Transthyretin monomers: a new plasma biomarker for pre-symptomatic transthyretin-related amyloidosis.

BACKGROUND: Genotyping and amyloid fibril detection in tissues are generally considered the diagnostic gold standard in transthyretin-related amyloidosis. Patients carry less stable TTR homotetramers prone to dissociation into non-native monomers, which rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Thus, the initial event of the amyloid cascade produces the smallest transthyretin species: the monomers. This creates engineering opportunities for diagnosis that remain unexplored. METHODS: We hypothesise that molecular sieving represents a promising method for isolating and concentrating trace TTR monomers from the tetramers present in plasma samples. Subsequently, immunodetection can be utilised to distinguish monomeric TTR from other low molecular weight proteins within the adsorbed fraction. A two-step assay was devised (ImmunoSieve assay), combining molecular sieving and immunodetection for sensing monomeric transthyretin. This assay was employed to analyse plasma microsamples from 10 individuals, including 5 pre-symptomatic carriers of TTR-V30M, the most prevalent amyloidosis-associated TTR variant worldwide, and 5 healthy controls. RESULTS: The ImmunoSieve assay enable sensitive detection of monomeric transthyretin in plasma microsamples. Moreover, the circulating monomeric TTR levels were significantly higher in carriers of amyloidogenic TTR mutation. CONCLUSIONS: Monomeric TTR can function as a biomarker for evaluating disease progression and assessing responses to therapies targeted at stabilising native TTR.

Inhibitors of Transthyretin Amyloidosis: How to Rank Drug Candidates Using X-ray Crystallography Data.

Amyloidosis is a group of protein misfolding diseases, which include spongiform encephalopathies, Alzheimer's disease and transthyretin (TTR) amyloidosis; all of them are characterized by extracellular deposits of an insoluble fibrillar protein. TTR amyloidosis is a highly debilitating and life-threatening disease. Patients carry less stable TTR homotetramers that are prone to dissociation into non-native monomers, which in turn rapidly self-assemble into oligomers and, ultimately, amyloid fibrils. Liver transplantation to induce the production of wild-type TTR was the only therapeutic strategy until recently. A promising approach to ameliorate transthyretin (TTR) amyloidosis is based on the so-called TTR kinetic stabilizers. More than 1000 TTR stabilizers have already been tested by many research groups, but the diversity of experimental techniques and conditions used hampers an objective prioritization of the compounds. One of the most reliable and unambiguous techniques applied to determine the structures of the TTR/drug complexes is X-ray diffraction. Most of the potential inhibitors bind in the TTR channel and the crystal structures reveal the atomic details of the interaction between the protein and the compound. Here we suggest that the stabilization effect is associated with a compaction of the quaternary structure of the protein and propose a scoring function to rank drugs based on X-ray crystallography data.

The mammalian peroxisomal membrane is permeable to both GSH and GSSG - Implications for intraperoxisomal redox homeostasis.

Despite the large amounts of H2O2 generated in mammalian peroxisomes, cysteine residues of intraperoxisomal proteins are maintained in a reduced state. The biochemistry behind this phenomenon remains unexplored, and simple questions such as "is the peroxisomal membrane permeable to glutathione?" or "is there a thiol-disulfide oxidoreductase in the organelle matrix?" still have no answer. We used a cell-free in vitro system to equip rat liver peroxisomes with a glutathione redox sensor. The organelles were then incubated with glutathione solutions of different redox potentials and the oxidation/reduction kinetics of the redox sensor was monitored. The data suggest that the mammalian peroxisomal membrane is promptly permeable to both reduced and oxidized glutathione. No evidence for the presence of a robust thiol-disulfide oxidoreductase in the peroxisomal matrix could be found. Also, prolonged incubation of organelle suspensions with glutaredoxin 1 did not result in the internalization of the enzyme. To explore a potential role of glutathione in intraperoxisomal redox homeostasis we performed kinetic simulations. The results suggest that even in the absence of a glutaredoxin, glutathione is more important in protecting cysteine residues of matrix proteins from oxidation by H2O2 than peroxisomal catalase itself.

