Biology subjects

Mueller, M.

Publications and source records attributed to Mueller, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2024

Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role in the nucleus during S phase, where it directly phosphorylates the replicative helicase, the minichromosome maintenance (MCM) complex. This leads to the initiation of chromosome replication. However, aside from the MCM complex, few other targets have been identified to date, leaving DDK an understudied kinase. Here, we describe a two-pronged mass spectrometry-based approach and define the nuclear DDK-dependent phosphoproteome, which consists of approximately 400 phosphorylation events. Within this network, we found that DDK directly phosphorylates the Arp8 subunit of the multi-subunit chromatin remodeler complex INO80. Arp8 phosphorylation stabilises INO80s intramolecular complex integrity, which finetunes its nucleosome spacing activity at replication origins. This adjustment of origin chromatin architecture stimulates replication and is important for the response to replication stress. Our results represent a significant advance in our understanding of the molecular mechanisms underlying the regulation of replication origins.

biochemistry↗

bioRxiv2024

A highly potent, orally bioavailable pyrazole-derived cannabinoid CB2 receptor-selective full agonist for in vivo studies

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (Ki 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent Kd values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

pharmacology and toxicology↗

bioRxiv2024

The transcription factor ZNF469 regulates collagen production in liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the worlds population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

molecular biology↗

bioRxiv2024

Real-time transcriptomic profiling in distinct experimental conditions

Nanopore technology offers real-time sequencing opportunities, providing rapid access to sequenced data and allowing researchers to manage the sequencing process efficiently, resulting in cost-effective strategies. Here, we present focused case studies demonstrating the versatility of real-time transcriptomics analysis in rapid quality control for long-read RNA-seq. We illustrate its utility through four experimental setups: 1) transcriptome profiling of distinct human cellular populations, 2) identification of experimentally enriched transcripts, 3) transcriptional analysis of cells under heat shock conditions and 4) identification of experimentally manipulated genes (knockout and overexpression) in several yeast strains. We show how to perform multiple layers of quality control as soon as sequencing has started, addressing both the quality of the experimental and sequencing traits. Real-time quality control measures assess sample/condition variability and determine the number of identified genes per sample/condition. Furthermore, real-time differential gene/transcript expression analysis can be conducted at various time points post-sequencing initiation (PSI), revealing dynamic changes in gene/transcript expression between two conditions. Using real-time analysis, which occurs in parallel to the sequencing run, we identified differentially expressed genes/transcripts as early as 1hr PSI. These changes were consistently observed throughout the entire sequencing process. We discuss the new possibilities offered by real-time data analysis, which have the potential to serve as a valuable tool for rapid and cost-effective quality checks in specific experimental settings and can be potentially integrated into clinical applications in the future.

bioinformatics↗

bioRxiv2024

Intrinsically disordered RNA-binding motifs cooperate to catalyze RNA folding and drive phase separation

RNA-binding proteins are essential for gene regulation and the spatial organization of cells. Here, we report that the yeast ribosome biogenesis factor Loc1p is an intrinsically disordered RNA-binding protein with eight repeating positively charged, unstructured nucleic acid binding (PUN) motifs. While a single of these previously undefined motifs stabilizes folded RNAs, multiple copies strongly cooperate to catalyze RNA folding. In the presence of RNA, these multivalent PUN motifs drive phase separation. Proteome-wide searches in pro-and eukaryotes for proteins with similar arrays of PUN motifs reveal a strong enrichment in RNA-mediated processes and DNA remodeling. Thus, PUN motifs are potentially involved in a large variety of RNA-and DNA-related processes by concentrating them in membrane-less organelles. The general function and wide distribution of PUN motifs across species suggests that in an ancient "RNA world" PUN-like motifs may have supported the correct folding of early ribozymes.

biochemistry↗

bioRxiv2024

High sensitivity cameras can lower spatial resolution in high-resolution optical microscopy

High-resolution optical fluorescence microscopies and, in particular, super-resolution fluorescence microscopy, are rapidly adopting highly sensitive cameras as their referred photodetectors. Camera-based parallel detection facilitates high-speed live cell imaging with the highest spatial resolution. Here, we show that the drive to use ever more sensitive, photon-counting image sensors in cameras can, however, have detrimental effects on spatial resolution that many researchers are not aware of. Typical parameters which influence the selection of image sensors are pixel size, quantum efficiency, signal-to-noise performance, dynamic range, and frame rate of the sensor. A parameter that is, however, often overlooked, is the sensors modulation transfer function (MTF). We have determined the wavelength-specific MTF of front- and back-illuminated image sensors and evaluated how it affects the spatial resolution that can be achieved in high fluorescence microscopy modalities. We find significant differences in image sensor performance for sensors that cause the resulting spatial resolution to vary with up to 28%. This result shows that the choice of image sensor has significant impact on the imaging performance of all camera-based optical microscopy modalities.

