A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.

Secondary Modification of S100B Influences Anti Amyloid-ß Aggregation Activity and Alzheimer's Disease Pathology.

Proteinaceous aggregates accumulate in neurodegenerative diseases such as Alzheimer's Disease (AD), inducing cellular defense mechanisms and altering the redox status. S100 pro-inflammatory cytokines, particularly S100B, are activated during AD, but recent findings reveal an unconventional molecular chaperone role for S100B in hindering Aß aggregation and toxicity. This suggests a potential protective role for S100B at the onset of Aß proteotoxicity, occurring in a complex biochemical environment prone to oxidative damage. Herein, we report an investigation in which extracellular oxidative conditions are mimicked to test if the susceptibility of S100B to oxidation influences its protective activities. Resorting to mild oxidation of S100B, we observed methionine oxidation as inferred from mass spectrometry, but no cysteine-mediated crosslinking. Structural analysis showed that the folding, structure, and stability of oxidized S100B were not affected, and nor was its quaternary structure. However, studies on Aß aggregation kinetics indicated that oxidized S100B was more effective in preventing aggregation, potentially linked to the oxidation of Met residues within the S100:Aß binding cleft that favors interactions. Using a cell culture model to analyze the S100B functions in a highly oxidative milieu, as in AD, we observed that Aß toxicity is rescued by the co-administration of oxidized S100B to a greater extent than by S100B. Additionally, results suggest a disrupted positive feedback loop involving S100B which is caused by its oxidation, leading to the downstream regulation of IL-17 and IFN-α2 expression as mediated by S100B.

M13 phage grafted with peptide motifs as a tool to detect amyloid-ß oligomers in brain tissue.

Oligomeric clusters of amyloid-ß (Aß) are one of the major biomarkers for Alzheimer's disease (AD). However, proficient methods to detect Aß-oligomers in brain tissue are lacking. Here we show that synthetic M13 bacteriophages displaying Aß-derived peptides on their surface preferentially interact with Aß-oligomers. When exposed to brain tissue isolated from APP/PS1-transgenic mice, these bacteriophages detect small-sized Aß-aggregates in hippocampus at an early age, prior to the occurrence of Aß-plaques. Similarly, the bacteriophages reveal the presence of such small Aß-aggregates in post-mortem hippocampus tissue of AD-patients. These results advocate bacteriophages displaying Aß-peptides as a convenient and low-cost tool to identify Aß-oligomers in post-mortem brain tissue of AD-model mice and AD-patients.

S100B chaperone multimers suppress the formation of oligomers during Aß42 aggregation.

Extracellular aggregation of the amyloid-ß 1-42 (Aß42) peptide is a major hallmark of Alzheimer's disease (AD), with recent data suggesting that Aß intermediate oligomers (AßO) are more cytotoxic than mature amyloid fibrils. Understanding how chaperones harness such amyloid oligomers is critical toward establishing the mechanisms underlying regulation of proteostasis in the diseased brain. This includes S100B, an extracellular signaling Ca2+-binding protein which is increased in AD as a response to neuronal damage and whose holdase-type chaperone activity was recently unveiled. Driven by this evidence, we here investigate how different S100B chaperone multimers influence the formation of oligomers during Aß42 fibrillation. Resorting to kinetic analysis coupled with simulation of AßO influx distributions, we establish that supra-stoichiometric ratios of dimeric S100B-Ca2+ drastically decrease Aß42 oligomerization rate by 95% and AßO levels by 70% due to preferential inhibition of surface-catalyzed secondary nucleation, with a concomitant redirection of aggregation toward elongation. We also determined that sub-molar ratios of tetrameric apo-S100B decrease Aß42 oligomerization influx down to 10%, while precluding both secondary nucleation and, more discreetly, fibril elongation. Coincidently, the mechanistic predictions comply with the independent screening of AßO using a combination of the thioflavin-T and X-34 fluorophores. Altogether, our findings illustrate that different S100B multimers act as complementary suppressors of Aß42 oligomerization and aggregation, further underpinning their potential neuroprotective role in AD.

Tau liquid-liquid phase separation is modulated by the Ca2+ -switched chaperone activity of the S100B protein.

