Biology subjects

Okkenhaug, H.

Publications and source records attributed to Okkenhaug, H..

11 recordsLinked to original sources

bioRxiv2026

Antigen accumulation in the B cell follicle is impaired in aged mice

Vaccine responses are impaired in aged mice and older people. An essential early step in mounting successful responses to vaccines is the delivery of antigens from the site of injection to the draining lymph node. This study tested the hypothesis that particle drainage to the lymph node is impaired in older mice after intramuscular injection. We observed fewer 20nm nanoparticles in the B cell follicle of the draining lymph node in aged mice after intramuscular injection. However, there was no significant difference in the abundance of larger 1000nm particles in the lymph nodes of aged mice, which are typically transported to the lymph node by antigen presenting cells. This data suggests that access of smaller antigens to the B cell follicle is impaired in ageing and may have implications for the initiation of T and B cell responses after vaccination.

immunology↗

bioRxiv2025

Declining intracellular proteostasis capacity drives misfolded protein secretion in senescent human cells.

Healthy protein homeostasis ( proteostasis) relies on tightly-regulated protein quality-control (PQC) circuits that co-ordinate sequestration and clearance of potentially toxic aggregation-prone proteins, arising from various internal or external stress throughout an organisms lifespan. At the protein level, proteotoxic stress responses typically involve extensive poly-ubiquitylation and sequestration of aggregation-prone proteins and PQC factors into various protective cytoplasmic and nuclear granules. However, much of our current understanding regarding this aspect of stress responses in humans stems from research in proliferating cells--despite growing evidence that stress responses vary considerably at the transcriptional level across cell proliferation states. Here, we show that the senescent cellular state--considered a major contributor to ageing-associated degeneration due to a chronic inflammatory phenotype--re-wires PQC and expels the misfolded protein load to mitigate proteotoxic stresses. Starting with a multi-dimensional transcriptomics and proteomics approach for measuring levels of total, poly-ubiquitylated, and granule-forming proteins, we have discovered a clear point of divergence between senescent and proliferating or quiescent human cell states in their responses to proteotoxic stress. Although the proteins that were poly-ubiquitylated and degraded during stress were largely conserved across states, the stress-induced sedimentation of a large number of disease-associated RNA-binding proteins (including TDP-43) was impaired only in the senescent state. Strikingly, TDP-43, as well as several other misfolded proteins, were actively secreted through the endo-lysosomal system by a diverse range of senescent cells during acute or chronic stress, through a process that requires the vesicle-associated HSP70 co-chaperone DNAJC5--an established risk factor for several neurodegenerative diseases. Misfolded protein secretion could be rescued by increasing intracellular HSP levels in shallow but not deep senescence, suggesting that secretion is a proteostatic adaptation that becomes less reversible over time. Our findings reveal an unappreciated aspect of the senescent-cell secretory phenotype, which may have important consequences for the non-cell-autonomous impact of senescence at the level of tissue resilience and frailty.

cell biology↗

bioRxiv2025

Minimal activation of the p53 DNA damage response by a modular cytosine base editor enables effective multiplexed gene knockout in induced pluripotent stem cells

Precise genome editing of induced pluripotent stem cells (iPSC) holds great promise for engineering advanced cell therapies. CRISPR-Cas systems have been widely adopted in genome engineering applications, however their dependence on genotoxic DNA double strand breaks (DSBs) presents challenges in hypersensitive iPSCs. Base editors are capable of both modifying and ablating gene function without generating DSBs making them an attractive solution for iPSC engineering. Here we report efficient and durable multiplexed target knockout with minimal impact on cell viability and expansion with a cytosine base editor composed of nCas9-UGI and Rat APOBEC1 assembled using the Pin-point platform (nCas9-UGI:rAPO). Minimal p53-mediated DNA damage signalling occurred independently of the number of simultaneous edits installed, and this could be further reduced by modulating the assembly of the base editor complex. Whereas non-homologous end-joining-mediated DNA damage repair led to p53-mediated selection against imprecise editing outcomes and an associated reduction in the on-target efficiency of multiplexed SpCas9 nuclease editing, p53 activity was dispensable for maintaining genome integrity during the base editing process. The Pin-point platform therefore enables the assembly of base editors optimised for high editing efficiency with substantially reduced risk of selecting for defective DNA damage responses inherent to DSB-dependent editing systems.

