Biology subjects

Frye, M.

Publications and source records attributed to Frye, M..

15 recordsLinked to original sources

bioRxiv2026

Non-invasive forecasting of skin cancer evolution through longitudinal hair sampling

The ability to longitudinally track clonal evolution non-invasively would transform cancer interception strategies, long before late-stage disease when most cancer genomes are analysed. Here, we demonstrate that repeated hair sampling from the same individual followed by exome sequencing enables tracking of somatic evolution in vivo over several months after chemically induced skin carcinogenesis. We found that hair follicles accumulate a higher mutation burden than spatially-matched skin and harbour mutations that spread into surrounding epidermis and persist throughout tumour progression. DNA-damaged follicles enter sustained quiescence that delays replication and repair, creating a reservoir for long-lived mutations. During premalignant progression, carcinogen-associated mutations become enriched as follicular clones expand into adjacent skin. Mutation tracking identified genes that may govern tumour predisposition and initiation, many of which are mutated at high incidence in human cutaneous squamous cell carcinoma cohorts. Hair follicles therefore provide a non-invasive readout to forecast the early development of skin cancer, enabling patient risk stratification.

cell biology↗

bioRxiv2026

Parkinson's disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

Mutations in several genes are known to cause familial forms of Parkinsons disease (PD), including mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene linked to late-onset, autosomal dominant PD. VPS35 encodes a core subunit of the retromer complex which functions in endosomal sorting and recycling. It remains unclear how the pathogenic D620N mutation in VPS35 disrupts retromer function to induce neurodegeneration in PD. Using cell-and rodent-based models expressing D620N VPS35, we performed interactome proteomics to identify alterations underlying the pathogenic effects of D620N VPS35 in PD. Using overexpression of VPS35 variants in HEK-293T cells, we conducted tandem affinity purification (TAP) or co-immunoprecipitation (co-IP) with protein chemical crosslinking to determine the native and non-native protein interactomes of wild-type (WT) and D620N VPS35, respectively. Notably, we can confirm the reduced interaction of D620N VPS35 with components of the WASH complex. Additionally, using a viral-mediated gene transfer model of human D620N VPS35 overexpression in adult rat brain, we identify the first brain-specific protein interactome of VPS35. These overexpression models reveal remarkably similar interaction profiles of WT and D620N VPS35, suggesting that the D620N mutation has a subtle effect on the overall VPS35 protein interactome. We also conducted proteomic analysis of brain tissue from a D620N VPS35 knockin (KI) mouse model that expresses VPS35 at endogenous levels. Using co-IP from hemi-brain or striatal extracts of WT and D620N VPS35 KI mice, we reveal a high degree of similarity between the brain interactomes of WT and D620N VPS35, further suggesting a subtle effect of the D620N mutation on VPS35 protein interactions. Notably, in both hemi-brain and striatum, we find a selective decrease in the interaction of two known interactors, TBC1D5 and VPS29, with D620N VPS35. We also performed global proteomic analysis of striatal tissue from D620N VPS35 KI mice and reveal a high degree of similarity between WT and D620N, further suggesting a subtle effect of this mutation. Together, our study provides a comprehensive evaluation of the VPS35 protein interactome and reveals a selective effect of the PD-linked D620N mutation in mammalian cells and brain. Our study provides key insight into the mechanisms of retromer dysfunction in VPS35-linked PD.

neuroscience↗

bioRxiv2026

Neprilysin inhibition reduces microtubule detyrosination in cardiomyocytes through a cGMP-PRKG1-VASH1 axis

