Biology subjects

Marzano, N. R.

Publications and source records attributed to Marzano, N. R..

9 recordsLinked to original sources

bioRxiv2026

Single-molecule visualisation of human Hsp70-driven conformational remodelling during stress

The Hsp70 chaperone system plays a central role in the folding of nascent polypeptides and in preventing protein misfolding and aggregation during cellular stress. However, the precise mechanism by which the human Hsp70, HspA8, remodels the conformations of individual chemically misfolded clients remains unclear. Moreover, it is unknown whether this mechanism differs from that used by Hsp70 to engage clients during heat stress to preserve native function. To address these questions, we report here the use of single-molecule fluorescence resonance energy transfer (smFRET) to temporally interrogate how the human Hsp70 system regulates the conformation of a heat-sensitive client protein, firefly luciferase (Fluc), following chemical denaturation and during heat stress. We find that Hsp70 recognises both chemically denatured and heat-induced misfolded states of Fluc and resolves them by conformational expansion. Release from a Hsp70-bound state, a process driven by the nucleotide exchange factor, Hsp110, guides Fluc toward productive folding trajectories that would otherwise be unlikely to occur spontaneously following collapse from a conformationally unfolded state. Moreover, we demonstrate that both temperature and the conformational state of misfolded Fluc dictate the ability of HspA8 to meaningfully resolve non-native structure within the protein. Collectively, this work provides direct visualisation of the mechanisms by which Hsp70 modulates client conformations under diverse stress conditions to preserve proteome integrity.

biophysics↗

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

Direct single-molecule visualization of Hsp90-mediated relief of a Hsp70-folding block

Hsp70 and Hsp90 are ubiquitous molecular chaperones that cooperate to promote the correct folding and maturation of client proteins. Despite their central role in proteostasis, the molecular mechanisms by which Hsp90 coordinates with Hsp70 to remodel clients remain poorly understood. In particular, how ATP hydrolysis by Hsp90 is coupled to client engagement and conformational change has been a long-standing question. This gap in understanding is largely due to the challenge of visualizing client conformations during the highly dynamic and heterogeneous interactions with the Hsp70/Hsp90 chaperone machinery. To address this, we used a combination of single-molecule fluorescence resonance energy transfer (smFRET) and total internal reflection fluorescence microscopy to observe individual firefly luciferase client proteins as they are sequentially engaged by Hsp70 and Hsp90. Here, we show that Hsp90 reduces rebinding of Hsp70 to folding intermediates while still allowing engagement to misfolded clients, thereby enabling productive refolding in the presence of typically inhibitory concentrations of Hsp70. Furthermore, Hsp90 uses ATP binding and hydrolysis to actively remodel the conformational landscape of the client, promoting controlled folding through localized compaction across multiple regions. These controlled folding events reduce misfolding and are essential for establishing native interdomain contacts. Using smFRET and kinetic simulations, we further demonstrate that heterogeneous Hsp70 binding generates region-specific folding kinetics and conformational dynamics, which are likely driven by variations in the number of available Hsp70-binding sites. This is then exploited during Hsp70/Hsp90-mediated folding to support localized folding of client subdomains, thereby reducing non-native interactions from distal regions to facilitate proper folding of multi-domain proteins.

biophysics↗

bioRxiv2025

Competing chaperone pathways in α-synuclein disaggregation and aggregation dynamics

The aggregation of the protein -synuclein into amyloid fibrils and their subsequent deposition into large proteinaceous inclusions is a pathological hallmark of several neurodegenerative diseases, including Parkinsons disease. Molecular chaperones, including the small heat shock proteins (sHsps) and the Hsp70 chaperone system, are known to interact with -synuclein fibrils, preventing further aggregation and disaggregating fibrillar species respectively. However, it remains unclear if sHsps co-operate with the Hsp70 chaperones to potentially improve the kinetics or effectiveness of Hsp70-mediated disaggregation and how disaggregation kinetics are influenced by aggregation-prone -synuclein monomers. Using thioflavin-T assays, we demonstrate that the sHsps Hsp27 (HSPB1) and B-crystallin (HSPB5) do not synergise with the Hsp70 chaperones during -synuclein seed fibril disaggregation. Moreover, the addition of monomeric -synuclein with fibril seeds results in increased aggregation that overwhelms Hsp70-mediated disaggregation. Upon addition of sHsps to this system, antagonism between the two chaperone classes is observed, likely due to these chaperones competing for productive binding to the ends of -synuclein fibrils. Overall, these results suggest that while Hsp70 and sHsp chaperones are independently capable of binding to and inhibiting fibril elongation, they do not have synergistic effects on disaggregation. Furthermore, Hsp70-mediated disaggregation is ineffectual in the presence of physiological concentrations of -synuclein monomers, conditions that actually lead to further -synuclein aggregation. Overall, these data may offer insight into factors that lead to the failure of the Hsp70 chaperones to clear cells of -synuclein aggregates that leads to neurodegenerative disease.

biochemistry↗

bioRxiv2024

Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons

Sensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.

