Biology subjects

Olsen, J. V.

Publications and source records attributed to Olsen, J. V..

At least 19 recordsLinked to original sources

bioRxiv2026

An auto-generated ADP-ribosyl-cysteine degron drives DTX2-dependent turnover of PARP7

The mono-ADP-ribosyltransferase PARP7 exerts negative feedback on androgen receptor (AR) signaling by generating an ADP-ribosyl-cysteine degron on AR that marks it for degradation. Here we show that the protein half-life of PARP7 is set by cysteine auto-ADP-ribosylation, providing a mechanism for temporal control of its feedback on AR-dependent transcription in prostate cancer cells. ADP-ribosyl-cysteine in the PARP7 catalytic domain constitutes a degron read by the E3 ligase DTX2, driving PARP7 polyubiquitination and proteasomal degradation. Thus, the same E3 is used to degrade both PARP7 and its substrate AR. Using label-free mass spectrometry, we identify the AP-1 factor c-JUN as a PARP7 substrate and show that ADP-ribosylated c-JUN accumulates upon DTX2 depletion. ADP-ribosyl degron formation is dependent on NAD+ availability since depleting NAD+ pharmacologically or under hypoxia prevents degron formation and stabilizes PARP7 in a catalytically constrained state that compromises its feedback activity. Correlations between PARP7 expression and hypoxia signatures in prostate tumors, together with sensitivity to the PARP7-selective inhibitor RBN-2397 across cancer lines, nominate hypoxic, NAD+ depleted contexts as heightened vulnerability to PARP7-targeting agents. Marking transcription factors for degradation by covalent ADP-ribosylation is emerging as a distinct mode of transcriptional control.

biochemistry↗

bioRxiv2026

High-fidelity bispecific protease KRysin for deeper plasma proteomics and phosphoproteomics

Bottom-up proteomics relies on efficient and reproducible proteolytic digestion. Yet trypsin, the most widely used protease, frequently misses cleavages, particularly near prolines, acidic and phosphorylated residues. Incomplete digestion inflates search space, dilutes peptide signal, and limits depth and reproducibility, especially in challenging matrices such as plasma and phosphoproteomics. Here we present KRysin, a recombinant bispecific protease that cleaves C-terminal to lysine and arginine with very high specificity and efficiency, generating tryptic peptides compatible with standard proteomics workflows. In HeLa digests, ~99% of KRysin-generated peptides ended in lysine or arginine, including at sites followed by proline, and only 0.2% of peptides contained an internal (missed-cleaved) arginine and 6% an internal lysine, compared with 9% and 25%, respectively, for trypsin. Using narrow-window DIA at 60 samples-per-day, KRysin quantified close to 50,000 class I phosphosites, >10,000 more than trypsin-based workflows, and identified up to 40% more plasma protein groups, uniquely revealing receptors, cytokines, and growth factors. KRysin therefore provides a direct alternative to trypsin wherever digestion completeness, speed and PTM coverage are limiting.

biochemistry↗

bioRxiv2026

Pulsed-SILAC in single mouse embryos reveals early embryonic protein synthesis dynamics and phosphosite regulation

Early embryogenesis relies extensively on maternally deposited products until zygotic genome activation, yet the dynamics for the synthesis of new proteins in mammalian embryos remains poorly characterized. To address this, we applied pulsed stable isotope labelling by amino acids in cell culture (pSILAC) combined with narrow-window data-independent acquisition mass spectrometry to single mouse oocytes and embryos to resolve de novo protein synthesis during early embryogenesis. This revealed that the maternal proteome is not a static reservoir, with components of the subcortical maternal complex and amino acid transporters SLC7A1/2 being actively synthesized during the earliest developmental stages. Furthermore, phosphoproteomic analysis identified hundreds of previously unreported phosphosites and extensive regulation during the oocyte-to-embryo transition. Notably, phosphorylation of the PRC2-interacting KLP motif of EZHIP emerged as a potential regulatory mechanism, with modification of this region reducing EZHIP-PRC2 interaction and coinciding with H3K27me3 remodelling. Together, single embryo pSILAC revealed a maternal proteome that is continuously synthesized, recycled, and post-translationally regulated during early embryogenesis.