Microtubule detyrosination and re-tyrosination on the C-terminus of -tubulin are mediated by the vasohibin (VASH)-small vasohibin-binding protein (SVBP) complex and tubulin tyrosine ligase (TTL), respectively. Elevated levels of detyrosinated -tubulin (dTyr-tub) are observed in heart failure, and reducing this modification improves cardiac function, suggesting that clinically used heart failure therapies may modulate microtubule detyrosination. We investigated whether sacubitrilat and valsartan, the active components of the angiotensin receptor-neprilysin inhibitor LCZ696, influence dTyr-tub levels in endothelin-1 (ET1)-induced hypertrophy in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). While both sacubitrilat and valsartan prevented hypertrophy, only sacubitrilat prevented ET1-induced dTyr-tub accumulation. RNA sequencing revealed that sacubitrilat normalized several ET1-induced dysregulated pathways. Sacubitrilat slightly increased cyclic guanosine 3,5-monophosphate (cGMP) levels and lowered dTyr-tub, whereas inhibition or knockdown of the cGMP-dependent protein kinase 1 (PRKG1) increased dTyr-tub level. Mechanistically, PRKG1 alpha phosphorylated native VASH1. Incubation of microtubules with the VASH1-SVBP complex containing wild-type VASH1 increased detyrosination, while incubation of the complex containing a VASH1 phosphomimic, in which seven C-terminal serine residues were mutated to glutamate (VASH1-7E) did not. Consistently, overexpression of VASH1-7E gave rise to lower dTyr-tub level than overexpression of a non-phosphorylatable form of VASH1 (VASH1-7A) in hiPSC-CMs deficient in VASH1. In conclusion, these findings identify a cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes. Our work provides mechanistic insight into how neprilysin inhibition may contribute to therapeutic benefit in heart failure. One Sentence SummaryWe establish a neprilysin-cGMP-PRKG1-VASH1 signaling axis that reduces microtubule detyrosination in cardiomyocytes.

cell biology↗

bioRxiv2025

De novo genome assembly of Ansell's mole-rat (Fukomys anselli)

Ansells mole-rat (Fukomys anselli) is an African rodent known for its subterranean lifestyle and unique phenotypic traits, including extreme longevity, magnetoreception, and a cooperative breeding social structure. Efforts to dissect the genetic architecture of these traits and to decipher their phylogenetic relationships within the broader African mole-rat family would greatly benefit from a reference-grade genome. Here, we report a first genome assembly of a male Ansells mole-rat. By combining Oxford Nanopore Technologies (ONT) long-reads and Illumina short-reads with Hi-C data, we generated a chromosome level assembly with a total length of 2.27 Gb, 412 scaffolds and a scaffold N50 of 72.4 Mb. We identified 99.54% of expected genes and annotated 29,094 transcripts using RNA sequencing data. This high-quality de novo genome of Fukomys anselli lays the foundation for dissecting the genetic and evolutionary basis of its extraordinary traits and resolving African mole-rat phylogeny. ARTICLE SUMMARYBekavac, Coimbra, and Busa et al. assemble a de novo genome of a male Fukomys anselli individual. They use short-read and long-read whole genome sequencing and Hi-C sequencing to achieve chromosome-level contiguity, and annotate the genome using RNA-seq from nine diverse tissues. This high-quality genome enables exploration of the genetic basis of Ansells mole-rats remarkable traits, including longevity, hypoxia tolerance, and magnetoreception. It also allows for comprehensive analysis of the currently-contested phylogenetic relationships within the mole-rat family.

genomics↗

bioRxiv2025

The two-step purification method ViREn identifies a single NSUN6-mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover

Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes including RNA stability and translation. Although advances in analytical methods have improved detection sensitivity, the precise mapping of RNA modifications at single-base resolution remains challenging. Especially for low abundant viral RNAs extracted from infected cells, requirements for sensitivity and purity limit accuracy and reproducibility. Here we report the two-step method ViREn for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a high-confidence 5-methylcytosine (m5C) site in DENV gRNA, orthogonally validated by Illumina-based bisulfite sequencing and direct RNA sequencing by Nanopore Oxford Technologies. Strikingly, this m5C modification was exclusively detected in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on low abundance viral gRNA with unprecedented precision and sensitivity and facilitates mechanistic studies into the role of RNA modification in virus replication.

