ApoE Isoforms Inhibit Amyloid Aggregation of Proinflammatory Protein S100A9.

Increasing evidence suggests that the calcium-binding and proinflammatory protein S100A9 is an important player in neuroinflammation-mediated Alzheimer's disease (AD). The amyloid co-aggregation of S100A9 with amyloid-ß (Aß) is an important hallmark of this pathology. Apolipoprotein E (ApoE) is also known to be one of the important genetic risk factors of AD. ApoE primarily exists in three isoforms, ApoE2 (Cys112/Cys158), ApoE3 (Cys112/Arg158), and ApoE4 (Arg112/Arg158). Even though the difference lies in just two amino acid residues, ApoE isoforms produce differential effects on the neuroinflammation and activation of the microglial state in AD. Here, we aim to understand the effect of the ApoE isoforms on the amyloid aggregation of S100A9. We found that both ApoE3 and ApoE4 suppress the aggregation of S100A9 in a concentration-dependent manner, even at sub-stoichiometric ratios compared to S100A9. These interactions lead to a reduction in the quantity and length of S100A9 fibrils. The inhibitory effect is more pronounced if ApoE isoforms are added in the lipid-free state versus lipidated ApoE. We found that, upon prolonged incubation, S100A9 and ApoE form low molecular weight complexes with stochiometric ratios of 1:1 and 2:1, which remain stable under SDS-gel conditions. These complexes self-assemble also under the native conditions; however, their interactions are transient, as revealed by glutaraldehyde cross-linking experiments and molecular dynamics (MD) simulation. MD simulation demonstrated that the lipid-binding C-terminal domain of ApoE and the second EF-hand calcium-binding motif of S100A9 are involved in these interactions. We found that amyloids of S100A9 are cytotoxic to neuroblastoma cells, and the presence of either ApoE isoforms does not change the level of their cytotoxicity. A significant inhibitory effect produced by both ApoE isoforms on S100A9 amyloid aggregation can modulate the amyloid-neuroinflammatory cascade in AD.

Plasma IAPP-Autoantibody Levels in Alzheimer's Disease Patients Are Affected by APOE4 Status.

Pancreas-derived islet amyloid polypeptide (IAPP) crosses the blood-brain barrier and co-deposits with amyloid beta (Aß) in brains of type 2 diabetes (T2D) and Alzheimer's disease (AD) patients. Depositions might be related to the circulating IAPP levels, but it warrants further investigation. Autoantibodies recognizing toxic IAPP oligomers (IAPPO) but not monomers (IAPPM) or fibrils have been found in T2D, but studies on AD are lacking. In this study, we have analyzed plasma from two cohorts and found that levels of neither immunoglobulin (Ig) M, nor IgG or IgA against IAPPM or IAPPO were altered in AD patients compared with controls. However, our results show significantly lower IAPPO-IgA levels in apolipoprotein E (APOE) 4 carriers compared with non-carriers in an allele dose-dependent manner, and the decrease is linked to the AD pathology. Furthermore, plasma IAPP-Ig levels, especially IAPP-IgA, correlated with cognitive decline, C-reactive protein, cerebrospinal fluid Aß and tau, neurofibrillary tangles, and brain IAPP exclusively in APOE4 non-carriers. We speculate that the reduction in IAPPO-IgA levels may be caused by increased plasma IAPPO levels or masked epitopes in APOE4 carriers and propose that IgA and APOE4 status play a specific role in clearance of circulatory IAPPO, which may influence the amount of IAPP deposition in the AD brain.

Tandem Ring Opening/Intramolecular [2 + 2] Cycloaddition Reaction for the Synthesis of Cyclobutane Fused Thiazolino-2-Pyridones.

Reaction of thiazoline fused 2-pyridones with alkyl halides in the presence of cesium carbonate opens the thiazoline ring via S-alkylation and generates N-alkenyl functionalized 2-pyridones. In the reaction with propargyl bromide, the thiazoline ring opens and subsequently closes via a [2 + 2] cycloaddition between an in situ generated allene and the α,ß-unsaturated methyl ester. This method enabled the synthesis of a variety of cyclobutane fused thiazolino-2-pyridones, of which a few analogues inhibit amyloid ß1-40 fibril formation. Furthermore, other analogues were able to bind mature α-synuclein and amyloid ß1-40 fibrils. Several thiazoline fused 2-pyridones with biological activity tolerate this transformation, which in addition provides an exocyclic alkene as a potential handle for tuning bioactivity.