Aggregation of the microtubule-associated protein tau is implicated in several neurodegenerative tauopathies including Alzheimer's disease (AD). Recent studies evidenced tau liquid-liquid phase separation (LLPS) into droplets as an early event in tau pathogenesis with the potential to enhance aggregation. Tauopathies like AD are accompanied by sustained neuroinflammation and the release of alarmins at early stages of inflammatory responses encompass protective functions. The Ca2+ -binding S100B protein is an alarmin augmented in AD that was recently implicated as a proteostasis regulator acting as a chaperone-type protein, inhibiting aggregation and toxicity through interactions of amyloidogenic clients with a regulatory surface exposed upon Ca2+ -binding. Here we expand the regulatory functions of S100B over protein condensation phenomena by reporting its Ca2+ -dependent activity as a modulator of tau LLPS induced by crowding agents (PEG) and metal ions (Zn2+ ). We observe that apo S100B has a negligible effect on PEG-induced tau demixing but that Ca2+ -bound S100B prevents demixing, resulting in a shift of the phase diagram boundary to higher crowding concentrations. Also, while incubation with apo S100B does not compromise tau LLPS, addition of Ca2+ results in a sharp decrease in turbidity, indicating that interactions with S100B-Ca2+ promote transition of tau to the mixed phase. Further, electrophoretic analysis and FLIM-FRET studies revealed that S100B incorporates into tau liquid droplets, suggesting an important stabilizing and chaperoning role contributing to minimize toxic tau aggregates. Resorting to Alexa488-labeled tau we observed that S100B-Ca2+ reduces the formation of tau fluorescent droplets, without compromising liquid-like behavior and droplet fusion events. The Zn2+ -binding properties of S100B also contribute to regulate Zn2+ -promoted tau LLPS as droplets are decreased by Zn2+ buffering by S100B, in addition to the Ca2+ -triggered interactions with tau. Altogether this work uncovers the versatility of S100B as a proteostasis regulator acting on protein condensation phenomena of relevance across the neurodegeneration continuum.

Relationship between Trace Elements and Matrix Metalloproteinases 2 and 9 and their Tissue Inhibitors in Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) constitutes around 5% of all thyroid cancers. Trace elements assessment has emerged as a useful strategy in the diagnostics of MTC combined with Matrix Metalloproteinases (MMPs) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) analysis. The aim of this study was to compare the presence and content of trace elements (i.e., Copper (Cu), Zinc (Zn), Iron (Fe), and Manganese (Mn)) in MTC with respect to control samples and their potential relationship with markers of MTC in tissues. The study included 26 patients who had undergone thyroidectomy, due to the diagnosis of MTC and 17 patients as control. We combined tumour pathology and staging, immunohistochemical analysis of calcitonin, MMPs, and TIMPs, with analytical biochemistry using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) to determine the levels of trace elements. No differences by MTC type for MMPs and their TIPMs, although strong TIMP-1 and TIMP-2 immunohistochemical expression of MTC were unveiled. Additionally, Zn, Fe, and Mn tended to be decreased, and Cu to be increased in samples presenting MTC with respect to controls. Moreover, Zn was the unique trace element which seemed to be correlated with MMPs and TIMPs. Trace elements such as Zn, Fe, and Mn are decreased in tissues affected by MTC. In addition, Zn may be the trace element which saves more relationship with the proportion and intensity of MMPs, being considered altogether useful biomarkers of MTC. We therefore suggest the analysis of novel and traditional markers of MTC as a novel approach in this pathology.

Matrix Metalloproteinases 2 and 9 and Their Tissue Inhibitors in the Diagnostics of Medullary Thyroid Carcinoma.

Medullary Thyroid Carcinoma (MTC) is a tumor of the neuroendocrine system. In recent years, the need to assess the MTC diagnostic-related parameters has emerged with the aim to elucidate the mechanisms involved in this pathology. The objective of this study was to evaluate the role of Matrix Metalloproteinases (MMPs) 2 and 9, their tissue inhibitors of matrix metalloproteinases (TIMPs), S100 protein, and amyloid in the diagnostic of MTC. Thirty-two samples with MTC (72% women) were included in this cross-sectional study and divided by groups: T category 1 (T1)≤20 mm and T category 2 (T2) 20 to 40 mm of tumor size. MMPs 2 and 9, TIMPs 2 and 1, S100 protein, and calcitonin in tissues were obtained by immunohistochemical techniques. The presence of amyloid in tissue sections was detected on Thioflavin T-stained slides under fluorescent microscope. Percentage of positive cells (P) observed for MMP-2 was higher in those samples presenting T2 MTC with respect to those with T1 MTC ( P <0.05). Moreover, P-MMP-2 showed a direct correlation with higher T category of MTC (Rho=0.439, P < 0.001), whereas P-MPP-9 was directly correlated with S100 protein and the intensity of calcitonin in tissues (Rho=0.419, P =0.017; Rho=0.422, P =0.016, respectively. Therefore, MMPs were directly correlated with some traditional biomarkers of MTC. In this regard, P-MMP-2 was more expressed in type 2 MTC. Combining the analysis of traditional and other useful biomarkers of MTC as MMPs 2 and 9 could be a useful strategy in the diagnostic of MTC.

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity.

Alzheimer's disease (AD) hallmarks include the aggregation of amyloid-ß (Aß), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aß42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aß42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aß client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aß42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aß42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

Zinc-binding metallophores protect Pseudomonas aeruginosa from calprotectin-mediated metal starvation.