cell biology↗

bioRxiv2026

Mapping the human sperm proteome across compartments, donors and individual cells

The human spermatozoon is among the most specialized cells in the body, yet where its proteins reside, where they come from and how they vary between and within men remain largely unresolved. Here we present the deepest proteomic atlas of human spermatozoa to date, identifying 10,828 proteins across six donors. Abundances correlate well between individuals and are anchored by a conserved core of mitochondrial energy-metabolism proteins, while a smaller fraction varies between donations from the same man. By mechanically separating heads from tails, we assigned thousands of previously unlocalized proteins to a compartment, and by comparing seminal fluid from vasectomized and non-vasectomized men we distinguished sperm- and epididymis-derived proteins from accessory-gland secretions. Using a single-cell analysis workflow we further show that the proteomes of individual spermatozoa can be measured, recovering up to ~1,000 protein groups per cell. In a cohort with total fertilization failure, we find recurrent mitochondrial rather than sperm-egg fusion dysregulation. Together or data provides novel insights into the dynamics of the sperm proteome and male infertility.

cell biology↗

bioRxiv2026

Tethering-mediated recruitment of pioneer factors by NuRD enables signal dependent gene activation and protects cell identity

Cell identity relies on precise control of transcription factor (TF) activity, localization and interpretation of signaling. Mitogen-activated protein kinase (MAPK) signaling regulates transcription via the ERK kinase, phosphorylating components of the Mediator complex to regulate its association with TF-bound enhancers. Here we describe two distinct forms of TF association related to signaling response, stable DNA recognition and tethering to active sites of transcription. We find that the tethering of TFs at these active sites is both essential for signaling response and cell identity. To identify tethering substrates, we focused on signaling dependent associations with the transcriptional coregulator, the Mediator, and find the NuRD complex is engaged with the Mediator at active promoters and enhancers. Here, NuRD functions as a molecular scaffold recruiting pioneer TFs like SOX2 independently of DNA sequence, enhancing ERK dependent transcription while sequestering these factors away from context inappropriate binding sites elsewhere in the genome. Balancing the recognition of a restricted spectrum of canonical DNA sites by pioneer factors while retaining them at high concentrations in the vicinity of transcription supports robust transcriptional response, ensures plasticity in differentiation and explains why these factors bind a fraction of their naturally occurring sites in any given cell type.

molecular biology↗

bioRxiv2026

A chemogenetic CRISPR knockout screen identifies a ubiquitin-mediated calcineurin-BMP pathway relay in T cell activation

T cell activation is dependent on calcineurin signalling, yet how this pathway integrates with other regulatory systems remains incompletely understood. Here, we exploit the dual pharmacology of FK506, which inhibits calcineurin while also releasing FKBP12-mediated repression of BMP receptors, to dissect signalling crosstalk during T cell responses. A comparative genome-wide CRISPR knockout screen using FK506 and Cyclosporin A revealed that FK506 uniquely engages BMP pathway components and ubiquitin regulatory networks. Functional analyses in Jurkat and primary human T cells showed that FK506 induces BMP receptor-dependent activation of SMAD1/5/8 and triggers a rapid remodelling of K48- and K29-linked ubiquitin chains. Proteomics further demonstrated selective ubiquitination of immune signalling proteins and ubiquitin regulators, linking BMP pathway activation to proteasome-dependent turnover and non-proteolytic ubiquitin signalling. In primary T cells, BMP receptor signalling enhance calcineurin-driven activation, while changes in ubiquitin conjugation modulate this response, thereby establishing an integrated calcineurin-BMP-ubiquitin relay. Together, our results uncover a context-dependent signalling axis in which BMP activation and ubiquitin dynamics fine-tune calcineurin-mediated T cell signalling.

cell biology↗

bioRxiv2026

Near-Zero Missed Cleavages with a High-Fidelity Recombinant Arg-C Zero for Mass Spectrometry-Based Proteomics

