Search

Molecular chaperones from the Hsp70 family can break up protein aggregates, including amyloids. Here, the authors utilize microfluidic diffusional sizing to assess the mechanism of α-synuclein (αS) disaggregation by the Hsc70–DnaJB1–Apg2 system, and show that single αS molecules are removed directly from the fibril ends.

Abstract Misfolded protein oligomers are increasingly recognized as highly cytotoxic agents in a wide range of human disorders associated with protein aggregation. In this study, we assessed the possible uptake and resulting toxic effects of model protein oligomers administered to C. elegans through the culture medium. We used an automated machine-vision, high-throughput screening procedure to monitor the phenotypic changes in the worms, in combination with confocal microscopy to monitor the diffusion of the oligomers, and oxidative stress assays to detect their toxic effects. Our results suggest that the oligomers can diffuse from the intestinal lumen to other tissues, resulting in a disease phenotype. We also observed that pre-incubation of the oligomers with a molecular chaperone (αB-crystallin) or a small molecule inhibitor of protein aggregation (squalamine), reduced the oligomer absorption. These results indicate that exogenous misfolded protein oligomers can be taken up by the worms from their environment and spread across tissues, giving rise to pathological effects in regions distant from their place of absorbance.

Joshi et al. identify two human metabolites, carnosine and kynurenic acid, that rescue a C. elegans model of Alzheimer’s disease by inhibiting the aggregation of the amyloid beta peptide in vivo. They find that these metabolites trigger a cytosolic unfolded protein response through the transcription factor HSF-1 and molecular chaperones DNJ-12 and DNJ-19, thus providing mechanistic links between metabolite homeostasis and protein homeostasis to further insights into interventions against neurodegenerative diseases.

Proteins from hyperthermophilic organisms are evolutionary optimised to adopt functional structures and dynamics under conditions in which their mesophilic homologues are generally inactive or unfolded. Understanding the nature of such adaptation is of crucial interest to clarify the underlying mechanisms of biological activity in proteins. Here we measured NMR residual dipolar couplings of a hyperthermophilic acylphosphatase enzyme at 80°C and used these data to generate an accurate structural ensemble representative of its native state. The resulting energy landscape was compared to that obtained for a human homologue at 37°C, and additional NMR experiments were carried out to probe fast (15N relaxation) and slow (H/D exchange) backbone dynamics, collectively sampling fluctuations of the two proteins ranging from the nanosecond to the millisecond timescale. The results identified key differences in the strategies for protein-protein and protein-ligand interactions of the two enzymes at the respective physiological temperatures. These include the dynamical behaviour of a β-strand involved in the protection against aberrant protein aggregation and concerted motions of loops involved in substrate binding and catalysis. Taken together these results elucidate the structure-dynamics-function relationship associated with the strategies of thermal adaptation of protein molecules.

The self-assembly of α-synuclein (αS) is a pathological feature of Parkinson’s disease. The αS species responsible for neuronal damage are not well characterized. Here, the authors show that αS fibrils release soluble prefibrillar oligomeric species responsible for neurotoxicity in vitro.

Cataldi et al. investigates the impact of the dopamine derivative DOPAL on the Aβ peptide oligomer formation. They report that DOPAL promotes the formation of stable Aβ oligomers that exert toxicity on neuroblastoma cells by increasing cytosolic calcium levels and generating reactive oxygen species. This study connects Aβ aggregation with processes regulating cellular homeostasis in the brain.

Promising treatments for neurogenerative disorders may involve targeting kinetic intermediates, including α-synuclein oligomers. Here a kinetic method for quantifying oligomer populations is used to screen small molecule inhibitors of oligomerisation and gain mechanistic insight into their modes of action.

Mitochondria are organelles with their own genomes, but they rely on the import of nuclear-encoded proteins that are translated by cytosolic ribosomes. Therefore, it is important to understand whether failures in the mitochondrial uptake of these nuclear-encoded proteins can cause proteotoxic stress and identify response mechanisms that may counteract it. Here, we report that upon impairments in mitochondrial protein import, high-risk precursor and immature forms of mitochondrial proteins form aberrant deposits in the cytosol. These deposits then cause further cytosolic accumulation and consequently aggregation of other mitochondrial proteins and disease-related proteins, including α-synuclein and amyloid β. This aggregation triggers a cytosolic protein homeostasis imbalance that is accompanied by specific molecular chaperone responses at both the transcriptomic and protein levels. Altogether, our results provide evidence that mitochondrial dysfunction, specifically protein import defects, contributes to impairments in protein homeostasis, thus revealing a possible molecular mechanism by which mitochondria are involved in neurodegenerative diseases.

