SnapShot: Intrinsic Structural Disorder.

Many proteins (intrinsically disordered proteins, IDPs) or regions of proteins (intrinsically disordered regions, IDRs) lack a well-defined 3D structure under physiological conditions. Albeit unfolded and highly dynamic, these proteins are not denatured; rather, intrinsic structural disorder is their native, functional state.

In vivo biotinylated calpastatin improves the affinity purification of human m-calpain.

Recently we established a novel affinity purification method for calpain by exploiting the specific and reversible binding properties of its intrinsically disordered protein inhibitor, calpastatin. The immobilization strategy relied on the strength and specificity of the biotin - streptavidin interaction. Here, we report an improved and optimized method that even enables the general applicability of in vivo biotinylated (intrinsically disordered) proteins in any affinity capture strategy. Since in vitro chemical biotinylation is only accomplished with reagents that lack exact site specificity, it can not only cause sample heterogeneity but it can also hamper the functionality of the biotinylated molecules. Therefore, we have developed a recombinant expression protocol to produce in vivo biotinylated human calpastatin domain 1 (hCSD1) in Escherichia coli. We have experimentally verified that the biotinylated polypeptide tag is compatible with the intrinsically disordered state of hCSD1 and that it does not influence the functional properties of this intrinsically disordered protein (IDP). The in vivo biotinylated hCSD1 was then used without the need of any prepurification step prior to the affinity capturing of its substrate, human m-calpain. This leads to a simplified purification strategy that allows capturing the calpain efficiently from a complex biological mixture with only a single chromatogaphic step and in a considerably reduced timeframe. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest, and its practical implementation will showcase the power to exploit the properties of IDPs in affinity capture strategies.

To be disordered or not to be disordered: is that still a question for proteins in the cell?

There is ample evidence that many proteins or regions of proteins lack a well-defined folded structure under native-like conditions. These are called intrinsically disordered proteins (IDPs) or intrinsically disordered regions (IDRs). Whether this intrinsic disorder is also their main structural characteristic in living cells has been a matter of intense debate. The structural analysis of IDPs became an important challenge also because of their involvement in a plethora of human diseases, which made IDPs attractive targets for therapeutic development. Therefore, biophysical approaches are increasingly being employed to probe the structural and dynamical state of proteins, not only in isolation in a test tube, but also in a complex biological environment and even within intact cells. Here, we survey direct and indirect evidence that structural disorder is in fact the physiological state of many proteins in the proteome. The paradigmatic case of α-synuclein is used to illustrate the controversial nature of this topic.

Could ecosystem management provide a new framework for Alzheimer's disease?

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that involves a plethora of molecular pathways. In the context of therapeutic treatment and biomarker profiling, the amyloid-beta (Aß) peptide constitutes an interesting research avenue that involves interactions within a complex mixture of Aß alloforms and other disease-modifying factors. Here, we explore the potential of an ecosystem paradigm as a novel way to consider AD and Aß dynamics in particular. We discuss the example that the complexity of the Aß network not only exhibits interesting parallels with the functioning of complex systems such as ecosystems but that this analogy can also provide novel insights into the neurobiological phenomena in AD and serve as a communication tool. We propose that combining network medicine with general ecosystem management principles could be a new and holistic approach to understand AD pathology and design novel therapies.

Binding of methylene blue to a surface cleft inhibits the oligomerization and fibrillization of prion protein.

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.

Mechanistic insights in glycation-induced protein aggregation.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

Self-assembly and conformational heterogeneity of the AXH domain of ataxin-1: an unusual example of a chameleon fold.

Ataxin-1 is a human protein responsible for spinocerebellar ataxia type 1, a hereditary disease associated with protein aggregation and misfolding. Essential for ataxin-1 aggregation is the anomalous expansion of a polyglutamine tract near the protein N-terminus, but the sequence-wise distant AXH domain modulates and contributes to the process. The AXH domain is also involved in the nonpathologic functions of the protein, including a variety of intermolecular interactions with other cellular partners. The domain forms a globular dimer in solution and displays a dimer of dimers arrangement in the crystal asymmetric unit. Here, we have characterized the domain further by studying its behavior in the crystal and in solution. We solved two new structures of the domain crystallized under different conditions that confirm an inherent plasticity of the AXH fold. In solution, the domain is present as a complex equilibrium mixture of monomeric, dimeric, and higher molecular weight species. This behavior, together with the tendency of the AXH fold to be trapped in local conformations, and the multiplicity of protomer interfaces, makes the AXH domain an unusual example of a chameleon protein whose properties bear potential relevance for the aggregation properties of ataxin-1 and thus for disease.

Structural basis for increased toxicity of pathological aß42:aß40 ratios in Alzheimer disease.