K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones.

We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-ß fibrils associated with Alzheimer's disease, while others bind to mature amyloid-ß and α-synuclein fibrils.

Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine Fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-ß Fibrils.

A BF3·OEt2 catalyzed intramolecular Povarov reaction was used to synthesize 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with several O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid ß fibrils in vitro. Analogues substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.

Morphological analysis of Apolipoprotein E binding to Aß Amyloid using a combination of Surface Plasmon Resonance, Immunogold Labeling and Scanning Electron Microscopy.

BACKGROUND: Immunogold labeling in combination with transmission electron microscopy analysis is a technique frequently used to correlate high-resolution morphology studies with detailed information regarding localization of specific antigens. Although powerful, the methodology has limitations and it is frequently difficult to acquire a stringent system where unspecific low-affinity interactions are removed prior to analysis. RESULTS: We here describe a combinatorial strategy where surface plasmon resonance and immunogold labeling are used followed by a direct analysis of the sensor-chip surface by scanning electron microscopy. Using this approach, we have probed the interaction between amyloid-ß fibrils, associated to Alzheimer's disease, and apolipoprotein E, a well-known ligand frequently found co-deposited to the fibrillar form of Aß in vivo. The results display a lateral binding of ApoE along the amyloid fibrils and illustrates how the gold-beads represent a good reporter of the binding. CONCLUSIONS: This approach exposes a technique with generic features which enables both a quantitative and a morphological evaluation of a ligand-receptor based system. The methodology mediates an advantage compared to traditional immunogold labeling since all washing steps can be monitored and where a high stringency can be maintained throughout the experiment.

Pyridine-Fused 2-Pyridones via Povarov and A3 Reactions: Rapid Generation of Highly Functionalized Tricyclic Heterocycles Capable of Amyloid Fibril Binding.

We here describe the use of three-component reactions to synthesize tricyclic pyridine ring-fused 2-pyridones. The developed protocols have a wide substrate scope and allow for the installation of diverse chemical functionalities on the tricyclic central fragment. Several of these pyridine-fused rigid polyheterocycles are shown to bind to Aß and α-synuclein fibrils, which are associated with neurodegenerative diseases.

Interspecies Variation between Fish and Human Transthyretins in Their Binding of Thyroid-Disrupting Chemicals.

Thyroid-disrupting chemicals (TDCs) are xenobiotics that can interfere with the endocrine system and cause adverse effects in organisms and their offspring. TDCs affect both the thyroid gland and regulatory enzymes associated with thyroid hormone homeostasis. Transthyretin (TTR) is found in the serum and cerebrospinal fluid of vertebrates, where it transports thyroid hormones. Here, we explored the interspecies variation in TDC binding to human and fish TTR (exemplified by Gilthead seabream ( Sparus aurata)). The in vitro binding experiments showed that TDCs bind with equal or weaker affinity to seabream TTR than to the human TTR, in particular, the polar TDCs (>500-fold lower affinity). Crystal structures of the seabream TTR-TDC complexes revealed that all TDCs bound at the thyroid binding sites. However, amino acid substitution of Ser117 in human TTR to Thr117 in seabream prevented polar TDCs from binding deep in the hormone binding cavity, which explains their low affinity to seabream TTR. Molecular dynamics and in silico alanine scanning simulation also suggested that the protein backbone of seabream TTR is more rigid than the human one and that Thr117 provides fewer electrostatic contributions than Ser117 to ligand binding. This provides an explanation for the weaker affinities of the ligands that rely on electrostatic interactions with Thr117. The lower affinities of TDCs to fish TTR, in particular the polar ones, could potentially lead to milder thyroid-related effects in fish.

Structure-Based Virtual Screening Protocol for in Silico Identification of Potential Thyroid Disrupting Chemicals Targeting Transthyretin.

