Biology subjects

Ryan, B. J.

Publications and source records attributed to Ryan, B. J..

12 recordsLinked to original sources

bioRxiv2026

Pathological levels of α-synuclein increase basal mitophagy in Parkinson's dopamine neurons

Elevated -synuclein results in mitochondrial dysfunction, however the exact mechanism is poorly understood. -Synuclein localises to multiple subcellular sites including mitochondria, where it binds anionic lipids which regulate mitochondrial dynamics through DRP1. We hypothesised that pathogenic levels of -synuclein lead to dysregulation of mitophagy and lipid-dependent mitochondrial dynamics, causing mitochondrial and neuronal dysfunction. Using induced pluripotent stem cell-dopamine neurons (iPSC-DANs) carrying the -synuclein gene triplication mutation (SNCA-Trp) which causes an early-onset form of Parkinson's disease (PD), we established that elevated -synuclein increased basal mitophagy compared to healthy controls and overall loss of mitochondrial mass. This was associated with accumulation of basal mitophagy receptors BNIP3 and NIX and decreased proteasome activity, and was independent of PINK1-Parkin mitophagy. We identified reduced mitochondrial gene expression by single cell RNAseq, mirroring heightened mitophagy and mitochondrial depletion. We found increased levels of DRP1-dependent mitochondrial fission and perturbed levels of anionic lipids, including cardiolipin and phosphatidic acid, two lipids which directly bind to -synuclein. Pharmacological inhibition of DRP1 reversed the excessive basal mitophagy in SNCA-Trp neurons and restored deficits in cellular ATP and calcium release. Finally, we assessed neuronal function and found that impairment of mitochondrial dysfunction by elevated -synuclein led to reductions in neuronal firing activity. Inhibition of DRP1 rescued the neuronal activity deficit in SNCA-Trp neurons, suggesting that relieving - instead of increasing - mitophagy pressure may be beneficial in -synuclein-driven PD.

cell biology↗

bioRxiv2026

Phosphorylated ubiquitin is a secondary messenger and an epigenetic mark mediating mitochondria to nucleus signaling

AbstractParkinsons disease (PD) is commonly associated with dysfunctional mitochondrial homeostasis. PINK1, a S/T kinase mutated in early-onset PD, generates phosphoserine 65 ubiquitin (pS65Ub) on damaged mitochondria facilitating their removal. Here, we show that pS65Ub translocates into the nucleus after generation at damaged mitochondria and is directly attached to substrates by resident E3 ligases. Histone H2A is a major substrate and is modified at lysine 119 (H2AK119) by the polycomb silencer, E3 ligase RING1B. At nucleosomes, pS65Ub simultaneously suppresses RING1B and potentiates H2A deubiquitinases USP16 and USP21. Epigenetic profiling and RNA sequencing reveal that pS65Ub is enriched at the promoters of poorly expressed yet dynamically regulated genes and is associated with H2AK119ub depletion. Functionally, we show that pS65Ub enrichment drives polycomb target gene expression, which accelerates the maturation of dopaminergic neurons. Importantly, post-mortem PD brains exhibit elevated nuclear pS65Ub, potentially linking nuclear pS65Ub accumulation with disease pathogenesis. Together, these data indicate that pS65Ub generated at damaged mitochondria regulates fundamental cellular processes at distant sites.

cell biology↗

bioRxiv2025

TFEB and TFE3 have cell-type specific expression in the brain and divergent roles in neurons

Lysosomal dysfunction occurs in many neurodegenerative diseases, including Parkinsons disease, and activating TFEB to enhance lysosomal biogenesis is a promising therapeutic strategy. To understand TFEB physiology in cells of the brain, we characterised TFEB expression using iPSC-derived models, and transcriptomic analysis of human and mouse brain tissue. Surprisingly, TFEB expression at the RNA and protein level was restricted to glia, whereas the related transcription factor, TFE3, was expressed ubiquitously. We identified HDAC1/2/3 as transcriptional repressors of neuronal TFEB and found the brain-penetrant HDAC inhibitor ACY-738 derepressed TFEB expression and enhanced TFE3 nuclear translocation in iPSC-dopaminergic neurons (iPSC-DaNs). We delineated the role of each transcription factor by genetic manipulation in iPSC-DaNs to reveal divergent roles in which TFEB activates mitochondrial biogenesis, whereas TFE3 enhances lysosomal biogenesis. Finally, we show TFE3 activation corrects the lysosomal dysfunction associated with GBA-N370S and SNCA-Triplication mutations in Parkinsons patient-derived iPSC-DaNs, demonstrating therapeutic utility in neurodegeneration.