Pseudomonas aeruginosa is known to exhibit considerable resistance to the antimicrobial activity of the metal-sequestering protein calprotectin (CP). In this study, we demonstrate that although CP induces zinc deficiency in P. aeruginosa, a strain unable to import zinc through the two most important metal acquisition systems, namely ZnuABC and ZrmABCD, maintains significant growth capacity in the presence of high concentrations of CP. Furthermore, we have shown that nicotianamine, a molecule structurally similar to the metallophore pseudopaline, can favor the acquisition of the metal even in the presence of CP. To gain insights into the mechanisms through which metallophores can promote zinc acquisition, we analyzed the effect of nicotianamine on the activity of the metallo-ß-lactamase VIM-1. Our data suggest that metallophores released by bacteria in response to zinc deficiency can extract the protein-bound metal. The ability to interfere with the binding of metals to proteins, as well as favoring the acquisition of zinc, may contribute to increasing the resistance of P. aeruginosa to the antimicrobial action of CP.

Deglutarylation of glutaryl-CoA dehydrogenase by deacylating enzyme SIRT5 promotes lysine oxidation in mice.

A wide range of protein acyl modifications has been identified on enzymes across various metabolic processes; however, the impact of these modifications remains poorly understood. Protein glutarylation is a recently identified modification that can be nonenzymatically driven by glutaryl-CoA. In mammalian systems, this unique metabolite is only produced in the lysine and tryptophan oxidative pathways. To better understand the biology of protein glutarylation, we studied the relationship between enzymes within the lysine/tryptophan catabolic pathways, protein glutarylation, and regulation by the deglutarylating enzyme sirtuin 5 (SIRT5). Here, we identify glutarylation on the lysine oxidation pathway enzyme glutaryl-CoA dehydrogenase (GCDH) and show increased GCDH glutarylation when glutaryl-CoA production is stimulated by lysine catabolism. Our data reveal that glutarylation of GCDH impacts its function, ultimately decreasing lysine oxidation. We also demonstrate the ability of SIRT5 to deglutarylate GCDH, restoring its enzymatic activity. Finally, metabolomic and bioinformatic analyses indicate an expanded role for SIRT5 in regulating amino acid metabolism. Together, these data support a feedback loop model within the lysine/tryptophan oxidation pathway in which glutaryl-CoA is produced, in turn inhibiting GCDH function via glutaryl modification of GCDH lysine residues and can be relieved by SIRT5 deacylation activity.

Hofmeister effects on protein stability are dependent on the nature of the unfolded state.

The interpretation of a salt's effect on protein stability traditionally discriminates low concentration regimes (<0.3 M), dominated by electrostatic forces, and high concentration regimes, generally described by ion-specific Hofmeister effects. However, increased theoretical and experimental studies have highlighted observations of the Hofmeister phenomena at concentration ranges as low as 0.001 M. Reasonable quantitative predictions of such observations have been successfully achieved throughout the inclusion of ion dispersion forces in classical electrostatic theories. This molecular description is also on the basis of quantitative estimates obtained resorting to surface/bulk solvent partition models developed for ion-specific Hofmeister effects. However, the latter are limited by the availability of reliable structures representative of the unfolded state. Here, we use myoglobin as a model to explore how ion-dependency on the nature of the unfolded state affects protein stability, combining spectroscopic techniques with molecular dynamic simulations. To this end, the thermal and chemical stability of myoglobin was assessed in the presence of three different salts (NaCl, (NH4)2SO4 and Na2SO4), at physiologically relevant concentrations (0-0.3 M). We observed mild destabilization of the native state induced by each ion, attributed to unfavorable neutralization and hydrogen-bonding with the protein side-chains. Both effects, combined with binding of Na+, Cl- and SO42- to the thermally unfolded state, resulted in an overall destabilization of the protein. Contrastingly, ion binding was hindered in the chemically unfolded conformation, due to occupation of the binding sites by urea molecules. Such mechanistic action led to a lower degree of destabilization, promoting surface tension effects that stabilized myoglobin according to the Hofmeister series. Therefore, we demonstrate that Hofmeister effects on protein stability are modulated by the heterogeneous physico-chemical nature of the unfolded state. Altogether, our findings evidence the need to characterize the structure of the unfolded state when attempting to dissect the molecular mechanisms underlying the effects of salts on protein stability.

S100B dysregulation during brain development affects synaptic SHANK protein networks via alteration of zinc homeostasis.

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.

Dynamic interactions and Ca2+-binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding.

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 ß 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.

Hypercholesterolemia and 27-Hydroxycholesterol Increase S100A8 and RAGE Expression in the Brain: a Link Between Cholesterol, Alarmins, and Neurodegeneration.