Abstract Protein aggregation is a pathological feature of neurodegenerative disorders. We previously demonstrated that protein inclusions in the brain are composed of supersaturated proteins, which are abundant and aggregation-prone, and form a metastable subproteome. It is not yet clear, however, whether this phenomenon is also associated with non-neuronal protein conformational disorders. To respond to this question, we analyzed proteomic datasets from biopsies of patients with genetic and acquired protein aggregate myopathy (PAM) by quantifying the changes in composition, concentration and aggregation propensity of proteins in the fibers containing inclusions and those surrounding them. We found that a metastable subproteome is present in skeletal muscle from healthy patients. The expression of this subproteome escalate as proteomic samples are taken more proximal to the pathologic inclusion, eventually exceeding its solubility limits and aggregating. While most supersaturated proteins decrease or maintain steady abundance across healthy fibers and inclusion-containing fibers, proteins within the metastable subproteome rise in abundance, suggesting that they escape regulation. Taken together, our results show in the context of a human conformational disorder that the supersaturation of a metastable subproteome underlies widespread aggregation and correlates with the histopathological state of the tissue.

Amyloid beta (Aβ42) peptides form heterogeneous mixtures of aggregates, which are closely linked to Alzheimer’s disease. This study shows how different types of Aβ42 aggregates are associated with distinct mechanisms of toxicity

Transient oligomeric species of the amyloid-β peptide (Aβ42) have been identified as key pathogenic agents in Alzheimer’s disease. Here the authors find that the aminosterol trodusquemine enhances Aβ42 aggregation and suppresses Aβ42-induced toxicity by displacing oligomers from cell membranes.

The aberrant aggregation of proteins is a key molecular event in the development and progression of a wide range of neurodegenerative disorders. We have shown previously that squalamine and trodusquemine, two natural products in the aminosterol class, can modulate the aggregation of the amyloid-β peptide (Aβ) and of α-synuclein (αS), which are associated with Alzheimer’s and Parkinson’s diseases. In this work, we expand our previous analyses to two squalamine derivatives, des-squalamine and α-squalamine, obtaining further insights into the mechanism by which aminosterols modulate Aβ and αS aggregation. We then characterize the ability of these small molecules to alter the physicochemical properties of stabilized oligomeric species in vitro and to suppress the toxicity of these aggregates to varying degrees toward human neuroblastoma cells. We found that, despite the fact that these aminosterols exert opposing effects on Aβ and αS aggregation under the conditions that we tested, the modifications that they induced to the toxicity of oligomers were similar. Our results indicate that the suppression of toxicity is mediated by the displacement of toxic oligomeric species from cellular membranes by the aminosterols. This study, thus, provides evidence that aminosterols could be rationally optimized in drug discovery programs to target oligomer toxicity in Alzheimer’s and Parkinson’s diseases.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Summary: The phenomenon of protein misfolding and aggregation is associated with a wide range of neurodegenerative conditions that cause progressive loss of function in specific regions of the human brain. To understand the causes of the selective cell and tissue vulnerability to the formation of these deposits, we analyzed the ability of different cell and tissue types to respond, in the absence of disease, to the presence of high levels of aggregation-prone proteins. By performing a transcriptional analysis, we found that the protein homeostasis system that regulates protein aggregation is weaker in neurons than in other cell types and in brain tissues than in other body tissues. These results suggest that the intrinsic level of regulation of protein aggregation in the healthy state is correlated with the selective vulnerability of cells and tissues to protein misfolding diseases. : Molecular Physiology; Neuroscience; Transcriptomics Subject Areas: Molecular Physiology, Neuroscience, Transcriptomics