Metal-Organic Frameworks as Sensors for Human Amyloid Diseases.

Metal-organic frameworks (MOFs) are versatile compounds with emergent applications in the fabrication of biosensors for amyloid diseases. They hold great potential in biospecimen protection and unprecedented probing capabilities for optical and redox receptors. In this Review, we summarize the main methodologies employed in the fabrication of MOF-based sensors for amyloid diseases and collect all available data in the literature related to their performance (detection range, limit of detection, recovery, time of analysis, among other parameters). Nowadays, MOF sensors have evolved to a point where they can, in some cases, outperform technologies employed in the detection of several amyloid biomarkers (amyloid ß peptide, α-synuclein, insulin, procalcitonin, and prolactin) present in biological fluids, such as cerebrospinal fluid and blood. A special emphasis has been given by researchers on Alzheimer's disease monitoring to the detriment of other amyloidosis that are underexploited despite their societal relevance (e.g., Parkinson's disease). There are still important obstacles to overcome in order to selectively detect the various peptide isoforms and soluble amyloid species associated with Alzheimer's disease. Furthermore, MOF contrast agents for imaging peptide soluble oligomers in living humans are also scarce (if not nonexistent), and action in this direction is unquestionably required to clarify the contentious link between the amyloidogenic species and the disease, guiding research toward the most promising therapeutic strategies.

Antibacterial and Antibiofilm Properties of Self-Assembled Dipeptide Nanotubes.

Over recent decades, multidrug-resistant pathogens have become a global concern, with WHO even considering it one of the biggest threats to global health, food security, and development today, which led to the search for alternative antibacterial agents. A special class is formed by peptides composed by the diphenylalanine motif whose antibacterial properties result from their supramolecular arrangement into nanotubes. However, several other dipeptides that also form nanotubes have been largely overlooked. Here, we present the antibacterial activity of four dipeptide nanotubes. The results point to diverse mechanisms through which dipeptide nanotubes exert their effect against bacteria. Antibacterial activity was similar for dipeptide nanotubes sufficiently wide to allow water flux while dipeptides displaying smaller channels were inactive. This suggests that two of the tested dipeptides, L-Phe-L-Phe (FF, diphenylalanine) and L-Leu-L-Ser (LS), are pore forming structures able to induce membrane permeation and affect cellular hydration and integrity. Of these two dipeptides, only FF demonstrated potential to inhibit biofilm formation. The amyloid-like nature and hydrophobicity of diphenylalanine assemblies are probably responsible for their adhesion to cell surfaces preventing biofilm formation and bacteria attachment.

Aß31-35 Decreases Neprilysin-Mediated Alzheimer's Amyloid-ß Peptide Degradation.

Alzheimer's disease is associated with the deposition of extracellular senile plaques, made primarily of amyloid-ß (Aß), particularly peptides Aß1-42 and Aß1-40. Neprilysin, or neutral endopeptidase (NEP), catalyzes proteolysis of the amyloid peptides (Aß) and is recognized as one of the major regulators of the levels of these peptides in the brain, preventing Aß accumulation and plaque formation. Here, we used a combination of techniques to elucidate the mechanism of Aß binding and cleavage by NEP. Our findings indicate that the Aß31-X cleavage products remain bound to the neprilysin active site, reducing proteolytic activity. Interestingly, it was already shown that this Aß31-35 sequence is also critical for recognition of Aß peptides by other targets, such as the serpin-enzyme complex receptor in neuronal cells.

Tetracyclic Thioxanthene Derivatives: Studies on Fluorescence and Antitumor Activity.

