We show that internalized astrocytic ApoE localizes mostly to lysosomes in neuroblastoma cells and astrocytes, whereas in neurons, it preferentially localizes to endosomes-autophagosomes of neurites. In AD transgenic neurons, astrocyte-derived ApoE intersects intracellularly with amyloid precursor protein/Aβ. Moreover, ApoE4 increases the levels of endogenous and internalized Aβ 42 in neurons. Taken together, we demonstrate differential localization of ApoE in neurons, astrocytes, and neuron-like cells, and show that internalized ApoE intersects with amyloid precursor protein/Aβ in neurons, which may be of considerable relevance to AD.
  •  
30.
  • Lahiri, Shibojyoti, et al. (författare)
  • MALDI-IMS combined with shotgun proteomics identify and localize new factors in male infertility.
  • 2021
  • Ingår i: Life science alliance. - 2575-1077. ; 4:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Spermatogenesis is a complex multi-step process involving intricate interactions between different cell types in the male testis. Disruption of these interactions results in infertility. Combination of shotgun tissue proteomics with MALDI imaging mass spectrometry is markedly potent in revealing topological maps of molecular processes within tissues. Here, we use a combinatorial approach on a characterized mouse model of hormone induced male infertility to uncover misregulated pathways. Comparative testicular proteome of wild-type and mice overexpressing human P450 aromatase (AROM+) with pathologically increased estrogen levels unravels gross dysregulation of spermatogenesis and emergence of pro-inflammatory pathways in AROM+ testis. In situ MS allowed us to localize misregulated proteins/peptides to defined regions within the testis. Results suggest that infertility is associated with substantial loss of proteomic heterogeneity, which define distinct stages of seminiferous tubuli in healthy animals. Importantly, considerable loss of mitochondrial factors, proteins associated with late stages of spermatogenesis and steroidogenic factors characterize AROM+ mice. Thus, the novel proteomic approach pinpoints in unprecedented ways the disruption of normal processes in testis and provides a signature for male infertility.
  •  
31.
  • Lindehell, Henrik, 1984-, et al. (författare)
  • Methylation of lysine 36 on histone H3 is required to control transposon activities in somatic cells
  • 2023
  • Ingår i: Life Science Alliance. - : NLM (Medline). - 2575-1077. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Transposable elements constitute a substantial portion of most eukaryotic genomes and their activity can lead to developmental and neuronal defects. In the germline, transposon activity is antagonized by the PIWI-interacting RNA pathway tasked with repression of transposon transcription and degrading transcripts that have already been produced. However, most of the genes required for transposon control are not expressed outside the germline, prompting the question: what causes deleterious transposons activity in the soma and how is it managed? Here, we show that disruptions of the Histone 3 lysine 36 methylation machinery led to increased transposon transcription in Drosophila melanogaster brains and that there is division of labour for the repression of transposable elements between the different methyltransferases Set2, NSD, and Ash1. Furthermore, we show that disruption of methylation leads to somatic activation of key genes in the PIWI-interacting RNA pathway and the preferential production of RNA from dual-strand piRNA clusters.
  •  
32.
  • Luu, TT, et al. (författare)
  • Short-term IL-15 priming leaves a long-lasting signalling imprint in mouse NK cells independently of a metabolic switch
  • 2021
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:4
  • Tidskriftsartikel (refereegranskat)abstract
    • IL-15 priming of NK cells is a broadly accepted concept, but the dynamics and underlying molecular mechanisms remain poorly understood. We show that as little as 5 min of IL-15 treatment in vitro, followed by removal of excess cytokines, results in a long-lasting, but reversible, augmentation of NK cell responsiveness upon activating receptor cross-linking. In contrast to long-term stimulation, improved NK cell function after short-term IL-15 priming was not associated with enhanced metabolism but was based on the increased steady-state phosphorylation level of signalling molecules downstream of activating receptors. Inhibition of JAK3 eliminated this priming effect, suggesting a cross talk between the IL-15 receptor and ITAM-dependent activating receptors. Increased signalling molecule phosphorylation levels, calcium flux, and IFN-γ secretion lasted for up to 3 h after IL-15 stimulation before returning to baseline. We conclude that IL-15 rapidly and reversibly primes NK cell function by modulating activating receptor signalling. Our findings suggest a mechanism by which NK cell reactivity can potentially be maintained in vivo based on only brief encounters with IL-15 trans-presenting cells.