Thyroid disruption by xenobiotics is associated with a broad spectrum of severe adverse outcomes. One possible molecular target of thyroid hormone disrupting chemicals (THDCs) is transthyretin (TTR), a thyroid hormone transporter in vertebrates. To better understand the interactions between TTR and THDCs, we determined the crystallographic structures of human TTR in complex with perfluorooctanesulfonic acid (PFOS), perfluorooctanoic acid (PFOA), and 2,2',4,4'-tetrahydroxybenzophenone (BP2). The molecular interactions between the ligands and TTR were further characterized using molecular dynamics simulations. A structure-based virtual screening (VS) protocol was developed with the intention of providing an efficient tool for the discovery of novel TTR-binders from the Tox21 inventory. Among the 192 predicted binders, 12 representatives were selected, and their TTR binding affinities were studied with isothermal titration calorimetry, of which seven compounds had binding affinities between 0.26 and 100 µM. To elucidate structural details in their binding to TTR, crystal structures were determined of TTR in complex with four of the identified compounds including 2,6-dinitro-p-cresol, bisphenol S, clonixin, and triclopyr. The compounds were found to bind in the TTR hormone binding sites as predicted. Our results show that the developed VS protocol is able to successfully identify potential THDCs, and we suggest that it can be used to propose THDCs for future toxicological evaluations.

The N-terminal region of amyloid ß controls the aggregation rate and fibril stability at low pH through a gain of function mechanism.

Alzheimer's disease is linked to a pathological polymerization of the endogenous amyloid ß-peptide (Aß) that ultimately forms amyloid plaques within the human brain. We used surface plasmon resonance (SPR) to measure the kinetic properties of Aß fibril formation under different conditions during the polymerization process. For all polymerization processes, a critical concentration of free monomers, as defined by the dissociation equilibrium constant (K(D)), is required for the buildup of the polymer, for example, amyloid fibrils. At concentrations below the K(D), polymerization cannot occur. However, the K(D) for Aß has previously been shown to be several orders of magnitude higher than the concentrations found in the cerebrospinal and interstitial fluids of the human brain, and the mechanism by which Aß amyloid forms in vivo has been a matter of debate. Using SPR, we found that the K(D) of Aß dramatically decreases as a result of lowering the pH. Importantly, this effect enables Aß to polymerize within a picomolar concentration range that is close to the physiological Aß concentration within the human brain. The stabilizing effect is dynamic, fully reversible, and notably pronounced within the pH range found within the endosomal and lysosomal pathways. Through sequential truncation, we show that the N-terminal region of Aß contributes to the enhanced fibrillar stability due to a gain of function mechanism at low pH. Our results present a possible route for amyloid formation at very low Aß concentrations and raise the question of whether amyloid formation in vivo is restricted to a low pH environment. These results have general implications for the development of therapeutic interventions.

Transthyretin is dysregulated in preeclampsia, and its native form prevents the onset of disease in a preclinical mouse model.

Preeclampsia is a major pregnancy complication with potential short- and long-term consequences for both mother and fetus. Understanding its pathogenesis and causative biomarkers is likely to yield insights for prediction and treatment. Herein, we provide evidence that transthyretin, a transporter of thyroxine and retinol, is aggregated in preeclampsia and is present at reduced levels in sera of preeclamptic women, as detected by proteomic screen. We demonstrate that transthyretin aggregates form deposits in preeclampsia placental tissue and cause apoptosis. By using in vitro approaches and a humanized mouse model, we provide evidence for a causal link between dysregulated transthyretin and preeclampsia. Native transthyretin inhibits all preeclampsia-like features in the humanized mouse model, including new-onset proteinuria, increased blood pressure, glomerular endotheliosis, and production of anti-angiogenic factors. Our findings suggest that a focus on transthyretin structure and function is a novel strategy to understand and combat preeclampsia.

The role of pro-inflammatory S100A9 in Alzheimer's disease amyloid-neuroinflammatory cascade.

Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer's disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aß. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aß and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aß protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aß, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aß. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aß aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.

Ca(2+) enhances Aß polymerization rate and fibrillar stability in a dynamic manner.

Identifying factors that affect the self-assembly of Aß (amyloid-ß peptide) is of utmost importance in the quest to understand the molecular mechanisms causing AD (Alzheimer's disease). Ca(2+) has previously been shown to accelerate both Aß fibril nucleation and maturation, and dysregulated Ca(2+) homoeostasis frequently correlates with development of AD. The mechanisms regarding Ca(2+) binding, as well as its effect on fibril kinetics, are not fully understood. Using a polymerization assay we show that Ca(2+) in a dynamic and reversible manner enhances both the elongation rate and fibrillar stability, where specifically the 'dock and lock' phase mechanism is enhanced. Through NMR analysis we found that Ca(2+) affects the fibrillar architecture. In addition, and unexpectedly, we found that Ca(2+) does not bind the free Aß monomer. This implies that Ca(2+) binding requires an architecture adopted by assembled peptides, and consequently is mediated through intermolecular interactions between adjacent peptides. This gives a mechanistic explanation to the enhancing effect on fibril maturation and indicates structural similarities between prefibrillar structures and mature amyloid. Taken together we show how Ca(2+) levels affect the delicate equilibrium between the monomeric and assembled Aß and how fluctuations in vivo may contribute to development and progression of the disease.

Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes.

Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

Structural topology and activation of an initial adenylate kinase-substrate complex.

Enzymatic activity is ultimately defined by the structure, chemistry, and dynamics of the Michaelis complex. A large number of experimentally determined structures between enzymes and substrates, substrate analogues, or inhibitors exist. However, transient, short-lived encounter and equilibrium structures also play fundamental roles during enzymatic reaction cycles. Such structures are inherently difficult to study with conventional experimental techniques. The enzyme adenylate kinase undergoes major conformational rearrangements in response to binding of its substrates, ATP and AMP. ATP is sandwiched between two binding surfaces in the closed and active enzyme conformation. Thus, adenylate kinase harbors two spatially distant surfaces in the substrate free open conformation, of which one is responsible for the initial interaction with ATP. Here, we have performed primarily nuclear magnetic resonance experiments on Escherichia coli adenylate kinase (AK(eco)) variants that allowed identification of the site responsible for the initial ATP interaction. This allowed a characterization of the structural topology of an initial equilibrium complex between AK(eco) and ATP. On the basis of the results, we suggest that the ATP binding mechanism for AK(eco) is a mixture between "induced fit" and "conformational selection" models. It is shown that ATP is activated in the initial enzyme-bound complex because it displays an appreciable rate of nonproductive ATP hydrolysis. In summary, our results provide novel structural and functional insights into adenylate kinase catalysis.

Modulation of α-synuclein fibrillization by ring-fused 2-pyridones: templation and inhibition involve oligomers with different structure.

In a recent study we discovered that a ring-fused 2-pyridone compound triggered fibrillization of a key protein in Parkinson's disease, α-synuclein. To reveal how variations in compound structure affect protein aggregation, we now prepared a number of strategic analogs and tested their effects on α-synuclein amyloid fiber formation in vitro. We find that, in contrast to the earlier templating effect, some analogs inhibit α-synuclein fibrillization. For both templating and inhibiting compounds, the key species formed in the reactions are α-synuclein oligomers that contain compound. Despite similar macroscopic appearance, the templating and inhibiting oligomers are distinctly different in secondary structure content. When the inhibitory oligomers are added in seed amounts, they inhibit fresh α-synuclein aggregation reactions. Our study demonstrates that small chemical changes to the same central fragment can result in opposite effects on protein aggregation.

Mechanisms of protein oligomerization: inhibitor of functional amyloids templates α-synuclein fibrillation.

Small organic molecules that inhibit functional bacterial amyloid fibers, curli, are promising new antibiotics. Here we investigated the mechanism by which the ring-fused 2-pyridone FN075 inhibits fibrillation of the curli protein CsgA. Using a variety of biophysical techniques, we found that FN075 promotes CsgA to form off-pathway, non-amyloidogenic oligomeric species. In light of the generic properties of amyloids, we tested whether FN075 would also affect the fibrillation reaction of human α-synuclein, an amyloid-forming protein involved in Parkinson's disease. Surprisingly, FN075 stimulates α-synuclein amyloid fiber formation as measured by thioflavin T emission, electron microscopy (EM), and atomic force microscopy (AFM). NMR data on (15)N-labeled α-synuclein show that upon FN075 addition, α-synuclein oligomers with 7 nm radius form in which the C-terminal 40 residues remain disordered and solvent exposed. The polypeptides in these oligomers contain ß-like secondary structure, and the oligomers are detectable by AFM, EM, and size-exclusion chromatography (SEC). Taken together, FN075 triggers oligomer formation of both proteins: in the case of CsgA, the oligomers do not proceed to fibers, whereas for α-synuclein, the oligomers are poised to rapidly form fibers. We conclude that there is a fine balance between small-molecule inhibition and templation that depends on protein chemistry.

Characterization of SPINK9, a KLK5-specific inhibitor expressed in palmo-plantar epidermis.

