Biology subjects

Yerbury, J. J.

Publications and source records attributed to Yerbury, J. J..

6 recordsLinked to original sources

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

Differential inclusion formation of an aggregation-prone protein reveals differences in the proteostasis capacity of neuronal cell lines

Maintaining proteome integrity is essential for cellular function and survival. Disruptions in proteostasis lead to the aggregation of proteins into inclusions, a process that underlies many neurodegenerative diseases. To quantitatively assess the proteostasis capacity of neuronal cells, we employed an aggregation-prone double mutant form of firefly luciferase (denoted FlucDM) as a reporter protein. We compared two commonly used neuronal cell lines, mouse neuroblastoma cells (Neuro-2a) and a motor neuron-like hybrid line (NSC-34), to evaluate their ability to prevent the aggregation of proteins into intracellular inclusions. We observed a significantly greater propensity of FlucDM to form inclusions in NSC-34 cells compared to Neuro-2a cells. This suggests a reduced capacity of NSC-34 cells for managing aggregation-prone proteins. Proteomic profiling of FlucDM inclusions purified from both cell types revealed cell-type-specific engagement of the proteostasis machinery with aggregation-prone proteins. Comparing the proteomic profiles of key arms of the proteostasis network between these two cell lines revealed that the endoplasmic reticulum (ER) unfolded protein response is differentially expressed. This study establishes a quantitative platform for assessing cellular proteostasis capacity and underscores the importance of cell-type context in proteome maintenance. These insights have implications for understanding the selective vulnerability of neurons in protein misfolding disorders.

cell biology↗

bioRxiv2021

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. Yet the exact processes that normally maintain proteostasis, but that are uniquely disturbed in motor neurons expressing diverse genetic mutations, remain to be established. Obtaining a better understanding of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/461468v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1d3a6c3org.highwire.dtl.DTLVardef@697319org.highwire.dtl.DTLVardef@1038930org.highwire.dtl.DTLVardef@123eb5b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDestabilised firefly luciferase (Fluc) mutants can be used in high-content analysis (HCA) assay format C_LIO_LIFluc HCA assay enables information-rich reporting on chaperone network activity in cell models of ALS C_LIO_LIExpression of SOD1A4V reduces chaperone network activity in NSC-34 cells C_LI

cell 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

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↗

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↗