  •  
33.
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34.
  • Mehmeti-Ajradini, Meliha, et al. (författare)
  • Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer
  • 2020
  • Ingår i: Life Science Alliance. - 2575-1077. ; 3:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients cotransplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.
  •  
35.
  • Mehmeti-Ajradini, Meliha, et al. (författare)
  • Human G-MDSCs are neutrophils at distinct maturation stages promoting tumor growth in breast cancer
  • 2020
  • Ingår i: Life Science Alliance. - : LIFE SCIENCE ALLIANCE LLC. - 2575-1077. ; 3:11
  • Tidskriftsartikel (refereegranskat)abstract
    • Myeloid-derived suppressor cells (MDSCs) are known to contribute to immune evasion in cancer. However, the function of the human granulocytic (G)-MDSC subset during tumor progression is largely unknown, and there are no established markers for their identification in human tumor specimens. Using gene expression profiling, mass cytometry, and tumor microarrays, we here demonstrate that human G-MDSCs occur as neutrophils at distinct maturation stages, with a disease-specific profile. G-MDSCs derived from patients with metastatic breast cancer and malignant melanoma display a unique immature neutrophil profile, that is more similar to healthy donor neutrophils than to G-MDSCs from sepsis patients. Finally, we show that primary G-MDSCs from metastatic breast cancer patients co-transplanted with breast cancer cells, promote tumor growth, and affect vessel formation, leading to myeloid immune cell exclusion. Our findings reveal a role for human G-MDSC in tumor progression and have clinical implications also for targeted immunotherapy.
  •  
36.
  • Mi, J, et al. (författare)
  • Efficient knock-in method enabling lineage tracing in zebrafish
  • 2023
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Here, we devised a cloning-free 3′ knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5′ AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92,nkx6.1,krt4, andid2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested thatnkx6.1+cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereasid2a+cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepaticid2a+duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.
  •  
37.
  • Mi, JR, et al. (författare)
  • Efficient knock-in method enabling lineage tracing in zebrafish
  • 2023
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Here, we devised a cloning-free 3′ knock-in strategy for zebrafish using PCR amplified dsDNA donors that avoids disrupting the targeted genes. The dsDNA donors carry genetic cassettes coding for fluorescent proteins and Cre recombinase in frame with the endogenous gene but separated from it by self-cleavable peptides. Primers with 5′ AmC6 end-protections generated PCR amplicons with increased integration efficiency that were coinjected with preassembled Cas9/gRNA ribonucleoprotein complexes for early integration. We targeted four genetic loci (krt92,nkx6.1,krt4, andid2a) and generated 10 knock-in lines, which function as reporters for the endogenous gene expression. The knocked-in iCre or CreERT2 lines were used for lineage tracing, which suggested thatnkx6.1+cells are multipotent pancreatic progenitors that gradually restrict to the bipotent duct, whereasid2a+cells are multipotent in both liver and pancreas and gradually restrict to ductal cells. In addition, the hepaticid2a+duct show progenitor properties upon extreme hepatocyte loss. Thus, we present an efficient and straightforward knock-in technique with widespread use for cellular labelling and lineage tracing.
  •  
38.
  • Moruzzi, N, et al. (författare)
  • Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology
  • 2022
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.
  •  
39.
  • Moruzzi, N, et al. (författare)
  • Mitochondrial impairment and intracellular reactive oxygen species alter primary cilia morphology
  • 2022
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
  • Tidskriftsartikel (refereegranskat)abstract
    • Primary cilia have recently emerged as cellular signaling organelles. Their homeostasis and function require a high amount of energy. However, how energy depletion and mitochondria impairment affect cilia have barely been addressed. We first studied the spatial relationship between a mitochondria subset in proximity to the cilium in vitro, finding similar mitochondrial activity measured as mitochondrial membrane potential compared with the cellular network. Next, using common primary cilia cell models and inhibitors of mitochondrial energy production, we found alterations in cilia number and/or length due to energy depletion and mitochondrial reactive oxygen species (ROS) overproduction. Finally, by using a mouse model of type 2 diabetes mellitus, we provided in vivo evidence that cilia morphology is impaired in diabetic nephropathy, which is characterized by ROS overproduction and impaired mitochondrial metabolism. In conclusion, we showed that energy imbalance and mitochondrial ROS affect cilia morphology and number, indicating that conditions characterized by mitochondria and radicals imbalances might lead to ciliary impairment.
