Biology subjects

Kuster, B.

Publications and source records attributed to Kuster, B..

32 records · Page 2Linked to original sources

bioRxiv2024

Mapping the Human Proteome with Physical Access to DNA

In a human cell, DNA is packed in histones, RNA, and chromatin-associated proteins, forming a cohesive gel. At any given moment, only a specific subset of the proteome has physical access to the DNA and organizes its structure, transcription, replication, repair and other molecular functions essential to the way the genome is read and maintained. We have developed a zero-distance photo-crosslinking approach to quantify proteins in direct contact with DNA in living cells. Collecting DNA interactomes from human breast cancer cells, we present an atlas of over one thousand proteins with physical access to DNA, and hundreds of peptide-nucleotide crosslinks pinpointing protein-DNA interfaces with single amino-acid resolution. Differential comparisons of DNA interactomes from cells undergoing treatment with estrogen or genotoxic chemotherapy recapitulated the recruitment of key transcription factors and DNA damage proteins. This opens a direct way to explore genomic regulation in a hypothesis-free manner, applicable to many organisms and systems.

molecular biology↗

bioRxiv2024

Gemcitabine and ATR inhibitors synergize to kill PDAC cells by blocking DNA damage response

The DNA-damaging agent gemcitabine (GEM) is a first-line treatment for pancreatic cancer but chemoresistance is frequently observed. Several clinical trials investigate the efficacy of GEM in combination with targeted drugs including kinase inhibitors but the experimental evidence for such rational is often unclear. Here, we phenotypically screened 13 human pancreatic adenocarcinoma (PDAC) cell lines against GEM in combination with 140 clinical kinase inhibitors and observed strong synergy for the ATR inhibitor Elimusertib in most cell lines. Dose-dependent phosphoproteome profiling of four ATR inhibitors following DNA damage induction by GEM revealed a strong block of the DNA damage response pathway including phosphorylated pS468 of CHEK1 as the underlying mechanism of drug synergy. The current work provides a strong rationale for why the combination of GEM and ATR inhibition may be useful for the treatment of PDAC patients and constitutes a rich phenotypic and molecular resource for further investigating effective drug combinations.

cancer biology↗

bioRxiv2024

Transport properties of canonical PIN-FORMED proteins and the role of the loop domain in auxin transport

Indole-3-acetic acid (IAA), the most abundant endogenous auxin is transported in plants in a polar fashion by PIN-FORMED (PIN) transporters and controls virtually all plant growth and developmental processes. Canonical PINs possess a long and largely disordered cytosolic loop domain which is shorter in non-canonical PINs. Auxin transport by canonical PINs is activated loop phosphorylation by kinases. While the structure of the transmembrane domains of these transporters was recently solved, their transport properties remained poorly characterized and particularly the relative roles of the transmembrane and loop domain therein. In this study we used flux studies to obtain quantitative kinetic parameters of IAA transport mediated by canonical PINs as well as of chimeras between transmembrane and loop domains of different PINs upon their activation by D6 PROTEIN KINASE or PINOID. We found that the transporters possess distinct transport properties that are due to both the transmembrane and loop domain. To demonstrate the physiological relevance of these distinct transport properties, we modelled root tip IAA distribution patterns and investigated the potential of different PINs to complement the agravitropic root growth phenotype of the pin2 mutant when expressed in the PIN2 domain. We found a strong correlation between transport parameters and physiological output indicating that in addition to PIN polarity a low transport rate in the PIN2 expression domain is required for gravitropic growth. Overall, the data show that the loop domain is not only required for activation of PIN-mediated auxin transport but has an additional role in the transport cycle by a currently unknown mechanism.

plant biology↗

bioRxiv2023

PTMNavigator: Interactive Visualization of Differentially Regulated Post-Translational Modifications in Cellular Signaling Pathways

Post-translational modifications (PTMs) play pivotal roles in regulating cellular signaling, fine-tuning protein function, and orchestrating complex biological processes. Despite their importance, the lack of comprehensive tools for studying PTMs from a pathway-centric perspective has limited our ability to understand how PTMs modulate cellular pathways on a molecular level. Here, we present PTMNavigator, a tool integrated into the ProteomicsDB platform, which offers an interactive interface for researchers to overlay experimental PTM data with pathway diagrams. PTMNavigator provides [~]3000 canonical pathways from manually curated databases and further enables users to modify and create custom diagrams, tailored to their data. Additionally, PTMNavigator automatically runs multiple kinase and pathway enrichment algorithms whose results are directly integrated into the visualization. This offers a comprehensive view of the intricate relationship between PTMs and signaling pathways. To demonstrate the utility of PTMNavigator, we applied it to two phosphoproteomics perturbation datasets. First, PTMNavigator enhanced pathway enrichment analysis by showing how the regulated peptides and proteins are distributed in the pathways with high enrichment scores. Second, it visualized how drug treatments result in a discernable flow of PTM-driven signaling within pathways. Third, PTMNavigator aided in proposing extensions to an existing pathway by suggesting putative new links between both PTMs and pathway components. By enhancing our understanding of cellular signaling dynamics and facilitating the discovery of novel PTM-pathway interactions, PTMNavigator advances our knowledge of PTM biology and its implications in health and disease.