Thioxanthones are bioisosteres of the naturally occurring xanthones. They have been described for multiple activities, including antitumor. As such, the synthesis of a library of thioxanthones was pursued, but unexpectedly, four tetracyclic thioxanthenes with a quinazoline-chromene scaffold were obtained. These compounds were studied for their human tumor cell growth inhibition activity, in the cell lines A375-C5, MCF-7 and NCI-H460. Photophysical studies were also performed. Two of the compounds displayed GI50 values below 10 µM for the three tested cell lines, and structure-activity relationship studies were established. Three compounds presented similar wavelengths of absorption and emission, characteristic of dyes with a push-pull character. The structures of two compounds were elucidated by X-ray crystallography. Two tetracyclic thioxanthenes emerged as hit compounds. One of the two compounds accumulated intracellularly as a bright fluorescent dye in the green channel, as analyzed by both fluorescence microscopy and flow cytometry, making it a promising theranostic cancer drug candidate.

Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53.

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1-3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1-3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

Metal-organic frameworks: a future toolbox for biomedicine?

The present review focuses on the use of Metal-Organic Frameworks, (MOFs) highlighting the most recent developments in the biological field. This review assesses, in the first instance, the cytotoxicity of MOFs (particularly those used for various biological applications described throughout this review), and shows that for standard MOFs based on metals already present in active molecules of the human body, toxicity is not a significant limitation. Here we underline the MIL-, UiO- and ZIF-series of MOFs which remain until now the most used materials in drug delivery of active pharmaceutical ingredients (APIs), such as antitumourals or retroviral drugs (with high loading and slow release time). Porosity remains undoubtedly the most studied key property of MOFs, that allows the protection of active biomolecules such as enzymes or the development of antimicrobial materials. Emphasis is given on the usage of MOFs for the detection of biomarkers in biological fluids such as urine and blood (detection of cystinuria, identification of penicillin anaphylaxis, urea, bilirubin, biomarkers related to human intoxication, tumoural indicators, among several others), for which a number of simple devices (such as paper strips) were developed. Despite the remarkable and promising results presented in recent years, the literature remains scarce (mostly non-existent) in terms of direct comparison of these novel technologies with the solutions presently available in the market. Action on this side may make the difference in the next years concerning research on MOFs, to see if some of these materials may reach the end-user as new and more efficient treatments or detection approaches.

Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer.

Transthyretin (TTR) is a homotetrameric protein involved in human amyloidosis, including familial amyloid polyneuropathy (FAP). Discovering small-molecule stabilizers of the TTR tetramer is a therapeutic strategy for these diseases. Tafamidis, the only approved drug for FAP treatment, is not effective for all patients. Herein, we discovered that benzbromarone (BBM), a uricosuric drug, is an effective TTR stabilizer and inhibitor against TTR amyloid fibril formation. BBM rendered TTR more resistant to urea denaturation, similarly to iododiflunisal (IDIF), a very potent TTR stabilizer. BBM competes with thyroxine for binding in the TTR central channel, with an IC50 similar to IDIF and tafamidis. Results obtained by isothermal titration calorimetry (ITC) demonstrated that BBM binds TTR with an affinity similar to IDIF, tolcapone and tafamidis, confirming BBM as a potent binder of TTR. The crystal structure of the BBM-TTR complex shows two molecules binding deeply in the thyroxine binding channel, forming strong intermonomer hydrogen bonds and increasing the stability of the TTR tetramer. Finally, kinetic analysis of the ability of BBM to inhibit TTR fibrillogenesis at acidic pH and comparison with other stabilizers revealed that benzbromarone is a potent inhibitor of TTR amyloidogenesis, adding a new interesting scaffold for drug design of TTR stabilizers.

Clinical and Genetic Analysis of Children with Kartagener Syndrome.

Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by dysfunction of motile cilia causing ineffective mucus clearance and organ laterality defects. In this study, two unrelated Portuguese children with strong PCD suspicion underwent extensive clinical and genetic assessments by whole-exome sequencing (WES), as well as ultrastructural analysis of cilia by transmission electron microscopy (TEM) to identify their genetic etiology. These analyses confirmed the diagnostic of Kartagener syndrome (KS) (PCD with situs inversus). Patient-1 showed a predominance of the absence of the inner dynein arms with two disease-causing variants in the CCDC40 gene. Patient-2 showed the absence of both dynein arms and WES disclosed two novel high impact variants in the DNAH5 gene and two missense variants in the DNAH7 gene, all possibly deleterious. Moreover, in Patient-2, functional data revealed a reduction of gene expression and protein mislocalization in both genes' products. Our work calls the researcher's attention to the complexity of the PCD and to the possibility of gene interactions modelling the PCD phenotype. Further, it is demonstrated that even for well-known PCD genes, novel pathogenic variants could have importance for a PCD/KS diagnosis, reinforcing the difficulty of providing genetic counselling and prenatal diagnosis to families.

