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Biology subjects

Marx, A.

Publications and source records attributed to Marx, A..

At least 19 recordsLinked to original sources

Large-scale, interpretable gene regulatory network inference through biologically informed matrix factorization

Gene regulatory networks (GRNs) provide a mechanistic framework for understanding how transcription factors coordinate gene expression to establish cellular identity and phenotype. Methods that integrate gene expression with motif-derived regulatory priors and other sources of biological information have substantially advanced gene regulatory network inference by reconstructing condition-specific regulatory architecture. These approaches estimate the evidence supporting regulatory interactions and have proven remarkably successful in a wide range of biological applications. A complementary view of regulatory networks, however, seeks to estimate the effect of those interactions on gene expression itself, providing a framework in which regulatory edges can be interpreted as activating or inhibitory influences on transcription. We developed Giraffe, a biologically informed matrix factorization framework that jointly estimates transcription factor activities and gene regulatory networks by integrating gene expression, motif-based regulatory priors, and transcription factor protein-protein interactions. Giraffe estimates signed partial regulatory effects whose magnitude and sign can be interpreted as the strength and direction of transcriptional regulation. Building directly on the biological framework established by methods such as PANDA, Giraffe provides a complementary representation of gene regulatory networks that emphasizes mechanistic interpretation while remaining scalable, flexible, and computationally efficient. Across synthetic benchmarks, six human tissues, yeast transcription factor perturbation experiments, and liver hepatocellular carcinoma, Giraffe accurately reconstructs regulatory interactions while distinguishing activating from inhibitory regulation with high accuracy. The inferred networks recover known features of tissue-specific regulation, correctly classify regulatory effects in transcription factor perturbation experiments, and identify biologically coherent changes in regulatory programs associated with liver cancer. Together, these results demonstrate that estimating the direction of transcriptional regulation provides a complementary perspective on gene regulatory networks that facilitates biological interpretation and hypothesis generation.

systems biology↗

The turn less taken: Investigating patterns in β-turn dynamics using large-scale molecular dynamics data

{beta}-turns are among the most common structural motifs in proteins, yet their conformational dynamics and sequence determinants remain incompletely understood. Here we present a data-driven classification and dynamic analysis of {beta}-turn conformations using large-scale molecular dynamics trajectories from the mdCATH database. Clustering of backbone dihedral angles using a cross-bond Ramachandran representation identifies six {beta}-turn types, including a previously uncharacterized hybrid I/I' cluster that combines geometric features of canonical type I and I' conformations. Time-resolved analysis indicates that this hybrid state acts as a transient intermediate state of {beta}-turns. Transitions observed in molecular dynamics simulations closely match NMR ensembles and altlocs detected in X-ray crystal structures, with the most dominant exchanges occurring between type I and II, and between type I' and II' turns. Sequence analysis shows that each turn type exhibits characteristic amino acid preferences at the central residues (i + 1 and i + 2). Within these overall preferences, specific residue pairs display distinct biases toward static or dynamic behavior. Targeted in silico substitutions that interchange dynamic- and static-enriched residue pairs shift the conformational behavior of turns accordingly, providing direct support for these sequence-dynamics relationships. Analysis of flanking secondary-structure environments reveals that structural context further modulates turn flexibility, with strand- and coil-associated turns exhibiting higher dynamic propensity than helix-associated turns. Together, these results reveal how sequence composition and structural context jointly shape the conformational landscape of {beta}-turns.

biochemistry↗

Statistical signals indicate a dependence between amino acid backbone conformation and the translated synonymous codon

Synonymous codons encode the same amino acid but can differ in their usage and translational properties. In previous work we reported statistical differences in backbone dihedral angle distributions associated with synonymous codons in the Escherichia coli proteome. This finding has been questioned due to concerns regarding the statistical methodology used. Here we revisit the dataset using corrected statistical procedures and alternative statistical tests. Across multiple frameworks, the real dataset consistently shows an excess of small p-values relative to randomized controls, indicating detectable codon-associated differences in backbone conformation.

bioinformatics↗

Synonymous coding significantly affects the domain swapping propensity of myoglobin

Co-translational folding is a critical, yet poorly understood, aspect of protein biogenesis due to its transient, heterogeneous, and experimentally inaccessible nature. Using a myoglobin variant engineered towards increased domain swapping, we show that stable dimers formed during heterologous E. Coli expression revert to the monomeric state following denaturation - renaturation and that domain swapping propensity is significantly affected by synonymous coding. Wider implications for the role of synonymous coding in aggregation and disease are discussed.