biochemistry↗

bioRxiv2024

Sepsis-induced NET formation requires MYD88 but is independent of GSDMD and PAD4

Neutrophils are peripheral blood-circulating leukocytes that play a pivotal role in host defense against bacterial pathogens which upon activation, they release web-like chromatin structures called neutrophil extracellular traps (NETs). Here, we analyzed and compared the importance of myeloid differentiation factor 88 (MYD88), peptidyl arginine deiminase 4 (PAD4), and gasdermin D (GSDMD) for NET formation in vivo following sepsis and neutrophilia challenge. Injection of lipopolysaccharide (LPS)/E. coli or the transgenic expression of granulocyte colony-stimulating factor (G-CSF), each induced NET-mediated lethal vascular occlusions in mice with combined genetic deficiency in Dnase1 and Dnase1l3 (D1/D1l3-/-). In accordance with the signaling of toll-like receptors, Myd88/D1/D1l3-/- animals were protected from the formation of lethal intravascular NETs during septic conditions. However, this protection was not observed during neutrophilia. It was unexpected to find that both Gsdmd/D1/D1l3-/- and Pad4/D1/D1l3-/- mice were fully capable of forming NETs upon LPS/E.coli challenge. Sepsis equally triggered a similar inflammatory response in these mice characterized by formation of DNA-rich thrombi, vessel occlusions, and mortality from pulmonary embolism, compared to D1/D1l3-/- mice. Pharmacologic GSDMD inhibitors did not reduce PMA-stimulated NET formation in ex vivo models either. Similarly, neither Pad4 nor GSDMD deficiency affected intravascular occlusive NET formation upon neutrophilia challenge. The magnitude of NET production, multi-organ damage, and lethality were comparable to those observed in challenged control mice. In conclusion, our data indicate that NET formation during experimental sepsis and neutrophilia is regulated by distinct stimulus-dependent pathways that may be independent of canonical PAD4 and GSDMD. Key points- Sepsis triggers vaso-occlusive NET formation in Dnase1/Dnase1l3-deficient mice in a myeloid differentiation factor 88-dependent manner - Peptidyl arginine deiminase 4 and gasdermin D are dispensable for NET formation in sepsis and neutrophilia models - Myeloid differentiation factor 88, peptidyl arginine deiminase 4 and gasdermin D differ in their importance for NET formation in vivo

immunology↗

bioRxiv2024

Unbiased functional genetic screens reveal essential RNA modifications in human cancer and drug resistance

RNA modification pathways are mis-regulated in multiple types of human cancer. To comprehensively identify cancer-relevant RNA modifications and their regulators, we screened all 150 annotated human RNA modifying proteins across 18 different normal and cancer cell lines using a CRISPR-based genetic knockout system. Fifty RNA modifying proteins were essential for survival of at least one cell type. A third of these essential genes were amplified in 38 different human primary cancer types and potentially drive cancer growth. Unexpectedly, the number of essential genes per cell line varied considerably, and this variation was not due to tissue of origin. Instead, we found that cancer cell-specific mitochondrial metabolic plasticity was responsible for the unique requirement of certain RNA modifications. For example, leukemia cells with high intrinsic drug tolerance required mitochondrial flexibility to survive treatment with the anti-leukemic drugs cytarabine and venetoclax. Synthetic lethality screens revealed that drug-resistance is abolished by deleting the mitochondrial methyltransferase TRMT5, which is responsible for the formation of N1-methylguanosine (m1G) in the tRNA anticodon loop. In summary, our study identifies cancer-relevant RNA modifying enzymes, and reveals a novel promising drug target for therapy-resistant acute myeloid leukemia.

