Biology subjects

Flynn, H. R.

Publications and source records attributed to Flynn, H. R..

9 recordsLinked to original sources

bioRxiv2026

CDKL5 phosphorylates neuronal ELAVL proteins to promote mRNA binding, protein synthesis and visual cortex development

Loss-of-function mutations in the X-linked CDKL5 gene lead to a severe neurodevelopmental disorder characterized by early-onset epilepsy, known as CDKL5 Deficiency Disorder (CDD). Despite its clinical significance, the physiological substrates of the serine/threonine kinase CDKL5 and its roles in neuronal development remain poorly understood. To address this, we performed quantitative phosphoproteomics analysis in Cdkl5 knockout (KO) mouse brains, identifying 22 CDKL5 substrates involved in diverse cellular functions. Among these, we focused on the neuronal RNA-binding proteins (nELAVLs) ELAVL2, ELAVL3, and ELAVL4, as these represented the only evolutionarily conserved phosphorylation and are known regulators of neuronal differentiation. Through kinase assays and individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP), we found that CDKL5 phosphorylates S119/131 in ELAVL2/3/4, promoting their cytoplasmic localization and enhancing their binding to target mRNAs at 3UTRs. Loss of CDKL5 activity in neurons caused reduced new protein synthesis, as measured by puromycin incorporation; this phenotype was rescued by knockdown of the nELAVL inhibitor long non-coding RNA, RNY3, revealing an essential function of CDKL5 in enhancing protein synthesis via nELAVL phosphorylation. To investigate the in vivo functions of nELAVL phosphorylations, we generated Elavl2/3/4 phosphomutant mice and found that collectively nELAVL phosphorylations are required for viability. Proteomic and transcriptomic analyses of Elavl2/3 homozygous phosphomutants, which exhibited sub-viability, revealed compensatory upregulation of ELAVL4 and synaptic proteins. Functionally, in Elavl2/3/4 triple heterozygous mice Neuropixels recordings in the primary visual cortex showed deficits in receptive field properties and orientation tuning, revealing the role of nELAVL phosphorylation for accurate cortical circuit formation. Our study uncovers a crucial role for CDKL5 in regulating nELAVL-mediated protein synthesis and the development of cortical circuits.

neuroscience↗

bioRxiv2026

The phosphoproteomic landscape of the neurological manifestations in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in TSC1 and TSC2, resulting in activation of mechanistic target of rapamycin complex 1 (mTORC1). Neurological manifestations in TSC patients include epilepsy, autism and intellectual disability. Two types of brain lesions, cortical tubers and subependymal giant cell astrocytomas (SEGAs), cause the majority of neurological manifestations in TSC. We have limited understanding of the molecular changes that occur in tubers and SEGAs and how these contribute to disease pathogenesis. To investigate this, we performed proteomic and phosphoproteomic analysis of TSC patient tuber and SEGA tissue. Tubers showed evidence of alterations in mitochondrial respiration, cytoskeleton organisation and neuronal function. However, we were unable to detect mTORC1 activation in tubers, likely due to the small number of cells with complete inactivation of TSC1 or TSC2. By contrast, SEGAs showed evidence of strong mTORC1 activation and large-scale changes in the proteome and phosphoproteome. SEGAs exhibited increased expression of ribosomal proteins and activation of a neuroinflammatory response. Phosphoproteomics identified 6060 phosphosites within 2154 proteins increased in SEGAs. Phosphorylation of multiple proteins involved in RNA-metabolism, including mRNA splicing, were increased in SEGAs. Consistent with this, we found evidence of extensive alterations in mRNA transcript splicing in SEGA tissue. These data greatly expand the repertoire of known mTORC1 target proteins in the human brain and reveal large-scale mis-regulation of mRNA splicing in SEGAs in TSC.

neuroscience↗

bioRxiv2025

Streptococcus pneumoniae accessory capsular genes modulate fitness, pathogenicity and immune evasion