  •  
40.
  • Muralidharan, Somsundar Veppil, et al. (författare)
  • Small molecule inhibitors and a kinase-dead expressing mouse model demonstrate that the kinase activity of Chk1 is essential for mouse embryos and cancer cells
  • 2020
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 3:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Chk1 kinase is downstream of the ATR kinase in the sensing of improper replication. Previous cell culture studies have demonstrated that Chk1 is essential for replication. Indeed, Chk1 inhibitors are efficacious against tumors with high-level replication stress such as Myc-induced lymphoma cells. Treatment with Chk1 inhibitors also combines well with certain chemotherapeutic drugs, and effects associate with the induction of DNA damage and reduction of Chk1 protein levels. Most studies of Chk1 function have relied on the use of inhibitors. Whether or not a mouse or cancer cells could survive if a kinase-dead form of Chk1 is expressed has not been investigated before. Here, we generate a mouse model that expresses a kinase-dead (D130A) allele in the mouse germ line. We find that this mouse is overtly normal and does not have problems with erythropoiesis with aging as previously been shown for a mouse expressing one null allele. However, similar to a null allele, homozygous kinase-dead mice cannot be generated, and timed pregnancies of heterozygous mice suggest lethality of homozygous blastocysts at around the time of implantation. By breeding the kinase-dead Chk1 mouse with a conditional allele, we are able to demonstrate that expression of only one kinase-dead allele, but no wild-type allele, of Chek1 is lethal for Myc-induced cancer cells. Finally, treatment of melanoma cells with tumor-infiltrating T cells or CAR-T cells is effective even if Chk1 is inhibited, suggesting that Chk1 inhibitors can be safely administered in patients where immunotherapy is an essential component of the arsenal against cancer.
  •  
41.
  • Nejedlá, Michaela, et al. (författare)
  • The actin regulator profilin 1 is functionally associated with the mammalian centrosome
  • 2021
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Profilin 1 is a crucial actin regulator, interacting with monomeric actin and several actin-binding proteins controlling actin polymerization. Recently, it has become evident that this profilin isoform associates with microtubules via formins and interferes with microtubule elongation at the cell periphery. Recruitment of microtubule-associated profilin upon extensive actin polymerizations, for example, at the cell edge, enhances microtubule growth, indicating that profilin contributes to the coordination of actin and microtubule organization. Here, we provide further evidence for the profilin-microtubule connection by demonstrating that it also functions in centrosomes where it impacts on microtubule nucleation.
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42.
  • Pan, Gang, et al. (författare)
  • Multifaceted regulation of hepatic lipid metabolism by YY1
  • 2021
  • Ingår i: Life Science Alliance. - : LIFE SCIENCE ALLIANCE LLC. - 2575-1077. ; 4:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Recent studies suggested that dysregulated YY1 plays a pivotal role in many liver diseases. To obtain a detailed view of genes and pathways regulated by YY1 in the liver, we carried out RNA sequencing in HepG2 cells after YY1 knockdown. A rigid set of 2,081 differentially expressed genes was identified by comparing the YY1-knockdown samples (n = 8) with the control samples (n = 14). YY1 knockdown significantly decreased the expression of several key transcription factors and their coactivators in lipid metabolism. This is illustrated by YY1 regulating PPARA expression through binding to its promoter and enhancer regions. Our study further suggest that down-regulation of the key transcription factors together with YY1 knockdown significantly decreased the cooperation between YY1 and these transcription factors at various regulatory regions, which are important in regulating the expression of genes in hepatic lipid metabolism. This was supported by the finding that the expression of SCD and ELOVL6, encoding key enzymes in lipogenesis, were regulated by the cooperation between YY1 and PPARA/RXRA complex over their promoters.
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43.