The ß-amyloid peptide (Aß) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aß(42):Aß(40) ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aß(42) in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aß(40) and Aß(42) individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aß(42):Aß(40) ratios by several techniques. We show that Aß(40) and Aß(42) directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aß(40) and Aß(42) mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aß(40) and Aß(42) influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aß(42):Aß(40) in synaptotoxicity assays and in AD patients.

Understanding the effect of the membrane-mimetic micelles on the interplay between α-synuclein and Cu(II)/Cu(I) cations.

α-Synuclein (αS) is a presynaptic protein whose aggregates are considered as a hallmark of Parkinson's disease (PD). Although its physiological function is still under debate, it is widely accepted that its functions are always mediated by its interaction with membranes. The association of αS with phospholipid membranes occurs concomitant to its folding from its monomeric, unfolded state towards an antiparallel amphipathic α-helix. Besides this, copper ions can also bind αS and modify its aggregation propensity. The effect of Cu(II) and Cu(I) on the lipid-αS affinity and on the structure of the membrane-bound αS have not yet been studied. This knowledge is relevant to understand the molecular pathogenesis of PD. Therefore, we have here studied the affinities between Cu(II) and Cu(I) and the micelle-bound αS, as well as the effect of these cations on the structure of micelle-bound αS. Cu(II) or Cu(I) did not affect the α-helical structure of the micelle-bound αS. However, while Cu(I) binds at the same sites of αS in the presence or in the absence of micelles, the micelle-bound αS displays different Cu(II) binding sites than unbound αS. In any case, sodium docecyl sulphate -micelles reduce the stability of the αS complexes with both Cu(II) and Cu(I). Finally, we have observed that the micelle-bound αS is still able to prevent the Cu(II)-catalysed oxidation of neuronal metabolites (e.g. ascorbic acid) and the formation of reactive oxygen species, thus this binding does not impair its biological function as part of the antioxidant machinery.

The intracerebral injection of Aß1-42 oligomers does not invariably alter seizure susceptibility in mice.

Objectives: Epileptiform activity and seizures are present in patients with Alzheimer's disease (AD) and genetic animal models of AD. Amyloid beta 1-42 (Aß1-42) oligomers are thought to be crucial in AD and can cause neuronal hyperexcitability in vitro. However, it is unclear whether these Aß1-42 oligomers cause the increased seizure susceptibility in vivo in people with AD and in AD animal models, nor via which mechanisms it would do so. We investigated this question by injecting Aß1-42 oligomers intracerebrally in mice and assessed its impact on seizure susceptibility. Materials and methods: We performed a single intracerebral injection of synthetic Aß1-42 oligomers or scrambled Aß1-42 in NMRI mice in three different cohorts and subjected them to an i.v. infusion of a chemoconvulsant. We evoked the seizures 1.5 h, 1 week, or 3 weeks after the intracerebral injection of Aß1-42 oligomers, covering also the timepoints and injection locations that were used by others in similar experimental set-ups. Results: With a thioflavine T assay and transmission electron microscopy we confirmed that Aß1-42 monomers spontaneously aggregated to oligomers. We did not find an effect of Aß1-42 oligomers on susceptibility to seizures - evoked 1.5 h, 1 week or 3 weeks - after their intracerebral injection. Significance: The lack of effect of Aß1-42 oligomers on seizure susceptibility in our experiments contrasts with recent findings in similar experimental set-ups. Contradicting conclusions are frequent in experiments with Aß1-42 and they are often attributed to subtle differences in the various aggregation forms of the Aß1-42 used in different experiments. We confirmed the presence of Aß1-42 oligomers with state-of-the-art methods but cannot ascertain that the protein aggregates we used are identical to those used by others. Whether our findings or those previously published best represent the role of Aß1-42 oligomers on seizures in AD remains unclear.

Prion fibrillization is mediated by a native structural element that comprises helices H2 and H3.

Aggregation and misfolding of the prion protein (PrP) are thought to be the cause of a family of lethal neurodegenerative diseases affecting humans and other animals. Although the structures of PrP from several species have been solved, still little is known about the mechanisms that lead to the misfolded species. Here, we show that the region of PrP comprising the hairpin formed by the helices H2 and H3 is a stable independently folded unit able to retain its secondary and tertiary structure also in the absence of the rest of the sequence. We also prove that the isolated H2H3 is highly fibrillogenic and forms amyloid fibers morphologically similar to those obtained for the full-length protein. Fibrillization of H2H3 but not of full-length PrP is concomitant with formation of aggregates. These observations suggest a "banana-peeling" mechanism for misfolding of PrP in which H2H3 is the aggregation seed that needs to be first exposed to promote conversion from a helical to a beta-rich structure.