cell biology↗

bioRxiv2025

Extraordinary activation of CALB by alkylammonium ions: a new paradigm for activity enhancement of enzymes

Candida antarctica lipase B (CALB) is widely used in biocatalysis with applications in plastics degradation and chemical synthesis. CALB is activated by hydrophobic matrices and, enigmatically, shows striking activation in polar, choline-based, Deep Eutectic Solvents (DES). Herein, we show that CALB activation and stabilisation by TAAs is caused by binding to cholines tetraalkylammonium (TAA) moiety. Several related TAA salts also caused CALB activation which was proportional to the hydrophobicity of their alkyl substituents. Remarkably, tetraoctylammonium bromide showed activation of [~]500% even at low micromolar levels. These TAA salts represent a new class of enzyme activator. Molecular modelling identified the alkylammonium binding location as a hydrophobic patch centred around Asp-145 of CALB. Binding at this site explains lipase activation in polar DES solvents and its relationship to other pathways of CALB activation. Herein, we also demonstrate that CALB, like many lipases, is activated by calcium. Intriguingly, mixed soluble activator experiments showed that calcium and choline bind to different CALB sites, suggesting a two-site model for CALB activation. These observations, along with previous findings, show that TAA activation is a widespread property of enzymes and constitutes a novel and potent means to enhance enzyme turnover and stability. HighlightsO_LICALB is activated by choline C_LIO_LISeveral tetraalkylammonium salts cause activation of CALB C_LIO_LIHyperactivation of CALB (5-fold) by tetraoctylammonium ions occurs at low micromolar concentrations. C_LIO_LITwo independent sites for CALB activation, by calcium and TAA ions, are identified C_LIO_LIActivation at the choline binding site stabilises CALB while calcium binding destabilises the enzyme C_LIO_LIA soluble activator is demonstrated, that can be used to probe the activation mechanism of CALB or other enzymes. C_LI

bioengineering↗

bioRxiv2025

USP30 inhibition improves mitochondrial health through both PINK1-dependent and independent mechanisms

Mitochondrial dysfunction is a key feature of many pathologies, including Parkinsons disease. The selective vulnerability of dopaminergic neurons is thought to be influenced by mitochondrial dysfunction and mutations in the mitophagy regulating proteins PINK1 and Parkin that are known to cause early-onset Parkinsonism in an autosomal recessive manner. Augmentation of mitophagy through inhibition of USP30 may be a viable therapeutic strategy for a number of diseases including Parkinsons. USP30 inhibition has been demonstrated to augment PINK1/PRKN mitophagy but also potentiate basal mitophagy to support the removal of dysfunctional mitochondria. Therefore, long-term de-regulation of mitophagy has been proposed to lead to mitochondrial depletion. We have used an integrated approach across cell lines, primary neurons and iPSC-derived dopaminergic neuronal cultures to assess the short and long-term effects of USP30 inhibition on mitochondrial health and neuronal activity. We investigated the dependence of USP30 inhibition phenotypes on the PINK1/Parkin pathway using genetic ablation and in iPSC-derived neurons from Parkinsons patients with PINK1 or PRKN mutations. Loss of USP30 through CRISPR/Cas9-mediated knockout resulted in increased basal and depolarisation-induced mitophagy in SH-SY5Y cells. Loss of USP30 or pharmacological inhibition altered mitochondrial morphology and led to increases in membrane potential and ATP levels with decreased oxygen consumption, suggesting that USP30 loss results in a more efficient mitochondrial network. These changes in morphology were found to be independent of PINK1 or Parkin. Chronic pharmacological inhibition of USP30 or CRISPRi-mediated knockdown of USP30 did not impact dopaminergic neuronal activity, as assessed by electrophysiological profiling, but did potentiate depolarisation-induced mitophagy in primary and iPSC-derived neuronal cultures. We observed minimal changes in mitophagy levels in iPSC-derived dopaminergic neurons from Parkinsons patients with PINK1 or PRKN mutations that were independent of the ability to produce p65Ub. Importantly, within this experimental paradigm, pharmacological USP30 inhibition increased depolarisation-induced mitophagy in both PINK1 and PRKN patients to the same extent as control neurons. These results support a role for USP30 in modulating the trigger threshold for mitophagy and suggest that USP30 inhibitors may be beneficial in patients with impairments in PINK1/Parkin-mediated mitophagy.

neuroscience↗

bioRxiv2024

M1-linked Ubiquitination by LUBAC Regulates AMPK Activity and the Response to Energetic Stress