Proteolytic digestion remains a critical step in bottom-up proteomics workflows, with enzyme specificity and efficiency directly impacting peptide identification and protein sequence coverage. Here, we present the comprehensive characterization of Arg-C Zero, a recombinant arginyl endopeptidase derived from Porphyromonas gingivalis that exhibits exceptional fidelity in cleaving specifically at the C-terminus of arginine residues. Unlike conventional serine proteases such as Trypsin, Arg-C Zero utilizes a histidine-cysteine catalytic dyad mechanism, achieving near-zero missed cleavage rates (>99% efficiency) under standard proteomics conditions. Through systematic evaluation using HeLa protein extracts, we demonstrate that Arg-C Zero maintains consistent performance across varying digestion times. The enzyme shows robust activity across a broad pH range and tolerates up to 4M urea, making it ideally suitable for a diverse range of proteomics sample preparation workflows. While Trypsin/LysC combinations remain superior for comprehensive proteome coverage, Arg-C Zero offers unique advantages for applications requiring high specificity and reproducible arginine-specific cleavage patterns, particularly for analysis of post-translational modifications (PTMs). Here, we demonstrate how Arg-C Zero aids comprehensive mapping of histone PTMs, and when used in low-pH workflows help preserve labile ADP-ribosylation sites, expanding the analytical capabilities of mass spectrometry for characterizing these challenging modifications. The enzymes resistance to proline-adjacent cleavage sites and compatibility with standard mass spectrometry buffers position it as a valuable addition to the proteomics enzyme toolkit.

biochemistry↗

bioRxiv2026

Molecular basis of the Druantia anti-phage defense system

Eukaryotes and prokaryotes have evolved diverse antiviral immune systems, many containing helicase modules central to defense. Druantia are widespread bacterial anti-phage defense systems, each built around a large helicase domain-containing protein, DruE, paired with variable subunits. Here, we investigate the molecular basis of a minimal two-protein module Druantia system, DruH-E. We demonstrate that DruH-E is sufficient to confer robust anti-phage defense. DruE exists in equilibrium between a monomer and an asymmetric dimer, with dimerization required for in vivo immunity. Cryo-EM structures define DruE asymmetric dimer assembly and its dsDNA unwinding mechanism, revealing a topologically closed architecture that is specifically activated by dsDNA substrates with a 3' overhang. We further identify DruH as an ssDNA-binding protein regulated by a metabolic switch, where its activity is inhibited by ATP at physiological concentrations through direct competition with ssDNA. Supported by mass spectrometry and single-cell microscopy data, we establish key determinants of the Druantia defense system and reveal how it mediates a direct antiviral immune mechanism.

microbiology↗

bioRxiv2026

The HSP90-CDC37 Chaperone System Orchestrates RAF1 Kinase Activation Through a Pre-Dimerization Mechanism

RAF kinases activate MEK in the RAS-MAPK signaling pathway, and changes in RAF kinase signaling have been linked to tumor formation. RAF1 requires the HSP90-CDC37 chaperone system for proper activation, but how the HSP90-CDC37 chaperone system regulates RAF kinase maturation remains enigmatic. We present novel cryo-EM structures of previously uncharacterized RAF1 chaperone complexes, including a 2:2:2 RAF1-HSP90-CDC37 complex (RRHCC), intermediate assemblies (RHCC), and a RAF1-HSP90-CDC37-p23 complex (RHCp23). These reveal an asymmetric stepwise folding mechanism unique among HSP90 kinase clients in which one RAF1 threads through the HSP90 lumen while another is captured in a "casting mold" formed by CDC37 and HSP90 that stabilizes the partially folded C helix of RAF1. The RHCp23 structure shows how p23 cooperates with CDC37 to regulate ATP hydrolysis and client release. The HSP90-CDC37 system supports pre-dimerization of RAF1 and BRAFV600E homodimers and RAF1 heterodimers, a mechanism unique to RAF among kinase clients of HSP90. Phosphoproteomics reveals selective activating phosphorylations within RRHCC. These RAF isoform complexes differentially activate MEK signaling and cell proliferation, establishing HSP90-CDC37 as not just a passive stabilizer but an active regulator of RAF signaling with therapeutic implications.

