Biology subjects

Bühler, M.

Publications and source records attributed to Bühler, M..

4 recordsLinked to original sources

bioRxiv2025

Advancing DIA-based Limited Proteolysis Workflows: introducing DIA-LiPA

Limited proteolysis coupled to mass spectrometry (LiP-MS) probes protein conformational dynamics, but interpretation of LiP-MS data is complicated by heterogenous proteolytic cleavage patterns and missing data. Recent advances in data-independent acquisition (DIA) and machine learning-based search engines promise improved sensitivity and reproducibility, yet their performance in LiP-MS workflows remains underexplored. We systematically evaluated selected library-free DIA workflows using a rapamycin-treated human cell lysate and a yeast heat shock dataset, benchmarking DIA-NN and Spectronaut for identification depth, reproducibility and false discovery rate control. Our results show that library-free approaches achieve high sensitivity, eliminating the experimental overhead and sample requirements associated with empirical libraries. Building on these advances, we introduce a DIA-based Limited Proteolysis data Analysis pipeline (DIA-LiPA), a data analysis workflow tailored for LiP-MS data that integrates semi-tryptic- and tryptic-level precursor data and accounts for missingness to enable structural interpretation. Validation across multiple datasets confirmed that DIA-LiPA reproduces known structural signatures and uncovers additional regulatory patterns, providing a robust framework for mechanistic insights into protein dynamics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/687786v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@19fc486org.highwire.dtl.DTLVardef@1f74b7org.highwire.dtl.DTLVardef@127db11org.highwire.dtl.DTLVardef@19f5b88_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration

Tissue regeneration relies on the ability of cells to undergo de novo patterning. While tissue patterning has been viewed as the transition from initially identical un-patterned cells to an arrangement of different cell types, recent evidence suggests that initial heterogeneities between cells modulate tissue-scale pattern formation. Yet, how such heterogeneities arise and, thereafter, regulate cell type emergence in a population of cells is poorly understood. Using in vivo and in vitro mouse regenerative systems, we identify a critical tissue density that is required to induce heterogeneous inactivation of the mechanosensor YAP1. Experimental and biophysical approaches demonstrate that YAP1 cell-to-cell heterogeneity pre-patterns the first cell fate decision, via both chromatin remodelling and a supracellular feedback between FOXA1 and Delta-Notch signalling. This feedback motif induces cell fate bistability endowing memory to the system and the maintenance of patterns during homeostasis. These findings reveal a generalisable framework in which transient cell-to-cell heterogeneity, regulated by tissue-scale properties, serves as a critical control parameter for the emergence of cell fate and stable patterning during regeneration.

cell biology↗

bioRxiv2024

ChAHP2 and ChAHP control diverse retrotransposons by complementary activities

Retrotransposon control in mammals is an intricate process that is effectuated by a broad network of chromatin regulatory pathways. We previously discovered ChAHP, a protein complex with repressive activity against SINE retrotransposons, composed of the transcription factor ADNP, chromatin remodeler CHD4, and HP1 proteins. Here we identify ChAHP2, a protein complex homologous to ChAHP, wherein ADNP is replaced by ADNP2. ChAHP2 is predominantly targeted to ERVs and LINEs, via HP1{beta}-mediated binding of H3K9 trimethylated histones. We further demonstrate that ChAHP also binds these elements in a mechanistically equivalent manner to ChAHP2, and distinct from DNA sequence-specific recruitment at SINEs. Genetic ablation of ADNP2 alleviates ERV and LINE1 repression, which is synthetically exacerbated by additional depletion of ADNP. Together, our results reveal that the ChAHP and ChAHP2 complexes function to control both non-autonomous and autonomous retrotransposons by complementary activities, further adding to the complexity of mammalian transposon control.

molecular biology↗

bioRxiv2023

BCAT1 inhibition affects CD8+ T cell activation, exhaustion, and tumoral immunity by altering iron homeostasis

The present study explores the role of the cytosolic branched chain amino acid aminotransferase (BCAT1) in CD8+ T cell activation, in general, and tumor immunity, in particular, and identifies a non-canonical function of the protein in iron homeostasis. Pharmacologic inhibition of BCAT1 using the novel drug ERG245 abrogates the effector functions of CD8+ T cells in vitro and metabolically reprograms the cells towards increased OXPHOS. In vivo, it suppresses activation of CD8+ T cells in DSS colitis leading to improved disease outcomes. Remarkably, withdrawal of BCAT1 inhibition further amplifies OXPHOS and gives rise to CD8+ T cells with increased cytotoxicity in vitro and in vivo. When combined with an anti-PD-1 treatment, temporal BCAT1 inhibition dramatically increases anti-PD-1 efficacy inducing complete and durable tumor regressions in the moderately immunogenic CT26 tumor model. Single cell RNA-seq data link expression of Bcat genes to exhausted T cells within the tumor microenvironment of human cancer patients, whereas in vitro assays indicate that BCAT1 inhibition partially prevents the adoption of a terminally exhausted phenotype by CD8+ T cells. We propose BCAT1 as a target for cancer combinatory therapies. SIGNIFICANCEThe study explores for the first time the role of BCAT1 in CD8+ T cell activation and proposes novel strategies for using BCAT1 inhibitors in cancer and beyond. It demonstrates that BCAT1 exerts its function without significantly altering branched chain amino acid (BCAA) levels through a mechanism that controls iron homeostasis, a novel non-canonical mechanism of action, and implicates BCAT1 in the adoption of an exhausted phenotype by T cells found in human cancers. While the majority of metabolic drugs temper OXPHOS, it demonstrates that an agent that increases OXPHOS in CD8+ T cells can be used successfully as an immune-oncology drug.

immunology↗