Structure of a membrane-based steric chaperone in complex with its lipase substrate.

Secretion via the type II secretion pathway in Gram-negative bacteria often relies crucially on steric chaperones in the periplasm. Here, we report the crystal structure of the soluble form of a lipase-specific foldase (Lif) from Burkholderia glumae in complex with its cognate lipase. The structure reveals how Lif uses a novel alpha-helical scaffold to embrace lipase, thereby creating an unusually extensive folding platform.

Aggregation mechanisms of cystatins: a comparative study of monellin and oryzacystatin.

Identification of diseases caused by protein misfolding has increased interest in the way proteins adopt non-native conformations and form aggregates. In this study we address the question of how proteins sharing the same fold respond to destabilizing environmental conditions. We have studied the behavior of two members of the cystatin superfamily, MNEI, a single chain monellin, and oryzacystatin_I, a plant cystatin. Despite the close similarity of their three-dimensional architecture, these two proteins aggregate in a different way: MNEI gives rise to amyloid aggregation whereas oryzacystatin_I yields amorphous aggregates. Mutants of oryzacystatin_I, designed to make it more similar to MNEI, generally behave like the parent protein, but a construct devoid of the disordered N- and C-terminal sequences does show a tendency to form amyloid fibers. Our results suggest that precise sequence details may be more important than the three-dimensional architecture in determining the type of aggregate formed. Oryzacystatin_I appears to be a very promising model system for further studies of protein aggregation.

Targeting an Intrinsically Disordered Protein by Covalent Modification.

Intrinsically disordered proteins (IDPs) play important roles in the regulation of cellular function and in disease, and thus they represent an important group of therapeutic targets. Yet, members of this "disorderome" have not yet been successfully targeted by drugs, primarily because traditional design principles cannot be applied to their highly dynamic, heterogeneous structural states. Binders developed against IDPs so far suffer from very weak binding and inability to act in a cellular context. Here, we describe a possible generic method for the targeting of IDPs via covalent modification, which could entail specific and strong binding and inhibitory potential, making such "warheads" reasonable starting points of drug-development efforts. We demonstrate this principle by addressing the cysteine-specific covalent modification of calpastatin, the IDP inhibitor of the calcium-dependent cysteine protease calpain. We describe the protocol for monitoring the covalent modification of the inhibitor, measuring the Ki of its inhibition and comparing it to the Kd of its interaction with the enzyme. Our premise is that the underlying principles can be easily adapted to screen for molecules targeting other, disease-related, IDPs in the future.

Calpain Purification Through Calpastatin and Calcium: Strategy and Procedures.

This chapter describes the strategy and procedures for the calcium-mediated affinity purification of calpain. The affinity capture method exploits the reversible binding properties of calpain's intrinsically disordered protein (IDP) inhibitor, calpastatin. IDPs are easily produced in heterologous expression hosts and purified to homogeneity. Combining these properties with in vivo biotinylation leads to a simplified purification strategy whereby biotinylated human calpastatin domain 1 (hCSD1) can capture calpain efficiently from a complex biological mixture with only a single chromatographic step and in a considerably reduced time. Our approach is generally applicable through the in vivo biotinylation of any IDP of interest in order to capture its binding partner in a calcium- and chelator-based protocol.

Affinity-based isolation of a bacterial lipase through steric chaperone interactions.

Lipases are important as additives in detergent formulations but their biocatalytic potential is increasingly exploited in the synthesis of high-added value chemicals, in fine-chemical production and in the pharmaceutical industry. Traditionally, conventional purification schemes comprise several chromatographic steps. Here we report a new purification procedure of the lipase (LipA) that is endogenously secreted by the Gram-negative bacterium Burkholderia glumae. This affinity purification combines the specific binding scaffold of a lipase-specific foldase (Lif) and the intrinsic resistance to chemical denaturation of LipA. The newly devised method is less labor-intensive, is fast, leads to a homogeneous preparation and can be easily scaled up. The novel and the conventional purification strategies were evaluated in parallel and characteristics of the B. glumae lipase were analyzed via CD spectroscopy. Lipopolysaccharide (LPS) was still present in the samples purified via the conventional purification scheme and was shown to increase the thermostability of the lipase.

Quantification of Intrinsically Disordered Proteins: A Problem Not Fully Appreciated.