neuroscience↗

bioRxiv2024

Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Pentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins that recognize specific ssRNA target sequences. There is significant interest in developing designer PPRs for use in diagnostics or as tools to detect and localize target RNA sequences. However, it is unclear how PPRs search for target sequences within complex transcriptomes and current models to predict PPR binding sites struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR (dPPR10) bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We demonstrate that longer RNA sequences were significantly slower to bind (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR10. Importantly, dPPR10 does not associate with non-target flanking sequences, binding specifically to its target sequence within longer ssRNA species. This data provides evidence that PPRs have limited to no capacity to scan RNA transcripts for target sequences and instead rely on diffusion for cognate searching. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus). These findings will inform improved prediction of PPR binding sites for the development of designer PPRs. SummaryPentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be designed to bind to a specific ssRNA nucleobase and thus any RNA sequence of interest. As such, there is substantial interest in developing designer PPRs for a range of biotechnology applications, including diagnostics or in vivo localisation of RNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. As such, we combined structure-based and single- molecule approaches and determined that PPRs bind only to their target sequences (i.e., they do not associate with non-target RNA sequences) and do not scan longer RNA oligonucleotides for the target sequence. Instead, target searching appears kinetically-constrained by random three-dimensional diffusion, providing an explanation as to why PPRs are found almost exclusively in organelle compartments that typically have smaller transcriptomes. Collectively, this work identifies several key considerations for future designer PPR developments.

biochemistry↗

bioRxiv2024

Single-molecule observations of human small heat shock proteins in complex with aggregation-prone client proteins

Small heat shock proteins (sHsps) are molecular chaperones that act to prevent the aberrant aggregation of misfolded proteins. Whilst it is widely suggested that sHsps prevent aggregation by binding to misfolded client proteins, the dynamic and heterogeneous nature of sHsps has hindered attempts to establish the mechanistic details of how sHsp-client protein complexes form. Single-molecule approaches have emerged as a powerful tool to investigate dynamic and heterogeneous interactions such as those that can occur between sHsps and their client proteins. Here, we use total internal reflection fluorescence microscopy to observe and characterise the complexes formed between model aggregation-prone client proteins [firefly luciferase (FLUC), rhodanese, and chloride intracellular channel 1 protein (CLIC)], and the human sHsps B-crystallin (B-c; HSPB1) and Hsp27 (HSPB5). We show that small (monomeric or dimeric) forms of both B-c and Hsp27 bind to misfolded or oligomeric forms of the client proteins at early stages of aggregation, resulting in the formation of soluble sHsp-client complexes. Stoichiometric analysis of these complexes revealed that additional B-c subunits accumulate onto pre-existing sHsp-client complexes to form larger species - this does not occur to the same extent for Hsp27. Instead, Hsp27-client interactions tend to be more transient than those of B-c. Elucidating these mechanisms of sHsp function is crucial to our understanding of how these molecular chaperones act to inhibit protein aggregation and maintain cellular proteostasis.

molecular biology↗

bioRxiv2020

An α-cyanostilbene derivative for the enhanced detection and imaging of amyloid fibril aggregates

The aggregation of proteins into amyloid fibrils has been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease. Benzothiazole dyes such as Thioflavin T (ThT) are well characterised and widely used fluorescent probes for monitoring amyloid fibril formation. However, existing dyes lack sensitivity and specificity to oligomeric intermediates formed during fibril formation. In this work we describe the use of an α-cyanostilbene derivative with aggregation-induced emission properties (called ASCP) as a fluorescent probe for the detection of amyloid fibrils. Similar to ThT, ASCP is fluorogenic in the presence of amyloid fibrils and upon binding and excitation at 460 nm produces a red-shifted emission with a large Stokes shift of 145 nm. ASCP has a higher binding affinity to fibrillar α-synuclein than ThT and likely shares the same binding sites to amyloid fibrils. Importantly, ASCP was found to also be fluorogenic in the presence of amorphous aggregates and can detect oligomeric species formed early during aggregation. Moreover, ASCP can be used to visualise fibrils via Total Internal Reflection Fluorescence (TIRF) microscopy and, due to its large Stokes shift, simultaneously monitor the fluorescence emission of other labelled proteins following excitation with the same laser used to excite ASCP. Consequently, ASCP possesses enhanced and unique spectral characteristics compared to ThT that make it a promising alternative for the in vitro study of amyloid fibrils and the mechanisms by which they form.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Single-molecule fluorescence-based approach reveals novel mechanistic insights into small heat shock protein chaperone function

Small heat shock proteins (sHsps) are a family of ubiquitous intracellular molecular chaperones that are up-regulated under stress conditions and play a vital role in protein homeostasis (proteostasis). It is commonly accepted that these chaperones work by trapping misfolded proteins to prevent their aggregation, however fundamental questions regarding the molecular mechanism by which sHsps interact with misfolded proteins remain unanswered. Traditionally, it has been difficult to study sHsp function due to the dynamic and heterogenous nature of the species formed between sHsps and aggregation-prone proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic protein complexes and we have therefore developed a novel single-molecule fluorescence-based approach to observe the chaperone action of human B-crystallin (Bc, HSPB5). Using this approach we have, for the first time, determined the stoichiometries of complexes formed between Bc and a model client protein, chloride intracellular channel 1 (CLIC1). By examining the polydispersity and stoichiometries of these complexes over time, and in response to different concentrations of Bc, we have uncovered unique and important insights into a two-step mechanism by which Bc interacts with misfolded client proteins to prevent their aggregation. Understanding this fundamental mechanism of sHsp action is crucial to understanding how these molecular chaperone function to protect the cell from protein misfolding and their overall role in the cellular proteostasis network.

biochemistry↗