biochemistry↗

Density-guided AlphaFold3 uncovers unmodelled conformations in β2-microglobulin

Although X-ray crystallography captures the ensemble of conformations present within the crystal lattice, models typically depict only the most dominant conformation, obscuring the existence of alternative states. Applying the electron density-guided AlphaFold3 approach to {beta}2-Microglobulin highlights how ensembles of alternate backbone conformations can be systematically modeled directly from crystallographic maps. This study also highlights how the detection of conformational ensembles is affected by the local quality of electron density and subtle variations in crystallization conditions and lattice packing. These results demonstrate that density-guided AlphaFold3 can uncover conformational heterogeneity missed by conventional refinement, offering a robust, systematic framework to capture the full structural landscape of proteins in crystals and enhancing the interpretive power of macromolecular crystallography. SynopsisElectron-density-guided AlphaFold3 reveals previously unmodeled conformational heterogeneity in {beta}2-Microglobulin and shows how crystal packing influences ensemble detection in X-ray crystallography.

bioinformatics↗

An RNA ligase shapes transcriptional profiles, neural function, and behaviour in the developing larval zebrafish

RNA ligases are essential for the repair, splicing, and editing of RNA across various biological systems. Recently, a new enzyme that catalyses 5-3 RNA ligation - RNA ligase 1 (Rlig1) - was identified in vitro. However, the in vivo biological functions of Rlig1 have remained elusive. Here, we reveal the role of Rlig1 during vertebrate development using embryonic and larval zebrafish as a model system. We found that rlig1 mRNA is maternally deposited and present ubiquitously during early embryogenesis, whereas at larval stages it localises to the brain and eyes. Interestingly, CRISPR/Cas9-generated rlig1 knockout zebrafish exhibited no overt morphological abnormalities, but showed reduced behavioural responsiveness to visual stimuli along with massively perturbed transcriptomes and widespread dysregulation of core metabolic and translational pathways. Brain-wide calcium imaging in rlig1 knockout larvae revealed decreased neuronal activity in key regions for visual processing, consistent with the observed behavioural defects. Together, our findings identify a role for Rlig1 in maintaining the integrity and function of the nervous system and uncover a new link between neuronal RNA processing, development, and sensory-motor computation.

developmental biology↗

Human RNA ligase 1 as a novel regulator of ribosome function and translation under oxidative stress

Human RNA ligase 1 (Rlig1) is a recently identified human 5-3 RNA ligase essential for maintaining 28S rRNA integrity and promoting cell survival under oxidative stress. Although the enzymatic activity of Rlig1 implies a role in RNA maintenance or repair, its broader molecular context remains insufficiently characterised. Here we identify cellular interactors of Rlig1 using affinity enrichment coupled to mass spectrometry in a HEK293 Rlig1-KO model. Our approach revealed several RNA and DNA surveillance, degradation and repair proteins as well as a diverse set of RNA binding and RNA processing enzymes as potential interactors. Notably, ribosomal proteins were highly enriched, suggesting a link between Rlig1 and the translational machinery. Cross-linking coupled to mass spectrometry confirmed that Rlig1 interacts with 80S ribosomes in vitro and preferentially binds the large ribosomal subunit near the 28S rRNA. Polysome profiling corroborated this interaction, as Rlig1 was recruited to ribosomal fractions containing the large ribosomal subunit in response to oxidative stress. Moreover, Rlig1-deficient cells exhibited earlier polysome loss and significantly impaired translational activity compared to their WT counterparts under these conditions. These results highlight a potential novel role for Rlig1 in safeguarding translation during oxidative stress. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/684095v2_ufig1.gif" ALT="Figure 1000"> View larger version (27K): org.highwire.dtl.DTLVardef@1d959f9org.highwire.dtl.DTLVardef@a21b98org.highwire.dtl.DTLVardef@c1b42dorg.highwire.dtl.DTLVardef@11c1db9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

Experiment-guided AlphaFold3 resolves accurate protein ensembles

AlphaFold3 predicts highly accurate protein structures from sequence, but tends to collapse to a single dominant conformation, even when the underlying structure is inherently heterogeneous. Moreover, its predictions are oblivious to experimental conditions that can alter local sequence conformation. In this work we show that AlphaFold3 can be guided to match data obtained by NMR spectroscopy, X-ray crystallography and cryo-EM experiments, and combinations thereof. Our approach can also incorporate data that explicitly report on dynamics, such as site-resolved order parameters. We demonstrate that this methodology can generate ensembles of conformations having less distance restraint violations than traditionally resolved NMR structures and uncover unmodelled alternate conformations detectable in electron density. This methodology paves the way for the development of experimentally aware predictive models that capture the ensemble nature of protein structures.

