Biology subjects

McAlary, L.

Publications and source records attributed to McAlary, L..

10 recordsLinked to original sources

bioRxiv2026

The folding, dimerization and allosteric landscapes of the ALS protein SOD1: a comprehensive mutational atlas

Mutations in superoxide dismutase 1 (SOD1) can disturb monomer folding and dimerization, ultimately leading to the development of amyotrophic lateral sclerosis (ALS). Genotype-phenotype knowledge of SOD1 monomer folding and dimerization is largely incomplete, impeding our ability to treat and understand SOD1-ALS. To address this issue, we performed multidimensional deep mutational scanning of [~]6,000 SOD1 variants (amino acid substitutions, insertions, deletions), quantifying folded monomer abundance, and measuring wild-type-variant heterodimerization. Importantly, both abundance and heterodimerization capture pathogenicity, with heterodimerization being a slightly better classifier of pathogenic variants. Notably, [~]80% of pathogenic variants and [~]70% of variants of uncertain significance in SOD1 perturb both monomer abundance and heterodimerization, pointing to protein destabilization as a major driver of SOD1-ALS. Orthogonal validation in mammalian cells shows that protein abundance strongly correlates with other disease-relevant phenotypes, including protein aggregation and toxicity. Although monomer abundance and heterodimerization are strongly coupled, residual effects identify variants that perturb the heterodimer beyond their impact on monomer folding. Mapping residual effects uncovers potential allosteric sites that modulate dimer stability independently of monomer folding, revealing mechanistic insights and potential therapeutic entry points to stabilize SOD1 as a dimer. Together, these results demonstrate that multidimensional phenotype variant mapping improves mechanistic understanding of SOD1 variants and clinical variant interpretation, while uncovering structural targets for SOD1-ALS treatment.

biophysics↗

bioRxiv2026

Lipid-Coated Water-in-Oil Droplets as a Passivation-Free Platform for Cost-Effective Fluorescence Spectroscopy

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@199c622org.highwire.dtl.DTLVardef@9f195eorg.highwire.dtl.DTLVardef@13a8969org.highwire.dtl.DTLVardef@138692_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

biochemistry↗

bioRxiv2025

Small heat shock proteins HspB1 and HspB5 differentially alter the condensation and aggregation of the TDP-43 low complexity domain

TAR DNA-binding protein 43 (TDP-43) is a nucleic acid-binding protein that regulates processes of mRNA metabolism, during which it undergoes condensation mediated by its C-terminal low complexity domain (TDP-43LCD). TDP-43 aggregation and condensation are associated with neurodegenerative disease. However, the proteostasis mechanisms that regulate these processes remain elusive. Some evidence has shown that the molecular chaperone small heat shock protein HspB1 binds to and regulates the cytoplasmic phase separation of TDP-43, indicating that other small heat shock proteins may have similar effects. Here, we demonstrate divergent behaviours for HspB1 and its homolog HspB5 on TDP-43LCD condensation and aggregation. In addition to inhibiting TDP-43LCD aggregation, HspB1 partitions into TDP-43LCD condensates and increases the dynamic exchange of TDP-43LCD within condensates and with the surrounding solution. These effects of HspB1 are enhanced by mutations that mimic phosphorylation. HspB5 inhibits TDP-43LCD aggregation more effectively than HspB1 and partitions into TDP-43LCD condensates, where it delays pathological transition of the condensate to a gel/solid. We localise the chaperone effects of HspB1 and HspB5 to the N- and C-terminal regions of the protein, emphasising the role of sequence diversity in these regions in defining small heat shock protein function. These findings demonstrate that HspB1 and HspB5 are regulators of TDP-43 phase separation and aggregation and may be potential therapeutic targets in mitigating toxic TDP-43 aggregation in neurodegenerative disease. StatementThis work describes how two small heat shock proteins (proteins that bind to misfolded proteins) impact the condensation and aggregation of TDP-43, a protein implicated in most cases of amyotrophic lateral sclerosis. In doing so, it highlights their divergent behaviours and therapeutic potential.

biochemistry↗

bioRxiv2025

Subcellular mass spectrometry imaging of lipids and nucleotides using transmission geometry ambient laser desorption and plasma ionisation

Demand for mass spectrometry imaging (MSI) technologies offering subcellular resolution for tissues and cell imaging is rapidly increasing. To accomplish this, efficient analyte ionisation is essential, given the small amounts of material sample in each pixel. Herein, we describe an atmospheric pressure transmission-geometry matrix-assisted laser desorption source equipped with plasma ionisation. By utilising a novel pre-staining method for sample preparation lipid signal intensities were enhanced by an order of magnitude compared to conventional matrix-only methods, while serendipitously enabling imaging of numerous nucleotides. The system enables detection of up to 200 lipids and nucleotides in tissues at 1 {micro}m-pixel size while informative MSI data is still obtained down to 250 nm pixel size. The use of sub-micron pixels is shown to discern subcellular features through combinations with fluorescence microscopy. This method provides a powerful tool for cellular and sub-cellular imaging of small molecules from tissues and cells for spatial biology applications.

