Biology subjects

Morse, M.

Publications and source records attributed to Morse, M..

5 recordsLinked to original sources

bioRxiv2025

Mechanism of SARS-CoV-2 Nucleocapsid Protein Phosphorylation-induced Functional Switch

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed WT Np formed discrete complexes on viral 5' UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1-4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs. WT Np also compacted and irreversibly bound single-stranded DNA; this activity was significantly reduced by phosphorylation. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants.

biochemistry↗

bioRxiv2025

SIRPγ modulates effector differentiation of human CD8 T Cells under suboptimal TCR stimulation: implications for immune homeostasis and autoimmunity

BackgroundAberrant CD8 T-cell differentiation contributes to the pathogenesis of autoimmune diseases, and immune-mediated tissue damage. However, the molecular mechanisms that prevent premature effector T cell programming in humans remain incompletely defined. Signal regulatory protein gamma (SIRP{gamma}) is selectively expressed on T-cells in the human immune system. Notably, genetic variants associated with reduced SIRP{gamma} expression have been linked to increased risk of immune-mediated diseases, including type 1 diabetes and multiple sclerosis, but the contribution of SIRP{gamma} to CD8 T-cell dysregulation in these contexts remains unclear. ObjectiveTo determine how inter-individual variation in SIRP{gamma} expression influences immune homeostasis and CD8 T-cell effector programming. MethodsPeripheral blood CD8 T-cells from healthy donors were analyzed for SIRP{gamma} expression and associated differentiation phenotypes. Naive CD8 T-cells were purified and subjected to siRNA-mediated knockdown of SIRPG, followed by suboptimal TCR stimulation. Differentiation status, transcription factor expression, and effector cytokine production were measured using flow cytometry. CD47 blockade was used to assess ligand dependency. ResultsLow SIRP{gamma} expression on CD8 T-cells was associated with increased frequencies of CD27-CD45RO effector-like and CD27-CD45RO- terminally differentiated CD8 T-cells. SIRPG knockdown induced effector-like differentiation, with increased CD45RO and T-bet expression and elevated TNF-, IFN-{gamma}, and Granzyme B production. This effect was not recapitulated by CD47 blockade, suggesting a CD47-independent regulatory mechanism. ConclusionSIRP{gamma} serves as a negative regulator of CD8 T-cell effector differentiation under suboptimal stimulation. Inter-individual variation in its expression may influence susceptibility to immune dysregulation, positioning it as a potential biomarker and therapeutic target.

immunology↗

bioRxiv2025

Heterochromatin fidelity is a therapeutic vulnerability in lymphoma and other human cancers

Genes involved in the regulation of chromatin structure are frequently disrupted in cancer, contributing to an aberrant transcriptome and phenotypic plasticity. Yet, therapeutics targeting mutant forms of chromatin-modifying enzymes have yielded only modest clinical utility, underscoring the difficulty of targeting the epigenomic underpinnings of aberrant gene regulatory networks. Here, we sought to identify novel epigenetic vulnerabilities in diffuse large B-cell lymphoma (DLBCL). Through phenotypic screens and biochemical analysis, we demonstrated that inhibition of the H3K9 demethylases KDM4A and KDM4C elicits potent, subtype-agnostic cytotoxicity by antagonizing transcriptional networks associated with B-cell identity and epigenetically rewiring heterochromatin. KDM4 demethylases associated with the KRAB zinc finger ZNF587, and their enzymatic inhibition led to DNA replication stress and DNA damage-induced cGAS-STING activation. Broad surveys of transcriptional data from patients also revealed KDM4 family dysregulation in several other cancer types. To explore this potential therapeutic avenue, we performed high-throughput small molecule screens with H3K9me3 nucleosome substrates and identified novel KDM4 demethylase inhibitors. AI-guided protein-ligand binding predictions suggested diverse modes of action for various small molecule hits. Our findings underscore the relevance of targeting fundamental transcriptional and epigenetic mechanisms for anti-cancer therapy. HIGHLIGHTSO_LIPhenotypic screens identified JIB-04 as a potent anti-cancer agent for multiple subtypes of diffuse large B-cell lymphoma C_LIO_LIJIB-04 binds and inhibits KDM4 demethylases resulting in epigenomic rewiring of heterochromatin C_LIO_LIKDM4 demethylases cooperate with KRAB zinc fingers to limit DNA replication stress, and KDM4 inhibition instigates DNA-damage and cGAS-STING activation in several human cancers C_LIO_LIHigh-throughput small molecule screens with semi-synthetic nucleosome substrates and AI-guided molecular docking simulations identify novel KDM4 inhibitors C_LI

genomics↗

bioRxiv2022

Epigenetic and transcriptional plasticity of myeloid cells in Cystic Fibrosis

The relative contributions of acute and chronic inflammatory mechanisms in diseases characterised by persistent bacterial infection are unclear, despite important consequences for the development of novel anti-inflammatory therapies. Here, we examined longitudinal transcriptional and epigenetic changes in patients with Cystic Fibrosis (CF), a genetic condition characterised by persistent bacterial lung infections that drive progressive inflammatory lung damage. We find that sudden clinical deteriorations in lung health, termed Acute Pulmonary Exacerbations (APEs), are linked to innate immune signalling driven by bacterial components, and accompanied by changes in macrophage function. Treatment of patients with intravenous antibiotics results in rapid modification of myeloid cell gene expression and epigenetic state, towards that of healthy volunteers, suggesting that repeated acute inflammatory episodes play an important role in CF inflammatory lung damage.

immunology↗

bioRxiv2019

Self-regulation of single-stranded DNA wrapping dynamics by E. coli SSB promotes both stable binding and rapid dissociation

E. coli SSB (EcSSB) is a model protein for studying functions of single-stranded DNA (ssDNA) binding proteins (SSBs), which are critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations in order to protect these transiently formed regions during processes such as replication and repair. Using optical tweezers, we measure the binding and wrapping of a single long ssDNA substrate under various conditions and free protein concentrations. We show that EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as protein density on the substrate passes a critical saturation. Increasing free EcSSB concentrations increase the fraction of EcSSBs in less-wrapped conformations, including a previously uncharacterized EcSSB8 bound state in which [~]8 nucleotides of ssDNA are bound by a single domain of the tetramer with minimal substrate deformation. When the ssDNA is over-saturated with EcSSB, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped EcSSB-ssDNA complexes. We develop a multi-step kinetic model in which EcSSB tetramers transition through multiple wrapped conformations which are regulated through nearest neighbor interactions and ssDNA occupancy. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers can be rapidly removed from an ssDNA substrate to allow for DNA maintenance and replication functions while still fully protecting ssDNA over a wide range of protein concentrations.

biophysics↗