α-Synuclein (αS) is a presynaptic protein whose aggregation is associated with Parkinson’s disease (PD). Although the physiological function of αS is still unclear, several lines of evidence indicate that this protein may play a role in the trafficking of synaptic vesicles (SVs) during neurotransmitter release, a task associated with its ability to bind SVs and promote their clustering. It is therefore crucial to identify the cellular factors that modulate this process. To address this question, using nuclear magnetic resonance (NMR) spectroscopy we have characterized the role of cholesterol, a major component of the membrane of SVs, in the binding of αS with synaptic-like vesicles. Our results indicate that cholesterol can act as a modulator of the overall affinity of αS for SVs by reducing the local affinity of the region spanning residues 65–97 in the non-amyloid-β component (NAC) of the protein. The increased population of bound states that expose the region 65–97 to the solvent was found to induce stronger vesicle-vesicle interactions by αS. These results provide evidence that cholesterol modulates the clustering of synaptic vesicles induced by (α)S, and supports the role of the disorder-to-order equilibrium of the NAC region in the modulation of the biological properties of the membrane-bound state of αS.

A wide variety of oligomeric structures are formed during the aggregation of proteins associated with neurodegenerative diseases. Such soluble oligomers are believed to be key toxic species in the related disorders; therefore, identification of the structural determinants of toxicity is of upmost importance. Here, we analysed toxic oligomers of α-synuclein and its pathological variants in order to identify structural features that could be related to toxicity and found a novel structural polymorphism within G51D oligomers. These G51D oligomers can adopt a variety of β-sheet-rich structures with differing degrees of α-helical content, and the helical structural content of these oligomers correlates with the level of induced cellular dysfunction in SH-SY5Y cells. This structure–function relationship observed in α-synuclein oligomers thus presents the α-helical structure as another potential structural determinant that may be linked with cellular toxicity in amyloid-related proteins.

Manual sample deposition on a substrate can introduce artifacts in quantitative AFM measurements. Here the authors present a microfluidic spray device for reliable deposition of subpicoliter droplets which dry out in milliseconds after landing on the surface, thereby avoiding protein self-assembly.

Many neurodegenerative disorders, including Alzheimer's, Parkinson's and the prion diseases, are characterized by a conformational conversion of normally soluble proteins or peptides into pathological species, by a process of misfolding and self-assembly that leads ultimately to the formation of amyloid fibrils. Recent studies support the idea that multiple intermediate species with a wide variety of degrees of neuronal toxicity are generated during such processes. The development of a high level of knowledge of the nature and structure of the pathogenic amyloid species would significantly enhance efforts to underline the molecular origins of these disorders and also to develop both accurate diagnoses and effective therapeutic interventions for these types of conditions. In this review, we discuss recent biophysical and structural information concerning different types of amyloid aggregates and the way in which such information can guide rational therapeutic approaches designed to target specific pathogenic events that occur during the development of these highly debilitating and increasingly common diseases.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid β (Aβ) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer’s and Parkinson’s diseases, respectively, a process that is intimately linked to the diseases’ progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aβ (Aβ40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aβ40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aβ40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt β-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aβ40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

One potential therapeutic strategy for Alzheimer’s disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-β peptide (Aβ); and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.

Abstract It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.

Silk fibres currently used in biotechnology are chemically reconstituted silk fibroins (RSF), which are more stable than native silk fibroin (NSF) but possess different biophysical properties. Here, the authors use microfluidic droplets to encapsulate and store NSF, preserving their native structure.

Abstract Background The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson’s disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. Results We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. Conclusions The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.

Neurodegenerative disorders progress across the brain in characteristic spatio-temporal patterns. A better understanding of the factors underlying the specific cell and tissue vulnerability responsible for such patterns could help identify the molecular origins of these conditions. To investigate these factors, based on the observation that neurodegenerative disorders are closely associated with the presence of aberrant protein deposits, we made the hypothesis that the vulnerability of cells and tissues is associated to the overall levels of supersaturated proteins, which are those most metastable against aggregation. By analyzing single-cell transcriptomic and subcellular proteomics data on healthy brains of ages much younger than those typical of disease onset, we found that the most supersaturated proteins are enriched in cells and tissues that succumb first to neurodegeneration. Then, by focusing the analysis on a metastable subproteome specific to Alzheimer's disease, we show that it is possible to recapitulate the pattern of disease progression using data from healthy brains. We found that this metastable subproteome is significantly enriched for synaptic processes and mitochondrial energy metabolism, thus rendering the synaptic environment dangerous for aggregation. The present identification of protein supersaturation as a signature of cell and tissue vulnerability in neurodegenerative disorders could facilitate the search for effective treatments by providing clearer points of intervention.