Alterations in cholesterol metabolism in the brain have a major role in the physiology of Alzheimer's disease (AD). Oxysterols are cholesterol metabolites with multiple implications in memory functions and in neurodegeneration. Previous studies have shown detrimental effects of cholesterol metabolites in neurons, but its effect in glial cells is unknown. We used a high-fat/high-cholesterol diet in mice to study the effects of hypercholesterolemia over the alarmin S100A8 cascade in the hippocampus. Using CYP27Tg, a transgenic mouse model, we show that the hypercholesterolemia influence on the brain is mediated by the excess of 27-hydroxycholesterol (27-OH), a cholesterol metabolite. We also employed an acute model of 27-OH intraventricular injection in the brain to study RAGE and S100A8 response. We used primary cultures of neurons and astrocytes to study the effect of high levels of 27-OH over the S100A8 alarmin cascade. We report that a high-fat/high-cholesterol diet leads to an increase in S100A8 production in the brain. In CYP27Tg, we report an increase of S100A8 and its receptor RAGE in the hippocampus under elevated 27-OH in the brain. Using siRNA, we found that 27-OH upregulation of RAGE in astrocytes and neurons is mediated by the nuclear receptor RXRγ. Silencing RXRγ in neurons prevented 27-OH-mediated upregulation of RAGE. These results show that S100A8 alarmin and RAGE respond to high levels of 27-OH in the brain in both neurons and astrocytes through RXRγ. Our study supports the notion that 27-OH mediates detrimental effects of hypercholesterolemia to the brain via alarmin signaling.

Computational Analysis of the Interactions between the S100B Extracellular Chaperone and Its Amyloid ß Peptide Client.

S100B is an astrocytic extracellular Ca2+-binding protein implicated in Alzheimer's disease, whose role as a holdase-type chaperone delaying Aß42 aggregation and toxicity was recently uncovered. Here, we employ computational biology approaches to dissect the structural details and dynamics of the interaction between S100B and Aß42. Driven by previous structural data, we used the Aß25-35 segment, which recapitulates key aspects of S100B activity, as a starting guide for the analysis. We used Haddock to establish a preferred binding mode, which was studied with the full length Aß using long (1 µs) molecular dynamics (MD) simulations to investigate the structural dynamics and obtain representative interaction complexes. From the analysis, Aß-Lys28 emerged as a key candidate for stabilizing interactions with the S100B binding cleft, in particular involving a triad composed of Met79, Thr82 and Glu86. Binding constant calculations concluded that coulombic interactions, presumably implicating the Lys28(Aß)/Glu86(S100B) pair, are very relevant for the holdase-type chaperone activity. To confirm this experimentally, we examined the inhibitory effect of S100B over Aß aggregation at high ionic strength. In agreement with the computational predictions, we observed that electrostatic perturbation of the Aß-S100B interaction decreases anti-aggregation activity. Altogether, these findings unveil features relevant in the definition of selectivity of the S100B chaperone, with implications in Alzheimer's disease.

Targeting S100B with Peptides Encoding Intrinsic Aggregation-Prone Sequence Segments.

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 ß-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.

Electron transfer flavoprotein and its role in mitochondrial energy metabolism in health and disease.

Electron transfer flavoprotein (ETF) is an enzyme with orthologs from bacteria to humans. Human ETF is nuclear encoded by two separate genes, ETFA and ETFB, respectively. After translation, the two subunits are imported to the mitochondrial matrix space and assemble into a heterodimer containing one FAD and one AMP as cofactors. ETF functions as a hub taking up electrons from at least 14 flavoenzymes, feeding them into the respiratory chain. This represents a major source of reducing power for the electron transport chain from fatty acid oxidation and amino acid degradation. Transfer of electrons from the donor enzymes to ETF occurs by direct transfer between the enzyme bound flavins, a process that is tightly regulated by the polypeptide chain and by protein:protein interactions. ETF, in turn relays electrons to the iron sulfur cluster of the inner membrane protein ETF:QO, from where they travel via the FAD in ETF:QO to ubiquinone, entering the respiratory chain at the level of complex III. ETF recognizes its dehydrogenase partners via a recognition loop that anchors the protein on its partner followed by dynamic movements of the ETF flavin domain that bring redox cofactors in close proximity, thus promoting electron transfer. Genetic mutations in the ETFA or ETFB genes cause the Mendelian disorder multiple acyl-CoA dehydrogenase deficiency (MADD; OMIM #231680). We here review the knowledge on human ETF and investigations of the effects of disease-associated missense mutations in this protein that have promoted the understanding of the essential role that ETF plays in cellular metabolism and human disease.

Cu2+-binding to S100B triggers polymerization of disulfide cross-linked tetramers with enhanced chaperone activity against amyloid-ß aggregation.

S100B is an extracellular protein implicated in Alzheimer's Disease and a suppressor of amyloid-ß 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.