Methionine-1 (M1)-linked ubiquitin chains, assembled by the ubiquitin ligase LUBAC and cleaved by the deubiquitinase OTULIN, are critical regulators of inflammation and immune homeostasis. Genetic loss of either LUBAC or OTULIN causes autoinflammatory syndromes, which are associated with defects in glycogen and lipid metabolism. However, how LUBAC and OTULIN regulate metabolic signalling remains unknown. Here, we demonstrate that LUBAC promotes, while OTULIN restricts, activation of the key metabolic regulator AMP-activated protein kinase (AMPK) in cells, mice, and human samples. LUBAC and OTULIN interact with AMPK, control its M1-ubiquitination, and regulate its activation in response to glucose starvation and allosteric activation. During starvation, LUBAC deficiency impairs autophagy induction and hinders the shift from oxidative phosphorylation to glycolysis. Strikingly, LUBAC-deficient Drosophila have a strongly reduced survival rate after starvation. Our work identifies LUBAC and OTULIN as physiological regulators of AMPK, providing the first mechanism by which M1-linked ubiquitin chains regulate metabolic signalling.

cell biology↗

bioRxiv2024

Structural basis of gap-filling DNA synthesis in the nucleosome by DNA Polymerase β

Single-strand breaks (SSBs) are one of the most prevalent forms of DNA damage found in the chromatinized genome and are repaired by direct single-strand break repair (SSBR) or base excision repair (BER). DNA polymerase beta (Pol {beta}) is the primary enzyme responsible for processing the 1-nt gap intermediate in chromatin during SSBR and BER. However, the mechanism used by Pol {beta} to process a 1-nt gap in the context of the nucleosome and chromatin remains poorly understood. Here, we use biochemical assays and cryogenic electron microscopy (cryo-EM) to determine the kinetic and structural basis of gap-filling DNA synthesis in the nucleosome by Pol {beta}. Kinetic analysis identified that gap-filling DNA synthesis in the nucleosome by Pol {beta} is position-dependent, where solvent exposed 1-nt gaps are processed more efficiently than histone-occluded 1-nt gaps. A series of cryo-EM structures of Pol {beta} bound to a solvent-exposed 1-nt gap in the nucleosome reveal a global DNA sculpting mechanism for 1-nt gap recognition, which is mediated by sequential engagement of the Pol {beta} lyase domain and polymerase domain. Finally, cryo-EM structures of Pol {beta} bound to 1-nt gaps at two additional positions in the nucleosomal DNA define the structural basis for position-dependent nucleotide insertion in the nucleosome. This work establishes the mechanism used by Pol {beta} for processing 1-nt gaps in the nucleosome during SSBR and BER, providing fundamental insight into DNA repair in chromatin.

biochemistry↗

bioRxiv2024

Molecular basis for the activation of the Fatty Acid Kinase complex of Staphylococcus aureus

Gram-positive bacteria utilize a Fatty Acid Kinase (FAK) complex to harvest fatty acids from the environment. The complex, consisting of the fatty acid kinase, FakA, and an acyl carrier protein, FakB, is known to impact virulence and disease outcomes. However, FAKs structure and enzymatic mechanism remain poorly understood. Here, we used a combination of modeling, biochemical, and cell-based approaches to establish critical details of FAK activity. Solved structures of the apo and ligand-bound FakA kinase domain captured the protein state through ATP hydrolysis. Additionally, targeted mutagenesis of an understudied FakA Middle domain identified critical residues within a metal-binding pocket that contribute to FakA dimer stability and protein function. Regarding the complex, we demonstrated nanomolar affinity between FakA and FakB and generated computational models of the complexs quaternary structure. Together, these data provide critical insight into the structure and function of the FAK complex which is essential for understanding its mechanism.

microbiology↗

bioRxiv2024

Biochemical and Structural Characterization of Fapy·dG Replication by Human DNA Polymerase β

N6-(2-deoxy-,{beta}-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy*dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2-deoxyguanosine (8-OxodGuo). Fapy*dG preferentially gives rise to G [->] T transversions and G [->] A transitions. However, the molecular basis by which Fapy*dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase {beta} (Pol {beta}), a model mammalian polymerase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer terminus. When Fapy*dG is present in the template, Pol {beta} incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy*dGTP is a poor substrate but is incorporated [~]3-times more efficiently opposite dA than dC. Extension from Fapy*dG at the 3-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy*dG is likely to be the source of the mutagenic effects of the lesion and not Fapy*dGTP. These experiments increase our understanding of the promutagenic effects of Fapy*dG.