molecular biology↗

bioRxiv2025

An Nlrp5-null mutation leads to attenuated de novo methylation in oocytes, accompanied by a significant reduction in DNMT3L

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5- null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

developmental biology↗

bioRxiv2025

RNA helicase DDX1 regulates germinal centre selection and affinity maturation by promoting tRNA ligase activity

Clonal expansion of antigen-specific B-cells defines effective germinal centre responses and is key for the generation of high-affinity antibodies. While positive selection in germinal centres has been associated with anabolic metabolism and cell growth, the downstream drivers of B-cell proliferation are not well understood. Here we report that the RNA helicase DDX1 is required for germinal centre maturation and accrual of dark-zone cellularity. Upon interaction with T-follicular helper cells, DDX1-deficient B-cells upregulate c-MYC but do not clonally expand. We show that positive selection is coupled with an increase in mRNA translation, that is dependent on DDX1. DDX1 endows B-cells with the protein biosynthetic capability that is required for rapid cell proliferation. It does so by modulating the activity of the tRNA ligase complex and tRNA splicing. Our data reveal that mRNA translation efficiency is a key determinant of B-cell fitness during germinal centre responses.

immunology↗

bioRxiv2023

A cyclic peptide toolkit reveals mechanistic principles of peptidylarginine deiminase IV (PADI4) regulation

Peptidylarginine deiminase IV (PADI4) deregulation promotes the development of autoimmunity, cancer, atherosclerosis and age-related tissue fibrosis. Genetic or pharmacological PADI4 inhibition is therapeutically efficacious in mice, but no clinically relevant inhibitors currently exist. PADI4 additionally mediates immune responses and cellular reprogramming, although the full extent of its physiological roles is unexplored. Despite detailed molecular knowledge of PADI4 activation in vitro, we lack understanding of its regulation within cells, largely due to lack of appropriate systems and tools. Here, we developed and applied a set of potent and selective PADI4 modulators. Using the mRNA- display-based RaPID system, we screened >1012 cyclic peptides for high-affinity, conformation-selective binders. We report PADI4_3, an inhibitor specific for the active conformation of PADI4; PADI4_7, an inert binder, which we functionalised for the isolation and study of cellular PADI4; and PADI4_11, a first-in-class activator. Using a newly developed method for the quantification of cellular PADI4 activity, we show that PADI4_3 and PADI4_11 are effective in cells. Structural studies with PADI4_11 reveal an allosteric binding mode that may reflect the mechanism that promotes cellular PADI4 activation. This work offers new understanding of PADI4 regulation and a toolkit for the study and modulation of PADI4 across physiological and pathophysiological contexts.

biochemistry↗

bioRxiv2023

Maternal SMARCA5 is required for major ZGA in mouse embryos

Zygotic genome activation (ZGA) in mice takes place in two waves, a minor wave in the one-cell embryo, and a major wave at the two-cell stage, both accompanied by global transcriptional and epigenetic reprogramming. However, the orchestration of these reprogramming events by maternal factors deposited in the oocyte is not yet entirely understood. We and others have recently shown that epigenetic modifiers such as SMARCA5 (the main ATPase in ISWI complexes) can initiate the ZGA transcriptional programme in vitro. So far, the role of SMARCA5 in ZGA in vivo has not been addressed, as constitutive knock-out mice lacking SMARCA5 are not viable. We have overcome this limitation by using the targeted protein-depletion system Trim Away to degrade SMARCA5 in early zygotes. We further harnessed the power of single cell multi-omics (scNMT-seq) and showed that in the absence of SMARCA5, major ZGA genes fail to be upregulated at the two-cell stage. This is explained by the lower accessibility and disrupted nucleosome positioning at their promoters and distal regulatory regions, compared to wild-type embryos. In contrast, we show that global chromatin accessibility at the two-cell stage is higher in SMARCA5 depleted embryos compared to control embryos, and this is accompanied by other global structural changes involving heterochromatic regions. Our results show that SMARCA5 has a global regulatory role at the two-cell stage, which includes the control of ZGA gene promoters and distal regulatory regions.