biophysics↗

bioRxiv2024

Enhanced cardiac mitochondrial biogenesis by nitro-oleic acid remedies diastolic dysfunction in a mouse model of heart failure with preserved ejection fraction

Prevalence of heart failure with preserved ejection fraction (HFpEF) is increasing, while treatment options are inadequate. Hypertension and obesity-related metabolic dysfunctions contribute to HFpEF progression. Nitro-oleic acid (NO2-OA) impacts metabolic processes by improving glucose tolerance and adipocyte function. In this study, 4 week treatment with NO2-OA ameliorated diastolic dysfunction in a HFpEF mouse model induced by high-fat diet and inhibition of the endothelial nitric oxide synthase. A proteomic analysis of left ventricular tissue revealed, that one third of the identified proteins, mostly mitochondrial proteins, were upregulated in hearts of NO2-OA-treated HFpEF mice compared to controls and vehicle-treated HFpEF mice, which was confirmed by immunoblot. Activation of the 5-adenosine-monophosphate-activated-protein-kinase (AMPK) signaling pathway mediated an enhancement of mitochondrial biogenesis in hearts of NO2-OA-treated HFpEF mice. In cardiomyocytes under metabolic stress, NO2-OA increased mitochondrial protein level accompanied by enhanced oxidative phosphorylation. In conclusion, targeting mitochondrial integrity in HFpEF leads to improved diastolic function.

pathology↗

bioRxiv2024

Discovery and binding mode of a small molecule inhibitor of the apo form of human TDO2

Tryptophan-2,3-dioxygenase (TDO2) and indoleamine-2,3-dioxygenase (IDO1) catalyze the conversion of L-tryptophan to N-formyl-kynurenine and play important roles in metabolism, inflammation, and tumor immune surveillance. Their enzymatic activities depend on their heme contents, which vary dynamically according to biological conditions. Inhibitors binding to heme-containing holo-TDO2 are known, but to date no inhibitor that binds to the heme-free state (apo-TDO2) has been reported. We describe the discovery of the first apo-TDO2 targeting inhibitors, to our knowledge, together with their co-crystal structures and inhibition of cellular TDO2 activity at low nanomolar concentrations.

biochemistry↗

bioRxiv2024

Image processing tools for petabyte-scale light sheet microscopy data

Light sheet microscopy is a powerful technique for high-speed 3D imaging of subcellular dynamics and large biological specimens. However, it often generates datasets ranging from hundreds of gigabytes to petabytes in size for a single experiment. Conventional computational tools process such images far slower than the time to acquire them and often fail outright due to memory limitations. To address these challenges, we present PetaKit5D, a scalable software solution for efficient petabyte-scale light sheet image processing. This software incorporates a suite of commonly used processing tools that are memory and performance-optimized. Notable advancements include rapid image readers and writers, fast and memory-efficient geometric transformations, high-performance Richardson-Lucy deconvolution, and scalable Zarr-based stitching. These features outperform state-of-the-art methods by over one order of magnitude, enabling the processing of petabyte-scale image data at the full teravoxel rates of modern imaging cameras. The software opens new avenues for biological discoveries through large-scale imaging experiments.

bioinformatics↗

bioRxiv2023

Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate binding pocket. Of 631 fragments soaked, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (2 hits) or at crystal packing interfaces (3 hits). Notably, two fragments with a pose sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. Our structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. SynopsisAn X-ray crystallographic screen on SARS-CoV-2 3CL protease resulted in 29 fragment hits, including two isatin-based reversible covalent binders, and revealed a strong influence of the crystal form used for fragment soaking on the bound conformation of three additional reference fragments.

biophysics↗

bioRxiv2023

Deep learning-based synaptic event detection

Quantitative information about synaptic transmission is key to our understanding of neural function. Spontaneously occurring synaptic events carry fundamental information about synaptic function and plasticity. However, their stochastic nature and low signal-to-noise ratio present major challenges for the reliable and consistent analysis. Here, we introduce miniML, a supervised deep learning- based method for accurate classification and automated detection of spontaneous synaptic events. Comparative analysis using simulated ground-truth data shows that miniML outperforms existing event analysis methods in terms of both precision and recall. miniML enables precise detection and quantification of synaptic events in electrophysiological recordings. We demonstrate that the deep learning approach generalizes easily to diverse synaptic preparations, different electrophysiological and optical recording techniques, and across animal species. miniML provides not only a comprehensive and robust framework for automated, reliable, and standardized analysis of synaptic events, but also opens new avenues for high-throughput investigations of neural function and dysfunction.