systems biology↗

bioRxiv2023

CurveCurator: A recalibrated F-statistic to assess, classify, and explore significance of dose-response curves

Dose-response curves are key metrics in pharmacology and biology to assess phenotypic or molecular actions of bioactive compounds in a quantitative fashion. Yet, it is often unclear whether or not a response significantly differs from a curve without regulation, particularly in high-throughput applications or unstable assays. Treating potency and effect size estimates from random and true curves with the same level of confidence can lead to incorrect hypotheses and issues in training machine learning models. Here, we present CurveCurator, an open-source software and interactive dashboard that provides reliable dose-response characteristics by computing p-values and false discovery rates based on a recalibrated F-statistic and a novel thresholding procedure. Application of CurveCurator to large-scale data sets demonstrates its scalable utility across several application areas.

bioinformatics↗

bioRxiv2023

The RALF Signaling Pathway Regulates Cell Wall Integrity during Pollen Tube Growth in Maize

Autocrine signaling pathways regulated by RAPID ALKALINIZATION FACTORs (RALFs) control cell wall integrity during pollen tube germination and growth in Arabidopsis. To investigate the role of pollen-specific RALFs in another plant species, we combined gene expression data with phylogenetic and biochemical studies to identify candidate orthologs in maize. We show that Clade IB ZmRALF2/3 mutants, but not that of Clade III ZmRALF1/5 caused cell wall instability in the sub-apical region of the growing pollen tube. ZmRALF2/3 are mainly located to the cell wall and are partially able to complement the pollen germination defect of their Arabidopsis orthologs AtRALF4/19. Mutations in ZmRALF2/3 compromise pectin distribution pattern leading to altered cell wall thickness, hyperphosphorylation of ZmPEX cell wall proteins and pollen tube burst. Clade IB, but not Clade III ZmRALFs are capable to interact with pollen-specific CrRLK1L receptor kinases ZmFERL4/7/9 and GPI-anchored co-receptors ZmLLG1/2 at similar binding affinities. In contrast, binding affinity to ZmPEX2/4 cell wall proteins is about five times higher. Based on these data, we now propose a dosage-dependent model showing how Clade IB RALFs act as extracellular sensors to regulate cell wall integrity and thickness during pollen tube growth in plants. One sentence summaryPollen-specific RALFs interact at different binding affinities with receptor kinases, GPI-anchored proteins and cell wall proteins to regulate cell wall integrity during pollen tube growth in maize.

developmental biology↗

bioRxiv2022

Proteogenomic analysis reveals RNA as an important source for tumor-agnostic neoantigen identification correlating with T-cell infiltration

Systemic pan-tumor analyses may reveal the significance of common features implicated in cancer immunogenicity and patient survival. Here, we provide a comprehensive multi-omics data set for 32 patients across 25 tumor types by combining proteogenomics with phenotypic and functional analyses. By using an optimized computational approach, we discovered a large number of novel tumor-specific and tumor-associated antigens including shared common target candidates. To create a pipeline for the identification of neoantigens in our cohort, we combined deep DNA and RNA sequencing with MS- based immunopeptidomics of tumor specimens, followed by the assessment of their immunogenicity. In fact, we could detect a broad variety of non-wild type HLA-binding peptides in the majority of patients and confirmed the immunogenicity of 24 neoantigens. Most interestingly, the majority of total and immunogenic neoantigens originated from variants identified in the RNA dataset, illustrating the importance of RNA as a still understudied source of cancer antigens. Moreover, the amount of these mainly RNA-based immunogenic neoantigens correlated positively with overall CD8+ tumor-infiltrating T cells. This study therefore underlines the importance of RNA-centered variant detection for the identification of shared biomarkers and potentially relevant neoantigen candidates. Statement of significanceThe significance of this study lies not only in the potential of our optimized proteogenomic workflow for the discovery of neoantigens (in particular RNA-derived neoantigens) for clinical application, but sheds light on the entity-agnostic prevalence of HLA class I peptide presentation of RNA processing events to be used for tumor targeting.

