Search

The microtubule-associated protein tau is implicated in the formation of oligomers and fibrillar aggregates that evade proteostasis control and spread from cell-to-cell. Tau pathology is accompanied by sustained neuroinflammation and, while the release of alarmin mediators aggravates disease at late stages, early inflammatory responses encompass protective functions. This is the case of the Ca2+-binding S100B protein, an astrocytic alarmin which is augmented in AD and which has been recently implicated as a proteostasis regulator, acting over amyloid β aggregation. Here we report the activity of S100B as a suppressor of tau aggregation and seeding, operating at sub-stoichiometric conditions. We show that S100B interacts with tau in living cells even in microtubule-destabilizing conditions. Structural analysis revealed that tau undergoes dynamic interactions with S100B, in a Ca2+-dependent manner, notably with the aggregation prone repeat segments at the microtubule binding regions. This interaction involves contacts of tau with a cleft formed at the interface of the S100B dimer. Kinetic and mechanistic analysis revealed that S100B inhibits the aggregation of both full-length tau and of the microtubule binding domain, and that this proceeds through effects over primary and secondary nucleation, as confirmed by seeding assays and direct observation of S100B binding to tau oligomers and fibrils. In agreement with a role as an extracellular chaperone and its accumulation near tau positive inclusions, we show that S100B blocks proteopathic tau seeding. Together, our findings establish tau as a client of the S100B chaperone, providing evidence for neuro-protective functions of this inflammatory mediator across different tauopathies., The calcium binding protein S100B is an abundantly expressed protein in the brain and has neuro-protective functions by inhibiting Aβ aggregation and metal ion toxicity. Here, the authors combine cell biology and biochemical experiments with chemical kinetics and NMR measurements and show that S100B protein is an extracellular Tau chaperone and further characterize the interactions between S100B and Tau.

S100B is an extracellular protein implicated in Alzheimer's Disease and a suppressor of amyloid-β aggregation. Herein we report a mechanism tying Cu2+ binding to a change in assembly state yielding disulfide cross-linked oligomers with higher anti-aggregation activity. This chemical control of chaperone function illustrates a regulatory process relevant under metal and proteostasis dysfunction as in neurodegeneration.

Autism Spectrum Disorders (ASD) are caused by a combination of genetic predisposition and nongenetic factors. Among the nongenetic factors, maternal immune system activation and zinc deficiency have been proposed. Intriguingly, as a genetic factor, copy-number variations in S100B, a pro-inflammatory damage-associated molecular pattern (DAMP), have been associated with ASD, and increased serum S100B has been found in ASD. Interestingly, it has been shown that increased S100B levels affect zinc homeostasis in vitro. Thus, here, we investigated the influence of increased S100B levels in vitro and in vivo during pregnancy in mice regarding zinc availability, the zinc-sensitive SHANK protein networks associated with ASD, and behavioral outcomes. We observed that S100B affects the synaptic SHANK2 and SHANK3 levels in a zinc-dependent manner, especially early in neuronal development. Animals exposed to high S100B levels in utero similarly show reduced levels of free zinc and SHANK2 in the brain. On the behavioral level, these mice display hyperactivity, increased stereotypic and abnormal social behaviors, and cognitive impairment. Pro-inflammatory factors and zinc-signaling alterations converge on the synaptic level revealing a common pathomechanism that may mechanistically explain a large share of ASD cases.

S100 proteins assume a diversity of oligomeric states including large order self-assemblies, with an impact on protein structure and function. Previous work has uncovered that S100 proteins, including S100B, are prone to undergo &beta, aggregation under destabilizing conditions. This propensity is encoded in aggregation-prone regions (APR) mainly located in segments at the homodimer interface, and which are therefore mostly shielded from the solvent and from deleterious interactions, under native conditions. As in other systems, this characteristic may be used to develop peptides with pharmacological potential that selectively induce the aggregation of S100B through homotypic interactions with its APRs, resulting in functional inhibition through a loss of function. Here we report initial studies towards this goal. We applied the TANGO algorithm to identify specific APR segments in S100B helix IV and used this information to design and synthesize S100B-derived APR peptides. We then combined fluorescence spectroscopy, transmission electron microscopy, biolayer interferometry, and aggregation kinetics and determined that the synthetic peptides have strong aggregation propensity, interact with S100B, and may promote co-aggregation reactions. In this framework, we discuss the considerable potential of such APR-derived peptides to act pharmacologically over S100B in numerous physiological and pathological conditions, for instance as modifiers of the S100B interactome or as promoters of S100B inactivation by selective aggregation.