Protein quantification is essential in a great variety of biochemical assays, yet the inherent systematic errors associated with the concentration determination of intrinsically disordered proteins (IDPs) using classical methods are hardly appreciated. Routinely used assays for protein quantification, such as the Bradford assay or ultraviolet absorbance at 280 nm, usually seriously misestimate the concentrations of IDPs due to their distinct and variable amino acid composition. Therefore, dependable method(s) have to be worked out/adopted for this task. By comparison to elemental analysis as the gold standard, we show through the example of four globular proteins and nine IDPs that the ninhydrin assay and the commercial QubitTM Protein Assay provide reliable data on IDP quantity. However, as IDPs can show extreme variation in amino acid composition and physical features not necessarily covered by our examples, even these techniques should only be used for IDPs following standardization. The far-reaching implications of these simple observations are demonstrated through two examples: (i) circular dichroism spectrum deconvolution, and (ii) receptor-ligand affinity determination. These actual comparative examples illustrate the potential errors that can be incorporated into the biophysical parameters of IDPs, due to systematic misestimation of their concentration. This leads to inaccurate description of IDP functions.

Protein Delivery into Plant Cells: Toward In vivo Structural Biology.

Understanding the biologically relevant structural and functional behavior of proteins inside living plant cells is only possible through the combination of structural biology and cell biology. The state-of-the-art structural biology techniques are typically applied to molecules that are isolated from their native context. Although most experimental conditions can be easily controlled while dealing with an isolated, purified protein, a serious shortcoming of such in vitro work is that we cannot mimic the extremely complex intracellular environment in which the protein exists and functions. Therefore, it is highly desirable to investigate proteins in their natural habitat, i.e., within live cells. This is the major ambition of in-cell NMR, which aims to approach structure-function relationship under true in vivo conditions following delivery of labeled proteins into cells under physiological conditions. With a multidisciplinary approach that includes recombinant protein production, confocal fluorescence microscopy, nuclear magnetic resonance (NMR) spectroscopy and different intracellular protein delivery strategies, we explore the possibility to develop in-cell NMR studies in living plant cells. While we provide a comprehensive framework to set-up in-cell NMR, we identified the efficient intracellular introduction of isotope-labeled proteins as the major bottleneck. Based on experiments with the paradigmatic intrinsically disordered proteins (IDPs) Early Response to Dehydration protein 10 and 14, we also established the subcellular localization of ERD14 under abiotic stress.

Affinity purification of human m-calpain through an intrinsically disordered inhibitor, calpastatin.

Calpains are calcium-activated proteases that have biomedical and biotechnological potential. Their activity is tightly regulated by their endogenous inhibitor, calpastatin that binds to the enzyme only in the presence of calcium. Conventional approaches to purify calpain comprise multiple chromatographic steps, and are labor-intensive, leading to low yields. Here we report a new purification procedure for the human m-calpain based on its reversible calcium-mediated interaction with the intrinsically disordered calpastatin. We exploit the specific binding properties of human calpastatin domain 1 (hCSD1) to physically capture human m-calpain from a complex biological mixture. The dissociation of the complex is mediated by chelating calcium, upon which heterodimeric calpain elutes while hCSD1 remains immobilized onto the stationary phase. This novel affinity-based purification was compared to the conventional multistep purification strategy and we find that it is robust, it yields a homogeneous preparation, it can be scaled up easily and it rests on a non-disruptive step that maintains close to physiological conditions that allow further biophysical and functional studies.

Glycation of Lysozyme by Glycolaldehyde Provides New Mechanistic Insights in Diabetes-Related Protein Aggregation.

Glycation occurs in vivo as a result of the nonenzymatic reaction of carbohydrates (and/or their autoxidation products) with proteins, DNA, or lipids. Protein glycation causes loss-of-function and, consequently, the development of diabetic-related diseases. Glycation also boosts protein aggregation, which can be directly related with the higher prevalence of aggregating diseases in diabetic people. However, the molecular mechanism connecting glycation with aggregation still remains unclear. Previously we described mechanistically how glycation of hen egg-white lysozyme (HEWL) with ribose induced its aggregation. Here we address the question of whether the ribose-induced aggregation is a general process or it depends on the chemical nature of the glycating agent. Glycation of HEWL with glycolaldehyde occurs through two different scenarios depending on the HEWL concentration regime (both within the micromolar range). At low HEWL concentration, non-cross-linking fluorescent advanced glycation end-products (AGEs) are formed on Lys side chains, which do not change the protein structure but inhibit its enzymatic activity. These AGEs have little impact on HEWL surface hydrophobicity and, therefore, a negligible effect on its aggregation propensity. Upon increasing HEWL concentration, the glycation mechanism shifts toward the formation of intermolecular cross-links, which triggers a polymerization cascade involving the formation of insoluble spherical-like aggregates. These results notably differ with the aggregation-modulation mechanism of ribosylated HEWL directed by hydrophobic interactions. Additionally, their comparison constitutes the first experimental evidence showing that the mechanism underlying the aggregation of a glycated protein depends on the chemical nature of the glycating agent.