biochemistry↗

bioRxiv2023

Transcriptional memory is conferred by combined heritable maintenance and local removal of selective chromatin modifications

Interferon-{gamma} (IFN{gamma}) transiently activates genes involved in inflammation and innate immunity. A subset of targets maintain a mitotically heritable memory of prior IFN{gamma} exposure resulting in hyperactivation upon reexposure. Here we discovered that the active chromatin marks H3K4me1, H3K14Ac and H4K16Ac are established during IFN{gamma} priming and selectively maintained on a cluster of GBP genes for at least 7 days in dividing cells in the absence of transcription. The histone acetyltransferase KAT7 is required for the accelerated GBP reactivation upon reexposure to IFN{gamma}. In naive cells, we find the GBP cluster is maintained in low-level repressive chromatin marked by H3K27me3 limiting priming in a PRC2-dependent manner. Unexpectedly, IFN{gamma} results in transient accumulation of this repressive mark but is then selectively depleted from primed GBP genes during the memory phase facilitating hyperactivation of primed cells. Furthermore, we identified a cis-regulatory element that makes transient, long-range contacts across the GBP cluster and acts as a repressor, primarily to curb the hyperactivation of previously IFN{gamma}-primed cells. Combined our results identify the putative chromatin basis for long-term transcriptional memory of interferon signalling that may contribute to enhanced innate immunity.

molecular biology↗

bioRxiv2023

Multi-modal Proteomic Characterization of Lysosomal Function and Proteostasis in Progranulin-Deficient Neurons

Progranulin (PGRN) is a lysosomal protein implicated in various neurodegenerative diseases. Over 70 mutations discovered in the GRN gene all result in reduced expression of PGRN protein. However, the detailed molecular function of PGRN within lysosomes and the impact of PGRN deficiency on lysosomal biology remain unclear. Here we leveraged multifaceted proteomic techniques to comprehensively characterize how PGRN deficiency changes the molecular and functional landscape of neuronal lysosomes. Using lysosome proximity labeling and immuno-purification of intact lysosomes, we characterized lysosome compositions and interactomes in both human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons (i3Neurons) and mouse brains. Using dynamic stable isotope labeling by amino acids in cell culture (dSILAC) proteomics, we measured global protein half-lives in i3Neurons for the first time and characterized the impact of progranulin deficiency on neuronal proteostasis. Together, this study indicated that PGRN loss impairs the lysosomes degradative capacity with increased levels of v-ATPase subunits on the lysosome membrane, increased catabolic enzymes within the lysosome, elevated lysosomal pH, and pronounced alterations in neuron protein turnover. Collectively, these results suggested PGRN as a critical regulator of lysosomal pH and degradative capacity, which in turn influences global proteostasis in neurons. The multi-modal techniques developed here also provided useful data resources and tools to study the highly dynamic lysosome biology in neurons.

neuroscience↗

bioRxiv2021

A novel post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons

The causes of Parkinsons disease (PD) likely involve complex interactions between environmental factors and susceptibility genes with variants at the GBA locus encoding the glucocerebrosidase (GCase) enzyme being the strongest common genetic risk factor for PD. To understand GBA-related disease mechanisms, we used a novel multipart-enrichment proteomics and post-translational modification workflow to simultaneously identify peptides with phosphorylation, reversible cysteine-modifications or sialylated N-linked glycosylation, alongside unmodified proteins. We identified large numbers of dysregulated proteins and post-translational modifications (PTMs) in heterozygous GBA-N370S PD patient induced pluripotent stem cells (iPSC)-derived dopamine neurons. Alterations in glycosylation status of lysosomal proteins identified disturbances in the autophagy-lysosomal pathway, concurrent with upstream perturbations in mTOR phosphorylation and activity in GBA-N370S iPSC-dopamine neurons. In addition, the strategy revealed several native and modified proteins encoded by PD-associated genes to be dysregulated in GBA-N370S neurons, enhancing our understanding of the wider role of GBA mutations on the neuronal proteome. Integrated pathway analysis of all datasets revealed impaired neuritogenesis in GBA-N370S PD iPSC-dopamine neurons and identified tau (MAPT) as a key mediator of this process. Using a functional assay, we confirmed neurite outgrowth deficits in GBA-N370S PD neurons and a central role for tau in this process. Furthermore, pharmacological restoration of GCase activity in GBA-N370S PD patient neurons rescued the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate novel neurodegeneration-associated pathways and identify phenotypes and potential drug targets in complex disease models.

neuroscience↗