S100B is an extracellular protein implicated in Alzheimer's Disease and a suppressor of amyloid-β aggregation. Herein we report a mechanism tying Cu2+ binding to a change in assembly state yielding disulfide cross-linked oligomers with higher anti-aggregation activity. This chemical control of chaperone function illustrates a regulatory process relevant under metal and proteostasis dysfunction as in neurodegeneration.

Despite the rapidly increasing number of patients suffering from type 2 diabetes, Alzheimer's disease, and diabetes-induced dementia, there are no disease-modifying therapies that are able to prevent or block disease progress. In this work, we investigate the potential of nature-inspired glucosylpolyphenols against relevant targets, including islet amyloid polypeptide, glucosidases, and cholinesterases. Moreover, with the premise of Fyn kinase as a paradigm-shifting target in Alzheimer's drug discovery, we explore glucosylpolyphenols as blockers of Aβ-induced Fyn kinase activation while looking into downstream effects leading to Tau hyperphosphorylation. Several compounds inhibit Aβ-induced Fyn kinase activation and decrease pTau levels at 10 μM concentration, particularly the per-O-methylated glucosylacetophloroglucinol and the 4-glucosylcatechol dibenzoate, the latter inhibiting also butyrylcholinesterase and β-glucosidase. Both compounds are nontoxic with ideal pharmacokinetic properties for further development. This work ultimately highlights the multitarget nature, fine structural tuning capacity, and valuable therapeutic significance of glucosylpolyphenols in the context of these metabolic and neurodegenerative disorders.

Amyloid beta (Aβ) aggregation and imbalance of metal ions are major hallmarks of Alzheimer's disease (AD). Indeed, amyloid plaques of AD patients are enriched in zinc and Aβ42, and AD related-cognitive decline is dependent on extracellular zinc concentration. In vitro, zinc induces the formation of polymorphic Aβ42 oligomers that delay the formation of amyloid fibers at the expense of increased cellular toxicity. S100B is an inflammatory alarmin and one of the most abundant proteins in the brain and is upregulated in AD and associated with amyloid plaques, where it exerts extracellular functions. Recent findings have uncovered novel neuroprotective functions for S100B as a suppressor of Aβ aggregation and toxicity and in the regulation of zinc homeostasis in neurons. Here we combine biophysical and kinetic approaches to demonstrate that such S100B protective functions converge, making the protein a dual-function chaperone capable of suppressing the formation of toxic Aβ oligomers through both chelation of zinc and inhibition of protein aggregation. From detailed kinetic analysis of Aβ42 aggregation monitoring ThT fluorescence, we show that substoichiometric S100B prevents the formation of toxic off-pathway oligomers that are formed by monomeric Aβ42 in the presence of zinc. Indeed, S100B is effective when added during the lag and transition phases of Aβ42 aggregation, and its action under these circumstances results from its ability to buffer zinc, as it perfectly mimics the effect obtained with the chelating agent EDTA. Further, bioimaging analysis combining transmission electron microscopy and atomic force microscopy confirms that catalytic amounts of S100B partly revert the formation of toxic oligomers. Taken together these results indicate a new role for S100B as a dual chaperone whose distinct functions are interrelated and depend on the relative levels of zinc, S100B, and Aβ, which dynamically evolve during AD.

Protein aggregation is a cornerstone in amyloid-forming neurodegenerative diseases that is largely due to altered conditions in the biochemistry of key components of the neuronal environment, including metal ions. Indeed, trace neurometals such as calcium, zinc, and copper are vital players in brain neurobiology, whose homeostasis is altered in most neurodegenerative conditions; further, metals ions are widely found within proteinaceous inclusions from patients and animal models. This chapter briefly gives an overview of the influence of trace metals in amyloid formation in connection to their homeostasis in the brain. In particular, the role of zinc in Alzheimer’s disease is more thoroughly discussed. Indeed, the deregulation of zinc ions has well-established mechanistic links to Alzheimer’s pathophysiology, including direct interaction effects with amyloid β and tau, the two amyloid-forming proteins involved in this neurodegenerative disease.