SPINK9, a Kazal-type serine protease inhibitor, is almost exclusively expressed in the palmo-plantar epidermis. SPINK9 selectively inhibits kallikrein-related peptidase 5 (KLK5), no other target enzyme is known at present. In this study, we defined the reactive loop to residues 48 and 49 of SPINK9 and characterized the inhibition and binding of different SPINK9 variants towards KLK5, KLK7, KLK8 and KLK14. Substitutions of single amino acids in the reactive loop had a large impact on both inhibitory efficiency and specificity. Binding studies showed that it is mainly the dissociation rate that is affected by the amino acid substitutions. The inhibitory effect of wild-type SPINK9 was clearly pH-dependent with an improved effect at a pH similar to that of the outer layers of the skin. Modeling of the enzyme-inhibitor complexes showed that the reactive loop of SPINK9 fits very well into the deep negatively charged binding pocket of KLK5. A decrease in pH protonates His48 of the wild-type protein resulting in a positively charged residue, thereby explaining the observed decreased dissociation rate. Interestingly, substitution with a positively charged amino acid at position 48 resulted in a more efficient inhibitor at higher pH.

The effect of capsular repair, bone block healing, and position on the results of the Bristow-Latarjet procedure (study III): long-term follow-up in 319 shoulders.

BACKGROUND: We evaluated the results of the May modification of the Bristow-Latarjet procedure ("coracoid in standing position") in 319 shoulders with respect to (1) coracoid healing and position and (2) surgical treatment of the joint capsule. METHODS: From 1980 until 2004, all shoulders with a Bristow-Latarjet repair were registered at our hospital. This study consists of 3 different cohorts with respect to follow-up. Series 1, 118 shoulders operated on during 1980 through 1985, had 15 years' radiographic and clinical follow-up. Series 2, 167 shoulders that had surgery during 1986 through 1999, underwent retrospective follow-up by a questionnaire and scores--Western Ontario Shoulder Instability Index; Disabilities of the Arm, Shoulder and Hand; and Subjective Shoulder Value--after 10 to 23 years. Series 3, 34 shoulders treated during 2000 through 2004, with an added modified Bankart repair ("capsulopexy") in 33 shoulders, were prospectively followed up for 5 to 8 years with the same questionnaire and scores as series 2. RESULTS: Of 319 shoulders, 16 (5%) had 1 or more redislocations and 3 of these (1%) had revision surgery because of remaining instability. One or more subluxations were reported in 41 shoulders (13%). The worst scores were found in 16 shoulders with 2 or more subluxations (P < .001). Radiographs showed bony healing in 246 of 297 shoulders (83%), fibrous union in 34 (13%), migration by 0.5 cm or more in 14 (5%), and no visualization in 3 (1%). Five of six shoulders that had the transplant positioned 1 cm or more medial to the glenoid rim had redislocations (83%, P = .001). Shoulders with migrated transplants did not differ from those with bony or fibrous healing with respect to redislocations and subluxations. When just a horizontal capsular shift was added to the transfer, the recurrence rate (redislocations or subluxations) decreased, with 2 of 53 (4%)compared with 37 of 208 (18%) with just anatomic closure of the capsule (P = .005), and the Western Ontario Shoulder Instability Index score improved (92 vs 85.6, P = .048). In total, for 307 of 319 shoulders (96%), patients were satisfied or very satisfied at final follow-up. CONCLUSION: The open Bristow-Latarjet procedure yields good and consistent results, with bony fusion of the coracoid in 83%. A position of the coracoid 1 cm or more medial to the rim meant significantly more recurrences. The rate of recurrences decreased and subjective results improved when a horizontal capsular shift was added to the coracoid transfer.

Endogenous Human Proteins Interfering with Amyloid Formation.

Amyloid formation is a pathological process associated with a wide range of degenerative disorders, including Alzheimer's disease, Parkinson's disease, and diabetes mellitus type 2. During disease progression, abnormal accumulation and deposition of proteinaceous material are accompanied by tissue degradation, inflammation, and dysfunction. Agents that can interfere with the process of amyloid formation or target already formed amyloid assemblies are consequently of therapeutic interest. In this context, a few endogenous proteins have been associated with an anti-amyloidogenic activity. Here, we review the properties of transthyretin, apolipoprotein E, clusterin, and BRICHOS protein domain which all effectively interfere with amyloid in vitro, as well as displaying a clinical impact in humans or animal models. Their involvement in the amyloid formation process is discussed, which may aid and inspire new strategies for therapeutic interventions.