cancer biology↗

bioRxiv2022

UHMK1 is a novel splicing regulatory kinase

The U2AF Homology Motif Kinase 1 (UHMK1) is the only kinase that contains the U2AF homology motif (UHM), a common protein interaction domain among splicing factors. Through this motif, UHMK1 interacts with the splicing factors SF1 and SF3B1, known to participate in the 3 splice site recognition during the early steps of spliceosome assembly. Although UHMK1 phosphorylates these splicing factors in vitro, the involvement of UHMK1 in RNA processing has not previously been demonstrated. Here, we identify novel putative substrates of this kinase and evaluate UHMK1 contribution to overall gene expression and splicing, by integrating global phosphoproteomics, RNA-seq, and bioinformatics approaches. Upon UHMK1 modulation, 163 unique phosphosites were differentially phosphorylated in 117 proteins, of which 106 are novel potential substrates of this kinase. Gene Ontology (GO) analysis showed enrichment of terms previously associated with UHMK1 function, such as mRNA splicing, cell cycle, cell division and microtubule organization. The majority of the annotated RNA-related proteins are components of the spliceosome, but are also involved in several steps of gene expression. Comprehensive analysis of splicing showed that UHMK1 affected over 200 alternative splicing events. Moreover, splicing reporter assay further supported UHMK1 function on splicing. Overall, RNA-seq data demonstrated that UHMK1 knockdown had a minor impact on gene expression and pointed to UHMK1 function in epithelial-mesenchymal transition. Finally, the functional assays demonstrated that UHMK1 modulation affects proliferation, colony formation, and migration of the cells. Taken together, our data implicate UHMK1 as a splicing regulatory kinase, connecting protein regulation through phosphorylation and gene expression in key cellular processes.

molecular biology↗

bioRxiv2020

Universal Spectrum Explorer: A standalone (web-)application for cross-resource spectrum comparison

Here we present the Universal Spectrum Explorer (USE), a web-based tool based on IPSA for cross-resource (peptide) spectrum visualization and comparison (https://www.proteomicsdb.org/use/). Mass spectra under investigation can either be provided manually by the user (table format), or automatically retrieved from online repositories supporting access to spectral data via the universal spectrum identifier (USI), or requested from other resources and services implementing a newly designed REST interface. As a proof of principle, we implemented such an interface in ProteomicsDB thereby allowing the retrieval of spectra acquired within the ProteomeTools project or real-time prediction of tandem mass spectra from the deep learning framework Prosit. Annotated mirror spectrum plots can be exported from the USE as editable scalable high quality vector graphics. The USE was designed and implemented with minimal external dependencies allowing local usage and integration into other websites (https://github.com/kusterlab/universal_spectrum_explorer).

bioinformatics↗

bioRxiv2020

Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

plant biology↗

bioRxiv2020

Stress-primed secretory autophagy drives extracellular BDNF maturation

The stress response is an essential mechanism that strives to maintain homeostasis, and its disruption is implicated in several psychiatric disorders. As a cellular response to stressors, autophagy is activated to regulate homeostasis through protein degradation and recycling. Secretory autophagy is a recently described pathway where autophagosomes fuse with the plasma membrane rather than lysosomes. In this study, we demonstrate that glucocorticoid-mediated stress enhances secretory autophagy, via the stress-responsive co-chaperone FK506-binding protein 51. We identified the matrix metalloproteinase 9 (MMP9) as one of the stress-induced secreted proteins. Using cellular assays and in vivo microdialysis, we further found that stress-enhanced MMP9 secretion increases the cleavage of pro-brain derived neurotrophic factor (proBDNF) to its mature form. BDNF is essential for adult synaptic plasticity and its pathway is associated with major depression and posttraumatic stress disorder. These findings unravel a novel mechanistic link between stress, stress adaptation and the development of psychiatric disorders, with possible therapeutic implications.

molecular biology↗

bioRxiv2019

Generating high-quality libraries for DIA-MS with empirically-corrected peptide predictions

Data-independent acquisition approaches typically rely on sample-specific spectrum libraries requiring offline fractionation and tens to hundreds of injections. We demonstrate a new library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa.

systems biology↗

bioRxiv2018

Quantification and discovery of sequence determinants of protein per mRNA amount in 29 human tissues

Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects is still lacking. Here we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We analyzed the contribution of known sequence determinants of protein synthesis and degradation and 15 novel mRNA and protein sequence motifs that we found by association testing. While the dynamic range of PTR ratios spans more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided significant functional support for two novel UTR motifs and a proteome-wide competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our direct comparison of protein to RNA levels led to a new metrics of codon optimality. Altogether, this study shows that a large fraction of PTR ratio variance across genes can be predicted from sequence and identified many new candidate post-transcriptional regulatory elements in the human genome.

systems biology↗

bioRxiv2018

A deep proteome and transcriptome abundance atlas of 29 healthy human tissues

Genome-, transcriptome- and proteome-wide measurements provide valuable insights into how biological systems are regulated. However, even fundamental aspects relating to which human proteins exist, where they are expressed and in which quantities are not fully understood. Therefore, we have generated a systematic, quantitative and deep proteome and transcriptome abundance atlas from 29 paired healthy human tissues from the Human Protein Atlas Project and representing human genes by 17,615 transcripts and 13,664 proteins. The analysis revealed that few proteins show truly tissue-specific expression, that vast differences between mRNA and protein quantities within and across tissues exist and that the expression levels of proteins are often more stable across tissues than those of transcripts. In addition, only ~2% of all exome and ~7% of all mRNA variants could be confidently detected at the protein level showing that proteogenomics remains challenging, requires rigorous validation using synthetic peptides and needs more sophisticated computational methods. Many uses of this resource can be envisaged ranging from the study of gene/protein expression regulation to protein biomarker specificity evaluation to name a few.

systems biology↗