  • Papaioannou, A., et al. (författare)
  • Stress-induced tyrosine phosphorylation of RtcB modulates IRE1 activity and signaling outputs
  • 2022
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:5
  • Tidskriftsartikel (refereegranskat)abstract
    • ER stress is mediated by three sensors and the most evolutionary conserved IRE1α signals through its cytosolic kinase and endoribonuclease (RNase) activities. IRE1α RNase activity can either catalyze the initial step of XBP1 mRNA unconventional splicing or degrade a number of RNAs through regulated IRE1-dependent decay. Until now, the biochemical and biological outputs of IRE1α RNase activity have been well documented; however, the precise mechanisms controlling whether IRE1α signaling is adaptive or pro-death (terminal) remain unclear. We investigated those mechanisms and hypothesized that XBP1 mRNA splicing and regulated IRE1-dependent decay activity could be co-regulated by the IRE1α RNase regulatory network. We identified that RtcB, the tRNA ligase responsible for XBP1 mRNA splicing, is tyrosine-phosphorylated by c-Abl and dephosphorylated by PTP1B. Moreover, we show that the phosphorylation of RtcB at Y306 perturbs RtcB interaction with IRE1α, thereby attenuating XBP1 mRNA splicing. Our results demonstrate that the IRE1α RNase regulatory network is dynamically fine-tuned by tyrosine kinases and phosphatases upon various stresses and that the extent of RtcB tyrosine phosphorylation determines cell adaptive or death outputs. © 2022 Papaioannou et al.
  •  
44.
  • Pillon, NJ, et al. (författare)
  • Palmitate impairs circadian transcriptomics in muscle cells through histone modification of enhancers
  • 2022
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:1
  • Tidskriftsartikel (refereegranskat)abstract
    • Obesity and elevated circulating lipids may impair metabolism by disrupting the molecular circadian clock. We tested the hypothesis that lipid overload may interact with the circadian clock and alter the rhythmicity of gene expression through epigenomic mechanisms in skeletal muscle. Palmitate reprogrammed the circadian transcriptome in myotubes without altering the rhythmic mRNA expression of core clock genes. Genes with enhanced cycling in response to palmitate were associated with post-translational modification of histones. The cycling of histone 3 lysine 27 acetylation (H3K27ac), a marker of active gene enhancers, was modified by palmitate treatment. Chromatin immunoprecipitation and sequencing confirmed that palmitate exposure altered the cycling of DNA regions associated with H3K27ac. The overlap between mRNA and DNA regions associated with H3K27ac and the pharmacological inhibition of histone acetyltransferases revealed novel cycling genes associated with lipid exposure of primary human myotubes. Palmitate exposure disrupts transcriptomic rhythmicity and modifies enhancers through changes in histone H3K27 acetylation in a circadian manner. Thus, histone acetylation is responsive to lipid overload and may redirect the circadian chromatin landscape, leading to the reprogramming of circadian genes and pathways involved in lipid biosynthesis in skeletal muscle.
  •  
45.
  • Ptasinski, Victoria, et al. (författare)
  • Modeling fibrotic alveolar transitional cells with pluripotent stem cell-derived alveolar organoids
  • 2023
  • Ingår i: Life Science Alliance. - 2575-1077. ; 6:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Repeated injury of the lung epithelium is proposed to be the main driver of idiopathic pulmonary fibrosis (IPF). However, available therapies do not specifically target the epithelium and human models of fibrotic epithelial damage with suitability for drug discovery are lacking. We developed a model of the aberrant epithelial reprogramming observed in IPF using alveolar organoids derived from human-induced pluripotent stem cells stimulated with a cocktail of pro-fibrotic and inflammatory cytokines. Deconvolution of RNA-seq data of alveolar organoids indicated that the fibrosis cocktail rapidly increased the proportion of transitional cell types including the KRT5-/KRT17+ aberrant basaloid phenotype recently identified in the lungs of IPF patients. We found that epithelial reprogramming and extracellular matrix (ECM) production persisted after removal of the fibrosis cocktail. We evaluated the effect of the two clinically approved compounds for IPF, nintedanib and pirfenidone, and found that they reduced the expression of ECM and pro-fibrotic mediators but did not completely reverse epithelial reprogramming. Thus, our system recapitulates key aspects of IPF and is a promising system for drug discovery.
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46.
  • Rovšnik, Urška, et al. (författare)
  • Dynamic closed states of a ligand-gated ion channel captured by cryo-EM and simulations
  • 2021
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 4:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. Biophysical and pharmacological characterization of these receptor proteins relies on high-quality structures in multiple, subtly distinct functional states. However, structural data in this family remain limited, particularly for resting and intermediate states on the activation pathway. Here, we report cryo-electr on microscopy (cryo-EM) structures of the proton-activated Gloeobacter violaceus ligandgated ion channel (GLIC) under three pH conditions. Decreased pH was associated with improved resolution and side chain rearrangements at the subunit/domain interface, particularly involving functionally important residues in the beta 1-beta 2 and M2-M3 loops. Molecular dynamics simulations substantiated flexibility in the closed-channel extracellular domains relative to the transmembrane ones and supported electrostatic remodeling around E35 and E243 in proton-induced gating. Exploration of secondary cryoEM classes further indicated a low-pH population with an expanded pore. These results allow us to define distinct protonation and activation steps in pH-stimulated conformational cycling in GLIC, including interfacial rearrangements largely conserved in the pentameric channel family.