Tegsedi (Inotersen): An Antisense Oligonucleotide Approved for the Treatment of Adult Patients with Hereditary Transthyretin Amyloidosis.

Tegsedi (Inotersen) is a chemically modified antisense oligonucleotide that inhibits the hepatic production of transthyretin (TTR). Several single-point mutations in TTR destabilize its structure, leading to the aggregation and accumulation of amyloid deposits in the nervous system, heart, kidneys and eyes. In July 2018, Tegsedi was approved by the European Commission for use in adults with stage one and two polyneuropathies. Later on, in October 2018, the FDA and Health Canada also approved its use for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (hATTR) in adults in the U.S. and Canada. Tegsedi was developed by Ionis Pharmaceuticals, the company that holds the global marketing license, together with its subsidiary Akcea Therapeutics.

Alzheimer's Aß1-40 peptide degradation by thermolysin: evidence of inhibition by a C-terminal Aß product.

The interaction of the amyloid-ß peptide (Aß) with thermolysin (TLN) was investigated by X-ray crystallography. Structural models of the complexes of TLN with several Aß fragments show that, despite the numerous possible cleavage sites of the Aß sequence, the C-terminal product of Ala30-Ile31 cleavage does not dissociate, thus inhibiting the enzyme. The high similarity between the TLN structural motif and neprilysin (NEP), the most extensively studied peptidase associated with Aß clearance, suggests that NEP should be more efficient against Aß polymorphs where Ala30-Ile31 is inaccessible, which is in agreement with studies in living mice that point to the limited role of NEP in degrading soluble Aß and its higher ability to degrade insoluble and/or oligomeric Aß forms, producing only the Aß10-37 intermediate.

Bis-Indolyl Benzenoids, Hydroxypyrrolidine Derivatives and Other Constituents from Cultures of the Marine Sponge-Associated Fungus Aspergillus candidus KUFA0062.

A previously unreported bis-indolyl benzenoid, candidusin D (2e) and a new hydroxypyrrolidine alkaloid, preussin C (5b) were isolated together with fourteen previously described compounds: palmitic acid, clionasterol, ergosterol 5,8-endoperoxides, chrysophanic acid (1a), emodin (1b), six bis-indolyl benzenoids including asterriquinol D dimethyl ether (2a), petromurin C (2b), kumbicin B (2c), kumbicin A (2d), 2″-oxoasterriquinol D methyl ether (3), kumbicin D (4), the hydroxypyrrolidine alkaloid preussin (5a), (3S, 6S)-3,6-dibenzylpiperazine-2,5-dione (6) and 4-(acetylamino) benzoic acid (7), from the cultures of the marine sponge-associated fungus Aspergillus candidus KUFA 0062. Compounds 1a, 2a-e, 3, 4, 5a-b, and 6 were tested for their antibacterial activity against Gram-positive and Gram-negative reference and multidrug-resistant strains isolated from the environment. Only 5a exhibited an inhibitory effect against S. aureus ATCC 29213 and E. faecalis ATCC29212 as well as both methicillin-resistant S. aureus (MRSA) and vancomycin-resistant enterococci (VRE) strains. Both 1a and 5a also reduced significant biofilm formation in E. coli ATCC 25922. Moreover, 2b and 5a revealed a synergistic effect with oxacillin against MRSA S. aureus 66/1 while 5a exhibited a strong synergistic effect with the antibiotic colistin against E. coli 1410/1. Compound 1a, 2a-e, 3, 4, 5a-b, and 6 were also tested, together with the crude extract, for cytotoxic effect against eight cancer cell lines: HepG2, HT29, HCT116, A549, A 375, MCF-7, U-251, and T98G. Except for 1a, 2a, 2d, 4, and 6, all the compounds showed cytotoxicity against all the cancer cell lines tested.