developmental biology↗

bioRxiv2023

PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate

The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2-phosphatase, PTEN. Despite huge investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signalling and constrained by pathway-feedback. In the absence of PTEN, the network is dramatically remodelled. A poorly understood, YXXM and PIP3/PI(3,4)P2-binding PH domain-containing, adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT-phosphorylation and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K and SRC-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1-mRNA and activating-Y419-phosphorylation of hSRC correlated with PI3K-pathway activity in human prostate cancers. We propose that in PTEN-null cells, receptor-independent, SRC-dependent tyrosine-phosphorylation of PLEKHS1 creates positive-feedback that escapes homeostasis, drives PIP3- signalling and supports tumour progression.

cell biology↗

bioRxiv2020

MSK phosphorylation of H3S28 is required for immediate early gene induction and cardiac hypertrophy

Heart failure is a leading cause of death that develops subsequent to deleterious hypertrophic cardiac remodelling. MAPK pathways play a key role in coordinating the induction of gene expression during hypertrophy. Induction of the immediate early gene (IEG) response is a necessary and early event in this process. How MAPK and IEG expression are coupled during cardiac hypertrophy is not yet resolved. Here, in vitro, in rodent models and in human samples, we demonstrate that MAPK-stimulated IEG induction depends on the Mitogen and Stress activated protein Kinase (MSK) and its phosphorylation of histone H3 at serine 28 (pH3S28). pH3S28 in IEG promoters in turn recruits Brg1, a BAF60 ATP-dependent chromatin remodelling complex component, initiating gene expression. Without MSK activity and IEG induction, the hypertrophic response is suppressed. These studies provide new mechanistic insights and highlight the role of signalling to the epigenome in gene expression regulation during cardiac hypertrophy. Brief summary one sentenceMSK1/2 phosphorylation of Histone 3 Serine 28 couples MAPK signalling with chromatin remodelling and immediate early gene expression to induce pro-hypertrophic cardiac transcriptional responses.

molecular biology↗

bioRxiv2019

A neuronal thermostat controls membrane fluidity in C. elegans

An organisms ability to adapt to heat can be key to its survival. Cells adapt to temperature shifts by adjusting lipid desaturation levels and the fluidity of membranes in a process that is thought to be controlled cell autonomously. We have discovered that subtle, step-wise increments in ambient temperature can lead to the conserved heat shock response being activated in head neurons of C. elegans. This response is exactly opposite to the expression of the lipid desaturase FAT-7 in the worms gut. We find that the over-expression of the master regulator of this response, Hsf-1, in head neurons, causes extensive fat remodeling to occur across tissues. These changes include a decrease in FAT-7 expression and a shift in the levels of unsaturated fatty acids in the plasma membrane. These shifts are in line with membrane fluidity requirements to survive in warmer temperatures. We have identified that the cGMP receptor, TAX-2/TAX-4, as well as TGF-{beta}/BMP signaling, as key players in the transmission of neuronal stress to peripheral tissues. This is the first study to suggest that a thermostat-based mechanism can centrally coordinate membrane fluidity in response to warm temperatures across tissues in multicellular animals.

genetics↗

bioRxiv2019

Follicular regulatory T cells can access the germinal centre independently of CXCR5

The germinal centre (GC) response is critical for generating high-affinity humoral immunity and immunological memory, which forms the basis of successful immunisation. Control of the GC response is thought to require follicular regulatory T (Tfr) cells, a subset of suppressive Foxp3+ Treg cells located within GCs. Relatively little is known about the exact role of Tfr cells within the GC, and the mechanism/s through which they exert their suppressive function. A unique feature of Tfr cells is their reported CXCR5-dependent localisation to the GC. Here we show that the lack of CXCR5 on Foxp3+ regulatory T cells resulted in a reduced frequency, but not an absence of, GC-localised Tfr cells. This demonstrates that additional, CXCR5-independent mechanisms facilitate Treg cell homing to the GC.

immunology↗