neuroscience↗

bioRxiv2023

Sodium chloride in the tumor microenvironment enhances T-cell metabolic fitness and cytotoxicity

Adoptive T-cell therapy has become a powerful weapon for cancer treatment. The efficacy of antitumor immunity is associated with the metabolic state of cytotoxic T cells, which is highly sensitive to the tumor microenvironment. It is therefore of considerable interest to bypass immunosuppressive signals in the tumor microenvironment and to identify factors that augment cytotoxic effector functions and ultimately tumor killing. Whether ionic signals serve as aberrant immune signals and influence the adaptive human antitumor immune response is still largely unexplored. We therefore investigated the effect of sodium on the phenotype, function and metabolic regulation of human CD8+ T cells using transcriptomic, metabolomic, high-dimensional flow cytometric and functional assays. We demonstrate a significant enrichment of sodium in solid tumors from patients with breast cancer, which leaves a transcriptomic imprint on intratumoral immune cells. Sodium chloride (NaCl) enhanced the activation state and effector functions of human CD8+ memory T cells. These functional alterations were associated with enhanced metabolic fitness, particularly increases in glycolysis, oxidative phosphorylation and overall nutrient uptake. These NaCl-induced effects translated into increased tumor cell killing in vitro and in a tumor mouse model in vivo. We therefore propose NaCl as a positive regulator of acute antitumor immunity that could be harnessed for ex vivo conditioning of adoptively transferred T cells, such as CAR T-cells.

immunology↗

bioRxiv2023

The eIF4A2 negative regulator of mRNA translation promotes extracellular matrix deposition to accelerate hepatocellular carcinoma initiation

Increased protein synthesis supports growth of established tumours. However, how mRNA translation contributes to early tumorigenesis remains unclear. Here we show that following oncogene activation, hepatocytes enter a non-proliferative/senescent-like phase characterized by 5{beta}1 integrin-dependent deposition of fibronectin-rich extracellular matrix (ECM) niches. These niches then promote exit from oncogene-induced senescence to permit progression to proliferating hepatocellular carcinoma (HCC). Removal of eIF4A2, a negative regulator of mRNA translation, boosts the synthesis of membrane/secretory proteins which drives a compensatory increase in the turnover/degradation of membrane proteins including 5{beta}1 integrin. This increased membrane protein degradation, in turn, compromises generation of ECM-rich tumour initiation niches, senescence-exit and progression to proliferating HCC. Consistently, pharmacological inhibition of mRNA translation following eIF4A2 loss restores ECM deposition and reinstates HCC progression. Thus, although inhibition of protein synthesis may be an effective way to reduce tumour biomass and the growth of established tumours, our results highlight how agents which reduce mRNA translation, if administered during early tumorigenesis, may awaken senescent cells and promote tumour progression.

cancer biology↗

bioRxiv2023

Acoustically evoked K-complexes are sufficient to boost verbal memory consolidation during sleep

Over the past decade, phase-targeted auditory stimulation (PTAS), a neuromodulation approach which presents auditory stimuli locked to the ongoing phase of slow waves during sleep, has shown potential to enhance specific aspects of sleep functions. However, the complexity of PTAS responses complicates the establishment of causality between specific electroencephalographic events and observed benefits. Here, we used down-PTAS during sleep to specifically evoke the early, K-complex (KC)-like response following PTAS without leading to a sustained increase in slow-wave activity throughout the stimulation window. Over the course of two nights, one with down-PTAS, the other without, high-density electroencephalography (hd-EEG) was recorded from 14 young healthy adults. The early response exhibited striking similarities to evoked KCs and was associated with improved verbal memory consolidation via stimulus-evoked spindle events nested into the up-phase of ongoing 1 Hz waves in a central region. These findings suggest that the early, KC-like response is sufficient to boost memory, potentially by orchestrating aspects of the hippocampal-neocortical dialogue.

neuroscience↗

bioRxiv2023

SARS-CoV-2 infection activates endogenous retroviruses of the LTR69 subfamily

Accumulating evidence suggests that endogenous retroviruses (ERVs) play an important role in the host response to infection and the development of disease. By combining RNA- and ChIP-sequencing analyses with RT-qPCR, we show that SARS-CoV-2 infection induces the LTR69 subfamily of ERVs, both in vitro and in vivo. Using functional assays, we identified one SARS-CoV-2-activated LTR69 locus, termed Dup69, which exhibits enhancer activity and is responsive to the transcription factors IRF3 and p65/RelA. LTR69-Dup69 is located about 500 bp upstream of a long non-coding RNA gene (ENSG00000289418) and within the PTPRN2 gene encoding a diabetes-associated autoantigen. Both ENSG00000289418 and PTPRN2 showed a significant increase in expression upon SARS-CoV-2 infection. Thus, our study sheds light on the interplay of exogenous with endogenous viruses and helps to understand how ERVs regulate gene expression during infection.