Globally, Streptococcus pneumoniae disproportionally affects children in resource-poor settings, older adults and people living with HIV. Frequently found as an asymptomatic colonizer of the nasopharynx, this versatile pathogen is a prominent cause of pneumonia, meningitis, bacteraemia and otitis media. Recently, a serotype 3 capsule variant (GPSC10-ST700) has expanded in Malawi with enhanced vaccine escape potential. Here, using a mutational and complementation approach, we show that loss of accessory capsular genes in GPSC10-ST700 contribute to increased opsonophagocytic resistance in this lineage. Although originally thought to be nonfunctional pseudogenes, we show that these genes modulate fitness and the global phosphoproteome in serotype 3 strains. These findings highlight that vaccine escape may be mediated through variations in the pneumococcal capsular locus that enhance fitness, pathogenicity and immune evasion, without capsule switching. IMPORTANCEPneumococcal polysaccharide-conjugate vaccines (PCV) target the polysaccharide capsule (CPS), which is a dominant virulence factor. However, current PCVs induce suboptimal protection against serotype 3 strains, which produce a thicker capsule that when released from the bacterial surface, interferes with antibody-mediated bacterial killing and protection. We recently described the clonal expansion of a sequence type (ST) 700-GPSC10 serotype 3 lineage in Malawi post-PCV13 introduction. This lineage is characterized by the absence of at least 6 genes in its cps locus and a distinct antimicrobial resistance (AMR) profile compared to other serotype 3 strains. Here we uncovered a functional role for the accessory capsular genes (acl) in serotype 3, previously considered to be pseudogenes, which modulate capsule production, shedding, serum tolerance, and bacterial fitness. By linking genotype to phenotype, our work provides new insights into the molecular basis of serotype 3 immune evasion, informing the design of more effective pneumococcal vaccines.

microbiology↗

bioRxiv2024

Plasmodium falciparum protein phosphatase PP7 is required for early ring-stage development

We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase which interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signalling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasise a role in calcium signalling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development.

molecular biology↗

bioRxiv2024

Activation loop phosphorylation and cGMP saturation of PKG regulate egress of malaria parasites.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

microbiology↗

bioRxiv2022

OxoScan-MS: Oxonium ion scanning mass spectrometry facilitates plasma glycoproteomics in large scale

Protein glycosylation is a complex and heterogeneous post-translational modification. Specifically, the human plasma proteome is rich in glycoproteins, and as protein glycosylation is frequently dysregulated in disease, glycoproteomics is considered an underexplored resource for biomarker discovery. Here, we present OxoScan-MS, a data-independent mass spectrometric acquisition technology and data analysis software that facilitates sensitive, fast, and cost-effective glycoproteome profiling of plasma and serum samples in large cohort studies. OxoScan-MS quantifies glycosylated peptide features by exploiting a scanning quadrupole to assign precursors to oxonium ions, glycopeptide-specific fragments. OxoScan-MS reaches a high level of sensitivity and selectivity in untargeted glycopeptide profiling, such that it can be efficiently used with fast microflow chromatography without a need for experimental enrichment of glycopeptides from neat plasma. We apply OxoScan-MS to profile the plasma glycoproteomic in an inpatient cohort hospitalised due to severe COVID-19, and obtain precise quantities for 1,002 glycopeptide features. We reveal that severe COVID-19 induces differential glycosylation in disease-relevant plasma glycoproteins, including IgA, fibrinogen and alpha-1-antitrypsin. Thus, with OxoScan-MS we present a strategy for quantitatively mapping glycoproteomes that scales to hundreds and thousands of samples, and report glycoproteomic changes in severe COVID-19.

systems biology↗

bioRxiv2021

The master energy homeostasis regulator PGC-1alpha couples transcriptional co-activation and mRNA nuclear export

PGC-1 plays a central role in maintaining the mitochondrial and energy metabolism homeostasis, linking external stimuli to the transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and a putative RNA recognition motif, however potential RNA-processing role(s) have remained elusive for the past 20 years. Here, we show that the RS domain of human PGC-1 directly interacts with RNA and the nuclear RNA export factor NXF1. Inducible depletion of endogenous PGC-1 and expression of RNAi-resistant RS-deleted PGC-1 further demonstrate that the RNA-binding activity is required for nuclear export of co-activated transcripts and mitochondrial homeostasis. Moreover, a quantitative proteomics approach confirmed PGC-1-dependent RNA transport and mitochondrial-related functions, identifying also novel mRNA nuclear export targets in age-related telomere maintenance. Discovering a novel function for a major cellular homeostasis regulator provides new directions to further elucidate the roles of PGC-1 in gene expression, metabolic disorders, ageing and neurodegenerative diseases.

molecular biology↗

bioRxiv2020

A multipass membrane protein interacts with the cGMP-dependent protein kinase to regulate critical calcium signals in malaria parasites

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in both Plasmodium falciparum asexual blood stages and P. berghei gametocytes. Phosphoproteomic analyses in both Plasmodium species reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion of P. berghei ICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss of P. falciparum ICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

microbiology↗

bioRxiv2020

A Structure-Based Mechanism for DNA Entry into the Cohesin Ring

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here, we combine biophysical approaches and cryo-EM to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design biochemical experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against a shut ATPase gate. ATP hydrolysis leads to ATPase gate opening to complete DNA entry. Whether DNA loading is successful, or rather results in loop extrusion, might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.

molecular biology↗