cell biology↗

bioRxiv2024

SRSF2 transcriptional function maintains genome integrity during cell division

Recurrent mutations in serine / arginine-rich splicing factor 2 (SRSF2), particularly the proline-to-histidine substitution at position 95 (P95H), have been proposed to drive neoplastic diseases. SRSF2 plays pivotal roles in pre-mRNA processing and gene transcription. However, the precise impact of these diverse functions of SRSF2 on cancer cell behaviour remains unclear. Here, we show that deletion or homozygous P95H mutation of SRSF2 both cause extensive DNA damage leading to cell cycle arrest in vitro and in vivo, regardless of cell genotype. We mechanistically demonstrate that SRSF2 is required for efficient bi-directional transcription of DNA replication and repair genes, independent of its function in splicing. In contrast, SRSF2 haploinsufficiency induces DNA damage without halting the cell cycle in cancer cells. Inducing the heterozygous Srsf2 P95H mutation leads to clonal expansion of epidermal cells in mouse skin, but tumor formation is inhibited following exposure to carcinogens. To survive carcinogen treatment, cells containing the Srsf2 P95H mutation undergo substantial transcription rewiring that restores bi-directional gene expression. Thus, our study reveals crucial roles of SRSF2 beyond splicing and underscores its importance in regulating transcription to orchestrate the cell cycle along with the DNA damage response.

cell biology↗

bioRxiv2023

An interactive cellular ecosystem blocks epithelial transformation in naked mole-rat

Long-lived species suppress cancer despite accumulating somatic mutations throughout life, but how this is achieved in renewing tissues remains unclear. Here, we show that naked mole-rat skin uncouples high cellular turnover from cancer risk through coordinated epithelial and stromal mechanisms that constrain clonal outgrowth and suppress tumor-promoting inflammation. Despite elevated epidermal proliferation and mutational burden, naked mole-rat skin maintains tissue integrity through an expanded pool of early committed progenitor (hybrid) cells and replication-coupled genome maintenance pathways. Under chronic carcinogenic stress, undifferentiated basal cells replenish the hybrid progenitor pool. This dilutes initiated clones and permits only limited selective expansion of rare cancer gene-mutant clones. Depletion of the hybrid compartment shifts this protective state toward clonal expansion and inflammatory activation. These expanding clones are further constrained by a highly tumor-suppressive stromal microenvironment, driven by fibroblasts that adopt a metabolically restricted, non-inflammatory program. Together, our data uncover a multi-layered tumor-suppressive strategy that couples turnover-driven regeneration with an anti-permissive stromal niche to prevent malignant progression under mutagenic stress.

cancer biology↗

bioRxiv2023

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC) enzyme. In platelets, the NO-GC/cGMP pathway inhibits aggregation. One aspect of the inhibitory mechanism involves changes in the cytoskeleton; however, the molecular mechanisms underlying platelet inhibition and its correlation with cytoskeletal cellular stiffness are poorly understood. We measured the cellular stiffness of individual platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat, using scanning ion conductance microscopy (SICM). We quantified changes in platelet shape using deep learning-based platelet morphometry. Cytoskeletal actin polymerization and platelet activation were measured by co-immunostaining F-actin and P-selectin, respectively. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulphate (ABC)-containing regimens, compared with HIV-negative volunteers. Stimulation of human and murine platelets with the NO-GC stimulator riociguat or with the NO-GC activator cinaciguat downregulated P-selectin expression, decreased F-actin polymerization, and decreased cellular stiffness by {approx}50%, compared to vehicle control. In addition, platelets became more circular, indicating decreased activation. Riociguat did not cause any change in platelet aggregation or circularity in HIV-positive patients taking ABC-containing regimens. These results corroborate a functional role of the NO-GC enzyme in platelet biomechanics (cellular stiffness) in correlation with the inhibition of platelet activation and morphological changes. The observed changes in stiffness and platelet shape therefore demonstrate the possibility of pharmacologically targeting the NO-GC/cGMP pathway.