cell biology↗

bioRxiv2023

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified throughout the primary sequence, indicating that common structural features responsible for aggregation and toxicity may be present. Here, we used in silico tools to predict amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding 7 regions spread throughout the protein structure. We found that the introduction of proline residues into {beta}-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in living cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in {beta}-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that {beta}-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

molecular biology↗

bioRxiv2021

A copper chaperone-mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS)-associated mutations in Cu/Zn superoxide dismutase (SOD1) reduce folding stability, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS, however, the results have been mixed. Here, we utilise a small-molecule polytherapy of CuATSM and ebselen to mimic the metal delivery and disulfide bond promoting activity of SOD1s cellular chaperone, the copper chaperone for SOD1 (CCS). We find that polytherapy using CuATSM and ebselen is highly effective at reducing inclusion formation in a cell model of SOD1 aggregation, reduces mutant SOD1-associated cell death, and promotes effective maturation of SOD1 beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may be an effective method of treating SOD1-associated ALS.

biochemistry↗

bioRxiv2020

Tryptophan residues in TDP-43 and SOD1 mediate the cross-seeding and toxicity of SOD1

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated wild-type TAR DNA-binding protein 43 (TDP-43). Co-expression of mutant or wild-type TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein G85R-GFP in HEK293FT cells, and promotes synergistic axonopathy in zebrafish. This pathological interaction is dependent upon natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce wild-type SOD1 misfolding in HEK293FT cells, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in HEK293FT cells, and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

cell biology↗

bioRxiv2020

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal-like cells

Protein aggregation that results in the formation of inclusions is strongly correlated with neuronal death and is a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Huntingtons disease. Cells are thought to dramatically up-regulate the levels of heat shock proteins during periods of cellular stress via induction of the heat shock response (HSR). Heat shock proteins are well-characterised molecular chaperones that interact with aggregation-prone proteins to either stabilise, refold, or traffic protein for degradation. The reason why heat shock proteins are unable to maintain the solubility of particular proteins in neurodegenerative disease is unknown. We sought to determine whether neurodegenerative disease-associated protein aggregates can induce the HSR. Here, we generated a neuroblastoma cell line that expresses a fluorescent reporter under conditions of HSR induction, for example heat shock. Using these cells, we show that the HSR is not induced by exogenous treatment with aggregated forms of Parkinsons disease-associated -synuclein or the ALS-associated G93A mutant of superoxide dismutase-1 (SOD1G93A). Furthermore, flow cytometric analysis revealed that intracellular expression of SOD1G93A or a pathogenic form of polyQ-expanded huntingtin (Htt72Q), similarly, results in no or low induction of the HSR. In contrast, expression of a non-pathogenic but aggregation-prone form of firefly luciferase (Fluc) did induce an HSR in a significantly greater proportion of cells. Finally, we show that HSR induction is dependent on the intracellular levels of the aggregation-prone proteins, but the pathogenic proteins (SOD1G93A and Htt72Q) elicit a significantly lower HSR compared to the non-pathogenic proteins (Fluc). These results suggest that pathogenic proteins either evade detection or impair induction of the HSR in neuronal-like cells. Therefore, defective HSR induction may facilitate the initiation of protein aggregation leading to inclusion formation in neurodegenerative diseases.

cell biology↗

bioRxiv2018

In Silico Determined Properties of Designed Superoxide Dismutase-1 Mutants Predict ALS-like Phenotypes In Vitro and In Vivo

The underlying physical causes of SOD1-related ALS are still not well-understood. We address this problem here by computationally designing two de novo mutants, A89R and K128N, which were predicted theoretically to be either significantly destabilizing or stabilizing respectively. We subjected these in silico designed mutants to a series of experimental tests, including in vitro measures of thermodynamic stability, cell-based aggregation and toxicity assays, and an in vivo developmental model of zebrafish motor neuron axonopathy. The experimental tests validated the theoretical predictions: A89R is an unstable, highly-deleterious mutant, and K128N is a stable, non-toxic mutant. Moreover, K128N is predicted computationally to form an unusually stable heterodimer with the familial ALS mutant A4V. Consistent with this prediction, co-injection of K128N and A4V into zebrafish shows profound rescue of motor neuron pathology. The demonstrated success of these first principles calculations to predict the physical properties of SOD1 mutants holds promise for rationally designed therapies to counter the progression of ALS.\n\nSignificanceMutations in the protein superoxide dismutase cause ALS, and many of these mutants have decreased folding stability. We sought to pursue this thread using a synthetic biology approach, where we designed two de novo mutations, one stabilizing and one destabilizing, as predicted using computational molecular dynamics simulations. We then tested these mutants using in vitro, cell-based, and in vivo zebrafish models. We found that the unstable mutant was toxic, and induced a severe ALS phenotype in zebrafish; the predicted stable mutant, on the other hand, behaved even better than WT. In fact, it was able to rescue the ALS phenotype caused by mutant SOD1. We propose a mechanism for this rescue, which may provide an avenue for therapeutic intervention.

biophysics↗

bioRxiv2017

SOD1A4V aggregation alters ubiquitin homeostasis in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving the selective death of upper and lower motor neurons in the primary motor cortex and spinal cord. A hallmark of ALS pathology is the accumulation of ubiquitinated protein inclusions within motor neurons. Previous studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here we show that Ub homeostasis is altered in a SOD1 cell model of ALS. Utilising fluorescently tagged Ub, we followed the distribution of Ub in living cells expressing SOD1 and show that Ub is present at the earliest stages of SOD1 aggregation. We also report that cells containing aggregates of mutant SOD1 have greater ubiquitin-proteasome system (UPS) dysfunction as measured by the accumulation of the fluorescent proteasome reporter tdTomatoCL1. Furthermore, SOD1 aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that mutant SOD1 is associated with a shift of Ub to a pool of supersaturated proteins including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.

molecular biology↗