Metal ions are well known modulators of protein aggregation and are key players in Alzheimer&rsquo, s Disease, being found to be associated to pathologic protein deposits in diseased brains. Therefore, understanding how metals influence amyloid aggregation is critical in establishing molecular mechanisms that underlie disease onset and progression. Here, we report data on the interaction of full-length human Tau protein with calcium and zinc ions, evidencing that Tau self-assembly is differently regulated, depending on the type of bound metal ion. We established that Tau binds 4 Zn2+ and 1 Ca2+ per monomer while using native mass spectrometry analysis, without inducing order or substantial conformational changes in the intrinsically disordered Tau, as determined by structural analysis using circular dichroism and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopies. However, Tau aggregation is found to proceed differently in the calcium- and -zinc bound forms. While the rate of aggregation, as determined from thioflavin-T (ThT) fluorescence kinetics, is highly increased in both cases, the reaction proceeds via different mechanisms, as evidenced by the absence of the lag phase in the reaction of zinc-bound Tau. Monitoring Tau aggregation using native mass spectrometry indeed evidenced a distinct distribution of Tau conformers along the reaction, as confirmed by dynamic light scattering analysis. We propose that such differences arise from zinc binding at distinct locations within the Tau sequence that prompt both the rapid formation of seeding oligomers through interactions at high affinity sites within the repeat domains, as well as amorphous aggregation, through low affinity interactions with residues elsewhere in the sequence, including at the fuzzy coat domain.

Dietary flavonoids and synthetic derivatives have a well-known potential for biomedical applications. In this perspective, we report herein new methodologies to access chrysin and 5,7-dihydroxychromone, and these structures were combined with those of naturally occurring quercetin, luteolin, (+)-dihydroquercetin and apigenin to assemble a set of polyphenols with structure variations for in vitro testing over the aggregation of Alzheimer’s disease (AD) amyloid peptide Aβ1−42. Using thioflavin-T (ThT) monitored kinetics and subsequent mechanistic analysis by curve fitting, we show that catechol-type flavonoids reduce Aβ1−42 fibril content by 30% at molar ratios over 10. Without affecting secondary nucleation, these compounds accelerate primary nucleation events responsible for early primary oligomer formation, putatively redirecting the latter into off-pathway aggregates. Atomic force microscopy (AFM) imaging of reaction end-points allowed a comprehensive topographical analysis of amyloid aggregate populations formed in the presence of each compound. Formation of Aβ1−42 small oligomers, regarded as the most toxic amyloid structures, seems to be limited by flavonoids with a C2 phenyl group, while flavonol 3-OH is not a beneficial structural feature. Overall, the diversity of structural variations within flavonoids opens avenues for their development as chemical tools in the treatment of AD by tackling the formation and distribution of neurotoxic oligomers species.

Proteins exhibit a remarkable structural plasticity and may undergo conformational changes resulting in protein misfolding both in a biological context and upon perturbing physiopathological conditions. Such nonfunctional protein conformers, including misfolded states and aggregates, are often associated to protein folding diseases. Understanding the biology of protein folding diseases thus requires tools that allow the structural characterization of nonnative conformations of proteins and their interconversions. Here we present detailed procedures to monitor protein conformational changes and aggregation based on spectroscopic and biophysical methods that include circular dichroism, ATR-Fourier-transformed infrared spectroscopy, fluorescence spectroscopy and dynamic light scattering. To illustrate the application of these methods we report to our previous studies on misfolding, aggregation and amyloid fibril formation by superoxide dismutase 1 (SOD1), a protein whose toxic deposition is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS).