New Polyketides and New Benzoic Acid Derivatives from the Marine Sponge-Associated Fungus Neosartorya quadricincta KUFA 0081.

Two new pentaketides, including a new benzofuran-1-one derivative (1) and a new isochromen-1-one (5), and seven new benzoic acid derivatives, including two new benzopyran derivatives (2a, b), a new benzoxepine derivative (3), two new chromen-4-one derivatives (4b, 7) and two new benzofuran derivatives (6a, b), were isolated, together with the previously reported 2,3-dihydro-6-hydroxy-2,2-dimethyl-4H-1-benzopyran-4-one (4a), from the culture of the marine sponge-associated fungus Neosartorya quadricincta KUFA 0081. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compounds 1, 2a, 4b, 5, 6a and 7, the absolute configurations of their stereogenic carbons were determined by an X-ray crystallographic analysis. None of the isolated compounds were active in the tests for antibacterial activity against Gram-positive and Gram-negative bacteria, as well as multidrug-resistant isolates from the environment (MIC > 256 µg/mL), antifungal activity against yeast (Candida albicans ATTC 10231), filamentous fungus (Aspergillus fumigatus ATTC 46645) and dermatophyte (Trichophyton rubrum FF5) (MIC > 512 µg/mL) and in vitro growth inhibitory activity against the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines (GI50 > 150 µM) by the protein binding dye SRB method.

Secondary Metabolites from the Culture of the Marine Sponge-Associated Fungi Talaromyces tratensis and Sporidesmium circinophorum.

Wortmin (1), meso-1,4-bis(4-methoxybenzyl)-2,3-butanediol (2), and a new isocoumarin derivative tratenopyrone (3) were isolated from the marine sponge-associated fungus Talaromyces tratensis KUFA 0091. A new diphenyl ether derivative, circinophoric acid (4), was isolated, together with the previously reported anthraquinones catenarin and physcion, the benzophenone monomethylsoluchrin, and ß-ergosterol-5,8-endoperoxide, from the marine sponge-associated fungus Sporidesmium circinophorum KUFA 0043. The structures of the new compounds were established based on an extensive analysis of 1D and 2D NMR spectra, and, in the case of compounds 2-4, also by X-ray analysis. All of the isolated compounds were tested for their antibacterial activity against Gram-positive and Gram-negative bacteria, and multidrug-resistant isolates from the environment, as well as for their anti-quorum sensing based on the pigment production of Chromobacterium violaceum ATCC 31523. None of the compounds exhibited either antibacterial (MIC > 256 µg/mL) or anti-quorum sensing activities. The compounds were also inactive in the antifungal (MIC > 512 µg/mL) and cancer cell line (GI50 > 150 µM) assays.

Structure-guided engineering of molinate hydrolase for the degradation of thiocarbamate pesticides.

Molinate is a recalcitrant thiocarbamate used to control grass weeds in rice fields. The recently described molinate hydrolase, from Gulosibacter molinativorax ON4T, plays a key role in the only known molinate degradation pathway ending in the formation of innocuous compounds. Here we report the crystal structure of recombinant molinate hydrolase at 2.27 Å. The structure reveals a homotetramer with a single mononuclear metal-dependent active site per monomer. The active site architecture shows similarities with other amidohydrolases and enables us to propose a general acid-base catalysis mechanism for molinate hydrolysis. Molinate hydrolase is unable to degrade bulkier thiocarbamate pesticides such as thiobencarb which is used mostly in rice crops. Using a structural-based approach, we were able to generate a mutant (Arg187Ala) that efficiently degrades thiobencarb. The engineered enzyme is suitable for the development of a broader thiocarbamate bioremediation system.

Molecular tweezers targeting transthyretin amyloidosis.