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47.
  • Sáinz-Jaspeado, Miguel, et al. (författare)
  • VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis
  • 2024
  • Ingår i: Life Science Alliance. - : Life Science Alliance. - 2575-1077. ; 7:3
  • Tidskriftsartikel (refereegranskat)abstract
    • The endothelial junction component vascular endothelial (VE)–cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2Y949F/Y949F) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.
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48.
  • Scherzer, M, et al. (författare)
  • Recruitment of Scc2/4 to double-strand breaks depends on γH2A and DNA end resection
  • 2022
  • Ingår i: Life science alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:5, s. 1-14
  • Tidskriftsartikel (refereegranskat)abstract
    • Homologous recombination enables cells to overcome the threat of DNA double-strand breaks (DSBs), allowing for repair without the loss of genetic information. Central to the homologous recombination repair process is the de novo loading of cohesin around a DSB by its loader complex Scc2/4. Although cohesin’s DSB accumulation has been explored in numerous studies, the prerequisites for Scc2/4 recruitment during the repair process are still elusive. To address this question, we combine chromatin immunoprecipitation-qPCR with a site-specific DSB in vivo, in Saccharomyces cerevisiae. We find that Scc2 DSB recruitment relies on γH2A and Tel1, but as opposed to cohesin, not on Mec1. We further show that the binding of Scc2, which emanates from the break site, depends on and coincides with DNA end resection. Absence of chromatin remodeling at the DSB affects Scc2 binding and DNA end resection to a comparable degree, further indicating the latter to be a major driver for Scc2 recruitment. Our results shed light on the intricate DSB repair cascade leading to the recruitment of Scc2/4 and subsequent loading of cohesin.
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49.
  • Schnerwitzki, Danny, et al. (författare)
  • Neuron-specific inactivation of Wt1 alters locomotion in mice and changes interneuron composition in the spinal cord
  • 2018
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 1:4
  • Tidskriftsartikel (refereegranskat)abstract
    • Locomotion is coordinated by neuronal circuits of the spinal cord. Recently, dI6 neurons were shown to participate in the control of locomotion. A subpopulation of dI6 neurons expresses the Wilms tumor suppressor gene Wt1. However, the function of Wt1 in these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed a slower walk with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-/hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0, and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and that loss of Wt1 is associated with alterations in locomotion.
  •  
50.
  • Ternet, Camille, et al. (författare)
  • Analysis of context-specific KRAS-effector (sub)complexes in Caco-2 cells
  • 2023
  • Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 6:5
  • Tidskriftsartikel (refereegranskat)abstract
    • Ras is a key switch controlling cell behavior. In the GTP-bound form, Ras interacts with numerous effectors in a mutually ex-clusive manner, where individual Ras-effectors are likely part of larger cellular (sub)complexes. The molecular details of these (sub)complexes and their alteration in specific contexts are not understood. Focusing on KRAS, we performed affinity puri-fication (AP)-mass spectrometry (MS) experiments of exoge-nously expressed FLAG-KRAS WT and three oncogenic mutants ("genetic contexts") in the human Caco-2 cell line, each exposed to 11 different culture media ("culture contexts") that mimic conditions relevant in the colon and colorectal cancer. We identified four effectors present in complex with KRAS in all genetic and growth contexts ("context-general effectors"). Seven effectors are found in KRAS complexes in only some contexts ("context-specific effectors"). Analyzing all interactors in complex with KRAS per condition, we find that the culture contexts had a larger impact on interaction rewiring than genetic contexts. We investigated how changes in the interactome impact functional outcomes and created a Shiny app for interactive visualization. We validated some of the functional differences in metabolism and proliferation. Finally, we used networks to evaluate how KRAS-effectors are involved in the modulation of functions by random walk analyses of effector-mediated (sub)complexes. Altogether, our work shows the impact of environmental contexts on network rewiring, which provides insights into tissue-specific signaling mechanisms. This may also explain why KRAS oncogenic mutants may be causing cancer only in specific tissues despite KRAS being expressed in most cells and tissues.
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