Proteins exhibit a remarkable structural plasticity and may undergo conformational changes resulting in protein misfolding both in a biological context and upon perturbing physiopathological conditions. Such nonfunctional protein conformers, including misfolded states and aggregates, are often associated to protein folding diseases. Understanding the biology of protein folding diseases thus requires tools that allow the structural characterization of nonnative conformations of proteins and their interconversions. Here we present detailed procedures to monitor protein conformational changes and aggregation based on spectroscopic and biophysical methods that include circular dichroism, ATR-Fourier-transformed infrared spectroscopy, fluorescence spectroscopy and dynamic light scattering. To illustrate the application of these methods we report to our previous studies on misfolding, aggregation and amyloid fibril formation by superoxide dismutase 1 (SOD1), a protein whose toxic deposition is implicated in the neurodegenerative disease amyotrophic lateral sclerosis (ALS).

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca

A novel role for S100B, a recognized brain distress marker, as a chaperone-like suppressor of Aβ42 aggregation and toxicity., Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer’s disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface–catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

Proteins containing EF-hand helix-loop-helix-binding motifs play essential roles in calcium homeostasis and signaling pathways. These proteins have considerable structural and functional diversity by virtue of their cation-binding properties, and occur as either Ca2+-bound or Ca2+-free states with distinct aggregation propensities. That is the case among β-parvalbumins and S100 proteins, which under certain conditions undergo Ca2+-dependent self-assembly reactions with the formation of oligomers, amyloid-type aggregates and fibrils. These phenomena may be particularly relevant in human S100A6 protein and in fish Gad m 1 allergenic protein, which are implicated in human disease processes. Here, we describe detailed methods to generate and monitor the formation of amyloidogenic assemblies and aggregates of these two EF-hand proteins in vitro.

Alzheimer’s disease (AD) is the most prevalent age-related dementia affecting millions of people worldwide. Its main pathological hallmark feature is the formation of insoluble protein deposits of amyloid-βand hyperphosphorylated tau protein into extracellular plaques and intracellular neurofibrillary tangles, respectively. Many of the mechanistic details of this process remain unknown, but a well-established consequence of protein aggregation is synapse dysfunction and neuronal loss in the AD brain. Different pathways including mitochondrial dysfunction, oxidative stress, inflammation, and metal metabolism have been suggested to be implicated in this process. In particular, a body of evidence suggests that neuronal metal ions such as copper, zinc, and iron play important roles in brain function in health and disease states and altered homeostasis and distribution as a common feature across different neurodegenerative diseases and aging. In this focused review, we overview neuronal proteins that are involved in AD and whose metal binding properties may underlie important biochemical and regulatory processes occurring in the brain during the AD pathophysiological process.

Superoxide dismutase 1 (SOD1) is a Cu/Zn metalloenzyme that aggregates in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. Correct metal insertion during SOD1 biosynthesis is critical to prevent misfolding; however Zn(2+) can bind to the copper-site leading to an aberrantly metallated protein. These effects of Zn(2+) misligation on SOD1 aggregation remain to be explored, even though Zn(2+) levels are upregulated in ALS motor neurons. Here we use complementary biophysical methods to investigate Zn(2+) binding and its effects on the aggregation of three immature metal-free SOD1 conformers that represent biogenesis intermediates: dimeric, monomeric and reduced monomeric SOD1. Using isothermal titration calorimetry we determined that Zn(2+) binds to all conformers both at the zinc- as well as to the copper-site; however Zn(2+) binding mechanisms to the zinc-site have distinct characteristics across immature conformers. We show that this 'zinc overload' of immature SOD1 promotes intermolecular interactions, as evidenced by dynamic light scattering and ThT fluorescence kinetic studies. Analysis of aged zinc-induced aggregates by energy-dispersive X-ray and electron energy-loss spectroscopy shows that aggregates integrate some Zn(2+). In addition, electron diffraction analysis identifies nano-scaled crystalline materials and amyloid fibril-like reflections. Transmission electron microscopy reveals that Zn(2+) diverts the SOD1 aggregation pathway from fibrils to amorphous aggregate, and electrophoretic analysis evidences an increase in insoluble materials. Overall, we provide evidence that aberrant zinc coordination to immature conformers broadens the population of SOD1 misfolded species at early aggregation stages and provide evidence for a high structural polymorphism and heterogeneity of SOD1 aggregates.

Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetallated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca(2+) over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca(2+) ions to apo SOD1, at pH7.5 and 37°C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca(2+) association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 β-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca(2+) binding locations reveals that among the most frequent ligands for Ca(2+) are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating β-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca(2+) interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.