biochemistry↗

bioRxiv2026

Archaeological preservation of amelogenesis pathways

Dental enamel, the hardest mineralised tissue in the human body, has proven to be an excellent source of ancient proteins, which have been found to survive within dental enamel for at least twenty million years. In archaeological and palaeontological contexts, the enamel proteome is generally considered to be rather small, consisting of about twelve proteins, most of which are unique to enamel. During amelogenesis these proteins undergo in vivo digestion by matrix metalloproteinase 20 (MMP20) and kallikrein 4 (KLK4) as well as serine phosphorylation by family with sequence similarity member 20-C (FAM20C) that alter their characteristics. Gaining knowledge of the previously understudied influence of amelogenesis on the archaeological human dental enamel proteome could benefit various palaeoproteomic analysis, especially in an human evolutionary context. Here we present archaeological dental enamel proteomes and explore protein cleavage patterns and sequence coverage to estimate the effects of in vivo digestion, as well as explore phosphorylation patterns. Additionally, we present a new marker based on phosphorylation to estimate genetic sex.

evolutionary biology↗

bioRxiv2026

Comparison of extracellular vesicles and mechanically induced vesicles for structure determination of membrane proteins

The structural and functional characteristics of membrane proteins can be influenced by the composition of the membrane. Consequently, native membranes are most relevant for the study of receptors and other membrane proteins. In this study, we investigated two types of cell-derived vesicles: natively shed extracellular vesicles (EVs) and mechanically derived vesicles (MVs). To this end, we utilized the human breast cancer cell line SKBR3, which strongly overexpresses the receptor HER2. We designed a protocol based on designed ankyrin repeat proteins (DARPins) to purify EVs and MVs enriched in HER2, and to ensure the native orientation of the HER2 receptors within the vesicle. The isolated HER2-containing EVs and MVs were characterized by cryo-EM, cryo-electron tomography (cryo-ET) and mass spectrometry (MS), which revealed fundamental differences between the different vesicle types. Our study highlights the greater structural diversity of EVs over MVs. A single particle cryo-EM analysis and classification of all visible receptors on the vesicle surface yielded electron density consistent with HER2 at modest resolution. Taken together, our results suggest that MVs can serve better than EVs as a suitable platform for the structure determination of membrane proteins within their native membrane environments.

biochemistry↗

bioRxiv2026

Early Identification of At-Risk Patients: ProteomicSignature Predicts Progression to DecompensatedCirrhosis

Background & AimsEarly identification of decompensation in patients with cirrhosis is important to enable timely detection, management of complications and for effective treatment. This study investigates the biology of decompensation and aim to identify protein biomarkers for identification of high-risk patients. MethodsThe primary analysis included plasma samples from 46 patients with metabolic dysfunction associated steatotic liver disease (MASLD) related cirrhosis. Plasma samples were depleted for the top 14 most abundant proteins and the proteome was measured by liquid chromatography tandem mass spectrometry. The dataset was divided into a training (14 compensated, 10 decompensated) and a test cohort of compensated patients (11 progressing to decompensation, 11 remaining compensated). Changes in protein levels were determined by ANCOVA and a prognostic model was developed using logistic regression. External validation was performed in an independent cohort of 120 patients with alcohol-related cirrhosis. Time-to-event analyses were conducted in this cohort using Cox regression. Results52 proteins involved in impaired hepatic function, fibrogenesis, immune activation, and metabolic changes were significantly different between compensated and decompensated patients. A prognostic model with four proteins (NBL1, LTBP4, APOC4, GHR), demonstrated predictive ability for future decompensation (AUC=0.93, 73% sensitivity, 100% specificity). In the external validation cohort, the model demonstrated generalizability (AUC=0.78, 72% sensitivity, 82% specificity). Validation cohort time-to-event analyses showed that higher baseline scores were associated with shorter time to liver-related events (HR 1.32; log-rank p = 0.027), underscoring the panels prognostic value. ConclusionOur study indicates that patients with decompensated cirrhosis are characterized by proteomic signatures of fibrogenesis and metabolic dysfunction. Capturing these signatures could help identify patients at risk of complications and potentially those eligible for aetiology directed treatment. Impact and ImplicationsAddressing a critical unmet need for early detection of cirrhosis decompensation, our proteomic study identifies a four-protein panel with predictive ability for decompensation. These findings hold significant implications for hepatologists, clinical researchers, and healthcare systems, offering a novel tool to enhance prognostication and refine treatment strategies, potentially facilitating targeted patient monitoring. However, considering the small discovery sample size and the distinct aetiology of the external validation cohort, further validation is essential before broad clinical integration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/709475v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@6620e2org.highwire.dtl.DTLVardef@f8dfe4org.highwire.dtl.DTLVardef@1331101org.highwire.dtl.DTLVardef@1a195ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2025