Removing or preventing the formation of [Formula: see text]-synuclein aggregates is a plausible strategy against Parkinson’s disease. To this end, we have engineered the [Formula: see text]-wrapin AS69 to bind monomeric [Formula: see text]-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of [Formula: see text]-synuclein and formation of visible [Formula: see text]-synuclein aggregates. In flies, AS69 reduced [Formula: see text]-synuclein aggregates and the locomotor deficit resulting from [Formula: see text]-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-[Formula: see text]-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes.

ABSTRACT A wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formation in vitro, in a model system of two common but unrelated biofilm-forming proteins, FapC from Pseudomonas fluorescens and CsgA from Escherichia coli, we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs. IMPORTANCE Biofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm's function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.

Although the aggregation of the amyloid-β peptide (Aβ) into amyloid fibrils is a well-established hallmark of Alzheimer's disease, the complex mechanisms linking this process to neurodegeneration are still incompletely understood. The nematode worm C. elegans is a valuable model organism through which to study these mechanisms because of its simple nervous system and its relatively short lifespan. Standard Aβ-based C. elegans models of Alzheimer's disease are designed to study the toxic effects of the overexpression of Aβ in the muscle or nervous systems. However, the wide variety of effects associated with the tissue-level overexpression of Aβ makes it difficult to single out and study specific cellular mechanisms related to the onset of Alzheimer's disease. Here, to better understand how to investigate the early events affecting neuronal signalling, we created a C. elegans model expressing Aβ42, the 42-residue form of Aβ, from a single-copy gene insertion in just one pair of glutamatergic sensory neurons, the BAG neurons. In behavioural assays, we found that the Aβ42-expressing animals displayed a subtle modulation of the response to CO2, compared to controls. Ca2+ imaging revealed that the BAG neurons in young Aβ42-expressing nematodes were activated more strongly than in control animals, and that neuronal activation remained intact until old age. Taken together, our results suggest that Aβ42-expression in this very subtle model of AD is sufficient to modulate the behavioural response but not strong enough to generate significant neurotoxicity, suggesting that slightly more aggressive perturbations will enable effectively studies of the links between the modulation of a physiological response and its associated neurotoxicity.

The Wnt signalling pathway plays an important role in cell proliferation, differentiation, and fate decisions in embryonic development and the maintenance of adult tissues. The twelve armadillo (ARM) repeat-containing protein β-catenin acts as the signal transducer in this pathway. Here, we investigated the interaction between β-catenin and the intrinsically disordered transcription factor TCF7L2, comprising a very long nanomolar-affinity interface of approximately 4800 Å2 that spans ten of the twelve ARM repeats of β-catenin. First, a fluorescence reporter system for the interaction was engineered and used to determine the kinetic rate constants for the association and dissociation. The association kinetics of TCF7L2 and β-catenin were monophasic and rapid (7.3 ± 0.1 × 107 M−1·s−1), whereas dissociation was biphasic and slow (5.7 ± 0.4 × 10−4 s−1, 15.2 ± 2.8 × 10−4 s−1). This reporter system was then combined with site-directed mutagenesis to investigate the striking variability in the conformation adopted by TCF7L2 in the three different crystal structures of the TCF7L2–β-catenin complex. We found that the mutation had very little effect on the association kinetics, indicating that most interactions form after the rate-limiting barrier for association. Mutations of the N- and C-terminal subdomains of TCF7L2 that adopt relatively fixed conformations in the crystal structures had large effects on the dissociation kinetics, whereas the mutation of the labile sub-domain connecting them had negligible effect. These results point to a two-site avidity mechanism of binding with the linker region forming a “fuzzy” complex involving transient contacts that are not site-specific. Strikingly, the two mutations in the N-terminal subdomain that had the largest effects on the dissociation kinetics showed two additional phases, indicating partial flux through an alternative dissociation pathway that is inaccessible to the wild type. The results presented here provide insights into the kinetics of the molecular recognition of a long intrinsically disordered region with an elongated repeat-protein surface, a process found to involve parallel routes with sequential steps in each.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-021-01680-7

Many super-resolution imaging techniques use fluorescence emission intensity to obtain precise positional information, but other spectral information is ignored. Here, the authors develop a method that records the spectrum and position of single dye molecules to map the hydrophobicity of a surface.