molecular biology↗

Non-hydrolyzable acetyllysine analogs to study protein acetylation in vitro and in cells

Lysine acetylation plays a prominent regulatory role in eukaryotic cells. Yet, determining the functional consequences of acetylation for a given protein represents a considerable challenge. For instance, lysine residues are subject to various posttranslational modifications, rendering interpretation of mutational studies difficult. The genetic code expansion technology enables site-specific incorporation of acetyllysine (AcK) into proteins, but the applicability of AcK is limited, as within cells, the acetyl group is removed by deacetylases. Here, we show that site-specific incorporation of the non-hydrolyzable AcK analog ketolysine (KeK) into ubiquitin closely resembles the structural and functional effects of AcK incorporation. Furthermore, AcK and KeK can be efficiently incorporated into the tumor suppressor p53 in cells. However, whereas AcK becomes deacetylated, KeK remains stable. Accordingly, incorporation of KeK, but not AcK, affects p53-mediated transcription. Thus, we propose that KeK is the AcK surrogate of choice for studying acetylation of a given protein in cells.

biochemistry↗

Recurrent DNA Break Clusters Regulated by Polymerase Theta Are Essential for Replication Stress Induced Copy Number Variation

Copy number variations (CNVs) are a form of genetic alteration strongly implicated in numerous neurological and psychiatric disorders, as well as brain cancer. Replication stress is a common cause of CNVs. Despite the prevailing model that CNVs arise from DNA double strand breaks (DSBs), there has been no assay that directly perturbs presumed DSB sources and measures CNV output. Here, we identified a subset of recurrent DNA break clusters (RDCs) as a causal factor for CNV formation. In murine neural progenitor cells under replication stress, mapping the formation of CNVs revealed their location in RDC regions that contain actively transcribed genes. CRISPR/Cas9-mediated transcriptional suppression abrogated both RDC and CNV formation, but does not alter their replication timing. We found that DNA polymerase theta (Pol {theta}), a protector against CNV formation, plays a critical but context dependent role upstream of RDC formation. Chemically inhibiting the activity of Pol {theta} reduced end filling and micro-homology-mediated end joining in XRCC4/P53-deficient cells. Conversely, Pol {theta} inhibition led to elevated DSB density detection at RDC-containing loci in wild-type neural stem and progenitor cells, suggesting its role in preventing transcription-replication conflicts. Our data identify RDCs as contributors to genomic heterogeneity with plausible downstream effects on brain disorders and malignancy.

molecular biology↗

The IFN I response in tumor cells is shaped by PARP7-p300/CBP interactions through distinct loss- and gain-of-function mechanisms

PARP7, a mono-ADP-ribosyl (MAR) transferase, is a key suppressor of the type I interferon (IFN-I) IFN{beta} in various tumor cells and a validated drug target. This negative regulation is reversed by small-molecule inhibitors of PARP7 catalytic activity, resulting in increased IFN-{beta} expression. Yet, the mechanism of action of PARP7 inhibitors remains unclear because the relevant substrates of PARP7-mediated MARylation are unknown. Using an optimized analog- sensitive chemical genetic (ASCG) approach, we identified the co-activators, p300 and CBP, as nuclear PARP7 substrates. We identified an -helical domain in PARP7 essential for p300/CBP interaction, MARylation, and proteasome degradation. Disrupting PARP7-p300/CBP interaction prevents PARP7s suppression of IFN{beta} in colorectal cancer cells. p300/CBP reciprocally regulate PARP7s activity and nuclear localization. Intriguingly, treatment with PARP7 inhibitors increased IFN{beta} expression more than PARP7 knockout in a p300/CBP-dependent manner. Our findings suggest that in some contexts, IFN{beta} induction by PARP7 inhibitors occurs via two mechanisms: inhibiting MARylation of p300/CBP (loss-of-function) and stabilizing the PARP7- p300/CBP complex (gain-of-function). TeaserChemical genetics discovery of p300 and CBP as substrates of PARP7 that are essential for PARP7-mediated regulation of IFN{beta} via a dual mechanism.