Acute IFN-γ elicits long-term proteomic IFN-γ signature in melanoma cells

Interferon-{gamma} (IFN-{gamma}) is a central mediator of antitumor immunity, and its downstream effects are widely assumed to require continuous cytokine exposure. Here, we applied state-of-the-art mass spectrometry (MS)-based proteomics to patient-derived melanoma cells exposed either continuously to IFN-{gamma} or to a 3-hour pulse followed by cytokine removal. A transient IFN-{gamma} exposure was sufficient to establish a stable proteomic state indistinguishable from continuous stimulation, demonstrating that IFN-{gamma}-induced remodeling is rapidly triggered and durably maintained independently of ligand persistence. Proximal JAK-STAT signaling was rapid, saturable at low cytokine concentrations, and fully reversible upon washout, yet downstream proteomic programs remained durably imprinted. In contrast, functional consequences such as cytostasis remained dose-dependent, revealing a disconnect between signaling activation and phenotypic sensitivity. Together, these findings challenge long-standing assumptions about IFN-{gamma} signaling, redefine the temporal requirements for cytokine-driven tumor cell programming, and provide a mechanistic framework for improving immunotherapies primarily working via IFN-{gamma}-signaling and physiologically relevant in vitro models of tumor-immune interactions.

biochemistry↗

bioRxiv2025

ProteoBench: the community-curated platform for comparing proteomics data analysis workflows

Mass spectrometry (MS)-based proteomics is a well-established strategy for analyzing complex biological mixtures. Many MS instruments and data acquisition strategies are available, and the data they acquire differ substantially, thus requiring tailored analysis algorithms. Hence, many dedicated bioinformatics workflows are developed. These are in constant evolution, and the community lacks a centralized platform for comparing their performance. Here, we propose ProteoBench, a single platform that brings together software developers and software users to provide an ever-evolving comparison of state-of-the-art proteomics data processing tools. ProteoBench is an open-source resource that enables the community to evaluate data analysis workflows, develop benchmarking modules dedicated to specific comparisons, and discuss the best methods to compare software tools. The platform ensures that the benchmark evolves alongside advances in proteomics data analysis workflows. ProteoBench guides researchers towards the best-suited tool and parameters for their specific project and data according to their needs, and developers can test their newly developed tools or workflows privately, before adding them as public references. This community-driven effort will increase transparency and reproducibility between MS data analysis workflows, as well as facilitate the development and publication of software workflows in the field.

bioinformatics↗

bioRxiv2025

The Human Cell Line Phosphoproteome Atlas: A Deep Empirical Resource Revealing Kinase Activity Landscapes

Protein phosphorylation orchestrates cellular signaling and controls diverse biological processes, with its dysregulation driving diseases, notably cancer. Comprehensive, high-throughput phosphoproteomics remains limited by detection sensitivity, data completeness, and computational bottlenecks, especially in low-input settings. We present the deepest empirical human phosphoproteome resource to date, regrouping over 200,000 class I phosphosites across 33 diverse human cell lines, and demonstrate that this spectral library dramatically improves single-shot phosphoproteomics with 30-fold faster data processing compared to library-free approaches and enhanced confidence in phosphosite localization even from minimal sample input. Integrating proteome and phosphoproteome data, we developed a combined kinase activity score, revealing cell line- and cancer-specific signaling vulnerabilities, many correlating with drug sensitivity. This resource accelerates deep, reproducible phosphoproteomics, enabling systematic functional mapping of cellular signaling networks, and empowers precision oncology by highlighting actionable kinase targets in diverse cell states.