α-synuclein, a protein associated to Parkinson's disease, is involved in synaptic vesicle interaction and assembly. Here, the authors use NMR spectroscopy and super-resolution microscopy to unveil the nature and molecular mechanism of α-synuclein-mediated synaptic vesicle clustering.

Molecular chaperones are recognized to interfere with protein aggregation, yet the underlying mechanisms are largely unknown. Here, the authors develop a kinetic model that reveals the variety of distinct microscopic mechanisms through which molecular chaperones act to suppress amyloid formation.

Basosquamous carcinoma (BSC) is an uncommon skin malignancy with significant invasive and metastatic potential. There are currently no clear management guidelines. This study evaluates the management and outcomes of patients diagnosed with BSC over a 7-year period. We present an evidence-based unit protocol for the management of BSC. All patients treated for BSC between 2009 and 2015 were reviewed. Data collected included patient demographics, tumour-specific information, management strategy, presence of recurrence or metastasis, and details of follow-up. 74 patients were identified, making this one of the largest cohorts of BSC patients reported. Mean age at diagnosis was 75.4 years, with a male:female ratio of 1.6:1. The most common tumour site was the head and neck (n=43, 58.1%). All tumours were graded at pT1 (n=51) or pT2 (n=23). Inadequate excision occurred in 17 patients (23%). Mean excision margins were >4mm peripherally and deep. Inadequately excised BSCs were further treated with wide local excision (n=6) or radiotherapy (n=5), or both (n=1). There were no cases of local recurrence or metastatic disease. This study demonstrates a cohort of patients with BSCs that appear less aggressive than previously reported. Current management with surgical excision appears to produce adequate results. However, an evidence-based guideline is still lacking.

Protein aggregation into amyloid deposits and oxidative stress are key features of many neurodegenerative disorders including Parkinson's and Alzheimer's disease. We report here the creation of four highly sensitive bifunctional fluorescent probes, capable of H2O2 and/or amyloid aggregate detection. These bifunctional sensors use a benzothiazole core for amyloid localization and boronic ester oxidation to specifically detect H2O2. We characterized the optical properties of these probes using both bulk fluorescence measurements and single-aggregate fluorescence imaging, and quantify changes in their fluorescence properties upon addition of amyloid aggregates of α-synuclein and pathophysiological H2O2 concentrations. Our results indicate these new probes will be useful to detect and monitor neurodegenerative disease.

The maintenance of protein solubility is a fundamental aspect of cellular homeostasis because protein aggregation is associated with a wide variety of human diseases. Numerous proteins unrelated in sequence and structure, however, can misfold and aggregate, and widespread aggregation can occur in living systems under stress or aging. A crucial question in this context is why only certain proteins appear to aggregate readily in vivo, whereas others do not. We identify here the proteins most vulnerable to aggregation as those whose cellular concentrations are high relative to their solubilities. We find that these supersaturated proteins represent a metastable subproteome involved in pathological aggregation during stress and aging and are overrepresented in biochemical processes associated with neurodegenerative disorders. Consequently, such cellular processes become dysfunctional when the ability to keep intrinsically supersaturated proteins soluble is compromised. Thus, the simultaneous analysis of abundance and solubility can rationalize the diverse cellular pathologies linked to neurodegenerative diseases and aging.

Misfolding, abnormal accumulation, and secretion of α-Synuclein (α-Syn) are closely associated with synucleinopathies, including Parkinson's disease (PD). VH14 is a human single domain intrabody selected against the non-amyloid component (NAC) hydrophobic interaction region of α-Syn, which is critical for initial aggregation. Using neuronal cell lines, we show that as a bifunctional nanobody fused to a proteasome targeting signal, VH14PEST can counteract heterologous proteostatic effects of mutant α-Syn on mutant huntingtin Exon1 and protect against α-Syn toxicity using propidium iodide or Annexin V readouts. We compared this anti-NAC candidate to NbSyn87, which binds to the C-terminus of α-Syn. NbSyn87PEST degrades α-Syn as well or better than VH14PEST. However, while both candidates reduced toxicity, VH14PEST appears more effective in both proteostatic stress and toxicity assays. These results show that the approach of reducing intracellular monomeric targets with novel antibody engineering technology should allow in vivo modulation of proteostatic pathologies.