biochemistry↗

bioRxiv2023

The phosphodiesterase 2A regulates lymphatic endothelial development via cGMP-mediated control of Notch signaling

During vascular development endothelial junctions mature and vessel integrity is established to form the endothelial barrier. The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we uncover the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic, but not blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, while large blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, while cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to downregulation of NOTCH signaling. Vice versa, DLL4-induced NOTCH activation restores junctional maturation in PDE2A-deficient lymphatic endothelial cells. Our data demonstrate that PDE2A selectively modulates a crosstalk between cGMP and NOTCH signaling to finetune lymphatic development and suggest that PDE2A may be a druggable target to control lymphatic leakage and regeneration.

developmental biology↗

bioRxiv2020

Defective NETs Clearance contributes to sustained FXII Activation in COVID-19-associated Pulmonary Thrombo-Inflammation

BackgroundCoagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. MethodsWe performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced Factor XIII (FXII) activation using a chromogenic substrate assay. FindingsFXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasma FXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro. InterpretationCollectively, our study supports that the NETs/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both, NETs and FXIIa, could provide a strategy to mitigate COVID-19-induced thrombo-inflammation. FundingThis study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

molecular biology↗

bioRxiv2020

Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6

Methylation is the most common internal modification in mRNA. While the highly abundant N6-methyladonsine (m6A) modification affects most aspects of mRNA function, the precise functions of the rarer 5-methylcytosine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed that only mRNA methylation sites containing the consensus motif depended on the presence of NSUN6. Furthermore, ribosome profiling demonstrated that NSUN6-specific consensus motifs marked translation termination. However, even though NSUN6-methylated mRNAs were reduced in NSUN6 knockout cells, NSUN6 was dispensable for mouse embryonic development. Thus, our study identifies NSUN6 as methyltransferase targeting mRNA in a sequence- and structure-specific manner.

molecular biology↗

bioRxiv2019

NSUN2 introduces 5-methylcytosines in mammalian mitochondrial tRNAs

Maintenance and expression of mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. Post-transcriptional modification of mitochondrial RNA has emerged as one of the key regulatory steps of human mitochondrial gene expression. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterised as an RNA methyltransferase that introduces 5-methylcytosine (m5C) in nuclear-encoded tRNAs, mRNAs, microRNA and noncoding RNAs. In these roles, NSUN2 has been associated with cell proliferation and differentiation. Pathogenic variants in NSUN2 have been linked with neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation - knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells - we show that NSUN2 in necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.

biochemistry↗

bioRxiv2018

Polygenic risk scores for major depressive disorder and neuroticism as predictors of antidepressant response: meta-analysis of three treatment cohorts.

There are currently no reliable approaches for correctly identifying which patients with major depressive disorder (MDD) will respond well to antidepressant therapy. However, recent genetic advances suggest that Polygenic Risk Scores (PRS) could allow MDD patients to be stratified for antidepressant response. We used PRS for MDD and PRS for neuroticism as putative predictors of antidepressant response within three treatment cohorts: The Genome-based Therapeutic Drugs for Depression (GENDEP) cohort, and 2 sub-cohorts from the Pharmacogenomics Research Network Antidepressant Medication Pharmacogenomics Study PRGN-AMPS (total patient number = 783). Results across cohorts were combined via meta-analysis within a random effects model. Overall, PRS for MDD and neuroticism did not significantly predict antidepressant response but there was a consistent direction of effect, whereby greater genetic loading for both MDD (best MDD result, p < 5*10-5 MDD-PRS at 4 weeks, {beta} = -0.019, S.E = 0.008, p = 0.01) and neuroticism (best neuroticism result, p < 0.1 neuroticism-PRS at 8 weeks, {beta} = -0.017, S.E = 0.008, p = 0.03) were associated with less favourable response. We conclude that the PRS approach may offer some promise for treatment stratification in MDD and should now be assessed within larger clinical cohorts.

genetics↗