molecular biology↗

bioRxiv2023

SARS-CoV-2 ORF3c suppresses immune activation by inhibiting innate sensing

SARS-CoV-2 proteins are translated from subgenomic RNAs (sgRNAs). While most of these sgRNAs are monocistronic, some viral mRNAs encode more than one protein. For example, the ORF3a sgRNA also encodes ORF3c, an enigmatic 4l-amino acid peptide. Here, we show that ORF3c is expressed in SARS-CoV-2 infected cells and suppresses RIG-I- and MDA5-mediated immune activation and IFN-{beta} induction. Mechanistic analyses revealed that ORF3c interacts with the signaling adaptor MAVS, induces its C-terminal cleavage and inhibits the interaction of RIG-I with MAVS. The immunosuppressive activity of ORF3c is conserved among members of the subgenus sarbecovirus, including SARS-CoV and coronaviruses isolated from bats. Notably, however, the SARS-CoV-2 delta and kappa variants harbor premature stop codons in ORF3c demonstrating that this reading frame is not essential for efficient viral replication in vivo and likely compensated by other viral proteins. In agreement with this, disruption of ORF3c did not significantly affect SARS-CoV-2 replication in CaCo-2 or CaLu-3 cells. In summary, we here identify ORF3c as an immune evasion factor of SARS-CoV-2 that suppresses innate sensing in infected cells.

microbiology↗

bioRxiv2022

STAT5 Gain-of-Function Variants Promote Precursor T-Cell Receptor Activation to Drive T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T-cell cancer. Hotspot mutations in JAK-STAT pathway members IL7R, JAK1 and JAK3 were analyzed in depth. However, the role of STAT5A or STAT5B mutations promoting their hyperactivation is poorly understood in the context of T-cell cancer initiation and acute leukemia progression. Importantly, the driver mutation STAT5BN642H encodes the most frequent activating STAT5 variant in T-ALL associated with poor prognosis. Here, we show that hyperactive STAT5 promotes early T-cell progenitor (ETP)-ALL-like cancer in mice and upregulated genes involved in T-cell receptor signaling (TCR), even in absence of surface TCR promoting. Importantly, these genes were also overexpressed in human T-ALL and other STAT5-dependent T-cell cancers. Moreover, human T-ALL cells were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers. Thus, we define STAT5 target genes in T-ALL that promote pre-TCR signaling mimicry. We propose therapeutic targeting using selective ZAP70 or STAT3/5 inhibitors in a subgroup of T-ALL patients with prominent IL-7R-JAK1/3-STAT5 activity. SignificanceWe provide detailed functional characterizations of hyperactive STAT5A or STAT5B in thymic T-cell development and transformation. We found that hyperactive STAT5 transcribes T-cell-specific kinases or pre-TCR signaling hubs to promote T-ALL. Biomolecular and next-generation-sequencing methods, transgenesis and pharmacologic interference revealed that hyperactive STAT5 is a key oncogenic driver that can be targeted in T-ALL using STAT3/5 or SYK family member tyrosine kinase inhibitors. Conflict of interestThe authors declare no potential conflicts of interest.

cancer biology↗

bioRxiv2022

Structural basis of RNA-induced autoregulation of the DExH-type RNA helicase maleless

Unwinding RNA secondary structures by RNA helicases is essential for RNA metabolism. How the basic unwinding reaction of DExH-type helicases is regulated by their accessory domains is unresolved. Here, we combine structural and functional analyses to address this challenge for the prototypic DExH RNA helicase maleless (MLE) from Drosophila. We captured the helicase cycle of MLE with multiple structural snapshots. We discovered that initially, dsRBD2 flexibly samples substrate dsRNA and aligns it with the open helicase tunnel. Subsequently, dsRBD2 releases RNA and associates with the helicase core, leading to closure of the tunnel around ssRNA. Structure-based MLE mutations confirm the functional relevance of the structural model in cells. We propose a molecular model in which the dsRBD2 domain of MLE orchestrates large structural transitions that depend on substrate RNA but are independent of ATP. Our findings reveal the fundamental mechanics of dsRNA unwinding by DExH helicases with high general relevance for dosage compensation and specific implications for MLEs human orthologue DHX9/RHA mechanisms in disease.

molecular biology↗