biochemistry↗

bioRxiv2025

Glucocorticoid-induced proteome and phosphoproteome changes in breast cancer cell lines

Glucocorticoids (GCs) are steroid hormones that bind to glucocorticoid receptor (GR) as ligands to initiate systemic anti-inflammatory effects. GCs are commonly administered alongside chemotherapy to reduce treatment-related side effects in breast cancer patients. However, GC administration has been shown to promote metastasis in breast cancer. In this study, we used quantitative mass spectrometry-based approaches to analyze proteome and phosphoproteome of three breast cancer cell lines following GC treatments. By comparing MCF7, MDA-MB-231, and MDA-MB-436 cells, we suggest that the level of GR significantly affects GC-mediated responses. Additionally, we identify noncanonical transcription factors (TFs) and kinases that are regulated by GCs in different cell lines. Together, our data present GC-induced modulations and modifications at protein level, indicating that breast cancer metastasis occurs via TF- and kinase-dependent signaling pathways. These findings highlight the need for careful consideration of GC use in breast cancer therapy and identify potential molecular targets for mitigating adverse effects. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/690354v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1c9ff8borg.highwire.dtl.DTLVardef@a8172forg.highwire.dtl.DTLVardef@18a4b85org.highwire.dtl.DTLVardef@6413cf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

From HeLa to human blood: extending single-cell proteomics to the smallest immune cells

Single-cell proteomics (SCP) holds the promise of decoding cellular heterogeneity at the functional level, yet achieving deep and reproducible proteome coverage from individual cells has remained a formidable challenge. Here, we established a high-sensitivity, label-free SCP platform that surpasses previous limits in depth and reproducibility. By integrating optimized low-input sample processing, refined liquid chromatography, and the Orbitrap Astral Zoom mass spectrometer, our approach routinely quantified over 7,000 proteins per individual HeLa cell, capturing thousands of low-abundance proteins that eluded prior SCP studies. Applied to very small human peripheral blood mononuclear cells (PBMCs), we identified up to 4,000 proteins per cell, including key markers distinguishing monocytes, T cells, and activated lymphocytes within heterogeneous populations, underscoring that single-cell proteomics can now directly elucidate clinically relevant primary samples with both depth and precision.

cell biology↗

bioRxiv2025

High-Throughput Spatial Proteomics at Cellular and Subcellular Resolution via a Hanging Droplet Workflow

Formalin-fixed paraffin-embedded (FFPE) samples are essential for clinical research and spatial proteomics (SP) but are technically challenging to analyze by liquid-chromatography coupled mass-spectrometry (LC-MS) due to losses during protein extraction, evaporation during decrosslinking and low-throughput sample preparation methods. Here, we present a streamlined micro-FFPE proteomics sample preparation protocol for laser-capture micro-dissection (LMD) that enables scalable and high-throughput processing in 96-well format, completing the entire process from tissue lysis to tryptic peptide mixtures on LC-MS-ready Evotips in just 2-hours. Our method integrates whole-slide imaging, LMD, and ultra-sensitive narrow-window data-independent acquisition (nDIA)-MS and facilitates enhanced lysis, decrosslinking, and proteolytic digestion directly within Teflon-based EVO96 chips. By utilizing nano-wells and the hanging droplet concept, our approach significantly reduces surface adsorption, thereby improving processing efficiency for both higher- and very low-input FFPE samples. This enables quantification of >5,500 protein groups (PGs) by nDIA-MS from 50,000 {micro}m{superscript 2} ([~]250 cells) with robust performance maintained down to 1000 {micro}m2 ([~]5 cells) with [~]2,500 PGs identified. Application to a panel of breast cancer (BC) tissues precisely discerned BC subtypes and their tumor micro-environment (TME) based on differential MS-based protein abundance of biomarkers including TP53, HER2 and PGR. Individually excised subcellular regions ([~]60 {micro}m{superscript 2}) yielded [~]1,500 PGs, demonstrating reliable micro-scale spatial proteomics profiling approaching near-organelle resolution. TeaserStreamlined micro-FFPE protocol enables scalable and high-throughput processing of LMD samples, from lysis to mass spectrometry in 2 hours.

biochemistry↗