Transthyretin (TTR) amyloidoses comprise a wide spectrum of acquired and hereditary diseases triggered by extracellular deposition of toxic TTR aggregates in various organs. Despite recent advances regarding the elucidation of the molecular mechanisms underlying TTR misfolding and pathogenic self-assembly, there is still no effective therapy for treatment of these fatal disorders. Recently, the "molecular tweezers", CLR01, has been reported to inhibit self-assembly and toxicity of different amyloidogenic proteins in vitro, including TTR, by interfering with hydrophobic and electrostatic interactions known to play an important role in the aggregation process. In addition, CLR01 showed therapeutic effects in animal models of Alzheimer's disease and Parkinson's disease. Here, we assessed the ability of CLR01 to modulate TTR misfolding and aggregation in cell culture and in an animal model. In cell culture assays we found that CLR01 inhibited TTR oligomerization in the conditioned medium and alleviated TTR-induced neurotoxicity by redirecting TTR aggregation into the formation of innocuous assemblies. To determine whether CLR01 was effective in vivo, we tested the compound in mice expressing TTR V30M, a model of familial amyloidotic polyneuropathy, which recapitulates the main pathological features of the human disease. Immunohistochemical and Western blot analyses showed a significant decrease in TTR burden in the gastrointestinal tract and the peripheral nervous system in mice treated with CLR01, with a concomitant reduction in aggregate-induced endoplasmic reticulum stress response, protein oxidation, and apoptosis. Taken together, our preclinical data suggest that CLR01 is a promising lead compound for development of innovative, disease-modifying therapy for TTR amyloidosis.

Pyranoxanthones: Synthesis, growth inhibitory activity on human tumor cell lines and determination of their lipophilicity in two membrane models.

The benzopyran and dihydrobenzopyran moieties can be considered as "privileged motifs" in drug discovery being good platforms for the search of new bioactive compounds. These moieties are commonly found fused to the xanthonic scaffold belonging to the biologically important family of the generally designated prenylated xanthones. Several pyranoxanthones have shown promising antitumor activity and since most of them are from natural origin, the biosynthetic pathway only allows a particular pattern of substitution which limits their structural diversity and renders any broad structure-activity study hard to be established. Accordingly, with the aim of rationalizing the importance of the fused ring orientation and oxygenation pattern in pyranoxanthones, this study describes the synthesis of 14 new pyranoxanthones and evaluation of their cell growth inhibitory activity in four human tumor cell lines as well as their lipophilicity in two membrane models. This systematic approach allowed establishing structure-activity and structure-lipophilicity relationships for the obtained compounds in combination with 6 previously described compounds. From this work an angular pyranoxanthone scaffold emerged as particularly promising, presenting a potent cell growth inhibitory activity and suitable drug-like lipophilicity.

Structural insights into a zinc-dependent pathway leading to Leu55Pro transthyretin amyloid fibrils.

Human transthyretin (TTR) is a homotetrameric protein that is responsible for the formation of amyloid in patients with familiar amyloidotic polyneuropathy (FAP), familiar amyloidotic cardiomyopathy (FAC) and senile systemic amyloidosis (SSA). Amyloid fibrils are characterized by a cross-ß structure. However, details of how TTR monomers are organized to form such an assembly remain unknown. The effect of Zn(2+) in increasing TTR L55P amyloidogenecity has been reported. Crystals of the TTR L55P-Zn(2+) complex were grown under conditions similar to those leading to higher amyloidogenic potential of the variant protein and the three-dimensional structure of the complex was determined by X-ray crystallography. Two different tetrahedral Zn(2+)-binding sites were identified: one cross-links two tetramers, while the other lies at the interface between two monomers in a dimer. The association of monomers involving the two Zn(2+)-binding sites leads to a bidimensional array with a cross-ß structure. The formation of this structure and subsequent organization into amyloid fibrils was monitored by fluorescence spectroscopy and electron microscopy. The TTR L55P-Zn(2+) structure offers the first molecular insights into the role of Zn(2+) as a mediator of cross-ß-type structure in TTR amyloidosis and the relevance of a Zn(2+)-dependent pathway leading to the production of early amyloidogenic intermediates is discussed.