cancer biology↗

The end of protein structure prediction: Improving prediction accuracy in chimeric proteins by windowed multiple sequence alignment

AlphaFold2 has predicted the structures of almost every known protein. A simple means to create proteins beyond those found in nature, is by unnaturally fusing together two known proteins. Here we demonstrate that dependence on multiple sequence alignment, limits the success with which AlphaFold and ESMfold capture such chimeric forms of otherwise well predicted, individual, proteins. Specifically we show that peptides are predicted with significantly reduced accuracy when added to the terminal ends of scaffold proteins. Appending the multiple sequence alignment for the individual peptide tags to that of the scaffold protein often restores prediction accuracy.

bioinformatics↗

Seeing Double: Molecular dynamics simulations reveal the stability of certain alternate protein conformations in crystal structures

Proteins jiggle around, adopting ensembles of interchanging conformations. Here we show through a large-scale analysis of the Protein Data Bank and using molecular dynamics simulations, that segments of protein chains can also commonly adopt dual, transiently stable conformations which is not explained by direct interactions. Our analysis highlights how alternate conformations can be maintained as non-interchanging, separated states intrinsic to the protein chain, namely through steric barriers or the adoption of transient secondary structure elements. We further demonstrate that despite the commonality of the phenomenon, current structural ensemble prediction methods fail to capture these bimodal distributions of conformations.

biochemistry↗

Spatial omics imaging of fresh-frozen tissue and routine FFPE histopathology on a single cancer needle core biopsy: freezing device and multimodal workflow

Complex molecular alterations underlying cancer pathophysiology are intensely studied with omics methods using bulk tissue extracts. For spatially resolved tissue diagnostics using needle biopsy cores, however, histopathological analysis using stained FFPE tissue and immuno-histochemistry (IHC) of few marker proteins is currently the main clinical focus. Today, spatial omics imaging using MSI or IRI are emerging diagnostic technologies for identification and classification of various cancer types. However, to conserve tissue-specific metabolomic states, fast, reliable and precise methods for preparation of fresh-frozen (FF) tissue sections are crucial. Such methods are often incompatible with clinical practice, since spatial metabolomics and routine histopathology of needle biopsies currently require two biopsies for FF and FFPE sampling, respectively. Therefore, we developed a device and corresponding laboratory and computational workflows for multimodal spatial omics analysis of fresh-frozen, longitudinally sectioned needle biopsies to accompany standard FFPE histopathology on the same biopsy core. As proof-of-concept, we analyzed surgical human liver cancer specimen by IRI and MSI with precise co-registration and, following FFPE processing, by sequential clinical pathology analysis on the same biopsy core. This workflow allowed spatial comparison between different spectral profiles and alterations in tissue histology, as well as direct comparison to histological diagnosis without the need of an extra biopsy. SIMPLE SUMMARYRoutine clinical approaches for cancer diagnosis demand fast, cost-efficient, and reliable methods, and their implementation within clinical settings. Currently, histopathology is the golden standard for tissue-based clinical diagnosis. Recently, spatially resolved molecular profiling techniques like mass spectrometry imaging (MSI) or infrared spectroscopy imaging (IRI) have increasingly contributed to clinical research, e.g., by differentiation of cancer subtypes using molecular fingerprints. However, adoption of the corresponding workflows in clinical routine remains challenging, especially for fresh-frozen tissue specimen. Here, we present a novel device based on 3D-printing technology, which facilitates sample preparation of needle biopsies for correlated clinical tissue analysis. It enables combination of MSI and IRI on fresh-frozen clinical samples with histopathological examination of the same needle core after formalin-fixation and paraffin-embedding (FFPE). This device and workflow can pave the way for a more profound understanding of biomolecular processes in cancer and, thus, aid more accurate diagnosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/528125v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@1da99aborg.highwire.dtl.DTLVardef@9ee706org.highwire.dtl.DTLVardef@5153daorg.highwire.dtl.DTLVardef@1581594_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

Water stabilizes an alternate turn conformation in horse heart myoglobin

Comparison of myoglobin structures reveals that protein isolated from horse heart consistently adopts an alternate turn conformation in comparison to its homologues. Analysis of hundreds of high-resolution structures discounts crystallization conditions or the surrounding amino acid protein environment as explaining this difference, that is also not captured by the AlphaFold prediction. Rather, a water molecule is identified as stabilizing the conformation in the horse heart structure, which immediately reverts to the whale conformation in molecular dynamics simulations excluding that structural water.

cell biology↗

Ythdf2 regulates cardiac remodeling through its m6A-mRNA target transcripts

m6A mRNA methylation controls cardiomyocyte function and increased overall m6A levels are a stereotyping finding in heart failure independent of the underlying etiology. However, it is largely unknown how the information is read by m6A reader proteins in heart failure. Here we show that the m6A reader protein Ythdf2 controls cardiac function and identified a novel mechanism how reader proteins control gene expression and cardiac function. Deletion of Ythdf2 in cardiomyocytes in vivo leads to cardiac hypertrophy, reduced heart function, and increased fibrosis during pressure overload as well as during aging. Similarly, in vitro the knockdown of Ythdf2 results in cardiomyocyte growth and remodeling. Mechanistically, we identified the eucaryotic elongation factor 2 as a major target of Ythdf2 using cell type specific Ribo-seq data. Our study expands our understanding on the regulatory functions of m6A methylation in cardiomyocytes and how cardiac function is controlled by the m6A reader protein Ythdf2.

molecular biology↗

Defining amino acid pairs as structural units suggests mutation sensitivity toadjacent residues

Proteins fold from chains of amino acids, forming secondary structures, -helices and {beta}-strands, that, at least for globular proteins, subsequently fold into a three-dimensional structure. A large-scale analysis of high-resolution protein structures suggests that amino acid pairs constitute another layer of ordered structure, more local than these conventionally defined secondary structures. We develop a cross-peptide-bond Ramachandran plot that captures the conformational preferences of the amino acid pairs and show that the effect of a particular mutation on the stability of a protein depends in a predictable manner on the adjacent amino acid context. One-Sentence SummaryLarge-scale protein backbone analysis reveals amino acid pair conformational preferences and predicts how sequence context affects mutant stability.

molecular biology↗

Combining molecular dynamics simulations and scoring method to computationally model ubiquitylated linker histones in chromatosomes

The chromatin in eukaryotic cells plays a fundamental role in all processes during a cells life cycle. This nucleoprotein is normally tightly packed but needs to be unpacked for expression and division. The linker histones are critical for such packaging processes and while most experimental and simulation works recognize their crucial importance, the focus is nearly always set on the nucleosome as the basic chromatin building block. Linker histones can undergo several modifications, but only few studies on their ubiquitylation have been conducted. Mono-ubiquitylated linker histones (HUb), while poorly understood, are expected to influence DNA compaction. The size of ubiquitin and the globular domain of the linker histone are comparable and one would expect an increased disorder upon ubiquitylation of the linker histone. However, the formation of higher order chromatin is not hindered and ubiquitylation of the linker histone may even promote gene expression. Structural data on chromatosomes is rare and HUb has never been modeled in a chromatosome so far. Descriptions of the chromatin complex with HUb would greatly benefit from computational structural data. In this study we generate molecular dynamics simulation data for six differently linked HUb variants with the help of a sampling scheme tailored to drive the exploration of phase space. We identify conformational sub-states of the six HUb variants using the sketch-map algorithm for dimensionality reduction and iterative HDBSCAN for clustering on the excessively sampled, shallow free energy landscapes. We present a highly efficient geometric scoring method to identify sub-states of HUb that fit into the nucleosome. We predict HUb conformations inside a nucleosome using on-dyad and off-dyad chromatosome structures as reference and show that unbiased simulations of HUb produce significantly more fitting than non-fitting HUb conformations. A tetranucleosome array is used to show that ubiquitylation can even occur in chromatin without too much steric clashes. Author summaryIn eukaryotic cells the linker histones play a crucial role in the formation of higher order nucleoprotein complex of DNA, especially for the arrangement of the nucleosomes. Histones can undergo several modifications, but modification of a linker histone with a single udiquitin (mono-ubiquitylation) remains one of the least understood epigenetic modifications. One reason is the inaccessibility of homogeneously modified linker histones for experimental methods, which are crucial for distinct studies. We combine molecular dynamics simulations with machine learning-based approaches to study the influence of mono-ubiquitylation in linker histones on DNA interaction and their ability to form higher order chromatin structures. We were able to determine the probable states in six differently linked histone-ubiquitin complexes via accelerating classical molecular dynamics simulations and using advanced state characterization techniques. As it is computationally unfeasible to simulate the whole chromatosome with different modified histones we developed efficient geometric scoring technique to select biologically relevant structures of all six mono-ubiquitylated linker histone that can bound to nucleosome.

bioinformatics↗