ABSTRACT
Deposition of amyloid is a common aging-associated phenomenon in several aging-related diseases. Osteoarthritis (OA) is the most prevalent joint disease, and aging is its major risk factor. Transthyretin (TTR) is an amyloidogenic protein that is deposited in aging and OA-affected human cartilage and promotes inflammatory and catabolic responses in cultured chondrocytes. Here, we investigated the role of TTR in vivo using transgenic mice overexpressing wild-type human TTR (hTTR-TG). Although TTR protein was detected in cartilage in hTTR-TG mice, the TTR transgene was highly overexpressed in liver, but not in chondrocytes. OA was surgically induced by destabilizing the medial meniscus (DMM) in hTTR-TG mice, wild-type mice of the same strain (WT), and mice lacking endogenous Ttr genes. In the DMM model, both cartilage and synovitis histological scores were significantly increased in hTTR-TG mice. Further, spontaneous degradation and OA-like changes in cartilage and synovium developed in 18-month-old hTTR mice. Expression of cartilage catabolic (Adamts4, Mmp13) and inflammatory genes (Nos2, Il6) was significantly elevated in cartilage from 6-month-old hTTR-TG mice compared with WT mice as was the level of phospho-NF-κB p65. Intra-articular injection of aggregated TTR in WT mice increased synovitis and significantly increased expression of inflammatory genes in synovium. These findings are the first to show that TTR deposition increases disease severity in the murine DMM and aging model of OA.
Subject(s)
Osteoarthritis/metabolism , Prealbumin/metabolism , Age Factors , Animals , Disease Models, Animal , Disease Progression , Humans , Immunohistochemistry , Male , Mice , Mice, Transgenic , Osteoarthritis/genetics , Osteoarthritis/pathology , Prealbumin/biosynthesis , Prealbumin/geneticsABSTRACT
Transthyretin (TTR) is a plasma homotetrameric protein implicated in fatal systemic amyloidoses. TTR tetramer dissociation precedes pathological TTR aggregation. Native state stabilizers are promising drugs to treat TTR amyloidoses. Here we repurpose tolcapone, an FDA-approved molecule for Parkinson's disease, as a potent TTR aggregation inhibitor. Tolcapone binds specifically to TTR in human plasma, stabilizes the native tetramer in vivo in mice and humans and inhibits TTR cytotoxicity. Crystal structures of tolcapone bound to wild-type TTR and to the V122I cardiomyopathy-associated variant show that it docks better into the TTR T4 pocket than tafamidis, so far the only drug on the market to treat TTR amyloidoses. These data indicate that tolcapone, already in clinical trials for familial amyloid polyneuropathy, is a strong candidate for therapeutic intervention in these diseases, including those affecting the central nervous system, for which no small-molecule therapy exists.
Subject(s)
Amyloid Neuropathies, Familial/drug therapy , Benzophenones/therapeutic use , Catechol O-Methyltransferase Inhibitors/therapeutic use , Nitrophenols/therapeutic use , Prealbumin/metabolism , Protein Aggregation, Pathological/drug therapy , Administration, Oral , Animals , Benzophenones/pharmacology , Catechol O-Methyltransferase Inhibitors/pharmacology , Cell Line , Dimerization , Drug Repositioning , Healthy Volunteers , Humans , Mice, Transgenic , Middle Aged , Nitrophenols/pharmacology , Prealbumin/drug effects , TolcaponeABSTRACT
OBJECTIVE: Amyloid deposits are prevalent in osteoarthritic (OA) joints. We undertook this study to define the dominant precursor and to determine whether the deposits affect chondrocyte functions. METHODS: Amyloid deposition in human normal and OA knee cartilage was determined by Congo red staining. Transthyretin (TTR) in cartilage and synovial fluid was analyzed by immunohistochemistry and Western blotting. The effects of recombinant amyloidogenic and nonamyloidogenic TTR variants were tested in human chondrocyte cultures. RESULTS: Normal cartilage from young donors did not contain detectable amyloid deposits, but 7 of 12 aged normal cartilage samples (58%) and 12 of 12 OA cartilage samples (100%) had Congo red staining with green birefringence under polarized light. TTR, which is located predominantly at the cartilage surfaces, was detected in all OA cartilage samples and in a majority of aged normal cartilage samples, but not in normal cartilage samples from young donors. Chondrocytes and synoviocytes did not contain significant amounts of TTR messenger RNA. Synovial fluid TTR levels were similar in normal and OA knees. In cultured chondrocytes, only an amyloidogenic TTR variant induced cell death as well as the expression of proinflammatory cytokines and extracellular matrix-degrading enzymes. The effects of amyloidogenic TTR on gene expression were mediated in part by Toll-like receptor 4, receptor for advanced glycation end products, and p38 MAPK. TTR-induced cytotoxicity was inhibited by resveratrol, a plant polyphenol that stabilizes the native tetrameric structure of TTR. CONCLUSION: These findings are the first to suggest that TTR amyloid deposition contributes to cell and extracellular matrix damage in articular cartilage in human OA and that therapies designed to reduce TTR amyloid formation might be useful.
Subject(s)
Amyloidogenic Proteins/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Osteoarthritis, Knee/metabolism , Prealbumin/metabolism , RNA, Messenger/metabolism , Synovial Fluid/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Amyloidogenic Proteins/pharmacology , Blotting, Western , Cells, Cultured , Chondrocytes/drug effects , Female , Humans , Immunohistochemistry , Male , Middle Aged , Prealbumin/genetics , Prealbumin/pharmacology , Receptor for Advanced Glycation End Products , Receptors, Immunologic/drug effects , Receptors, Immunologic/metabolism , Recombinant Proteins/pharmacology , Toll-Like Receptor 4/drug effects , Toll-Like Receptor 4/metabolism , Young Adult , p38 Mitogen-Activated Protein Kinases/drug effects , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
TTR (transthyretin) amyloidoses are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membrane protein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a KD near 650 nM. Only amyloidogenic V122I TTR compete with fluorescent V122I for cell-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase 3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.
Subject(s)
Amyloid/genetics , Amyloidosis/genetics , Myocytes, Cardiac/pathology , Prealbumin/genetics , Protein Aggregation, Pathological/genetics , Amyloid/metabolism , Amyloidosis/metabolism , Amyloidosis/pathology , Caspase 3/metabolism , Caspase 7/metabolism , Cell Line , Humans , Myocytes, Cardiac/metabolism , Point Mutation , Prealbumin/metabolism , Protein Aggregation, Pathological/metabolism , Protein Aggregation, Pathological/pathology , Protein Transport , Proteolysis , Superoxides/metabolismABSTRACT
The quaternary structure stability of proteins is typically studied under conditions that accelerate their aggregation/unfolding processes on convenient laboratory time scales. Such conditions include high temperature or pressure, chaotrope-mediated unfolding, or low or high pH. These approaches have the limitation of being nonphysiological and that the concentration of the protein in solution is changing as the reactions proceed. We describe a methodology to define the quaternary structure stability of the amyloidogenic homotetrameric protein transthyretin (TTR) under physiological conditions. This methodology expands from a described approach based on the measurement of the rate of subunit exchange of TTR with a tandem flag-tagged (FT2) TTR counterpart. We demonstrate that subunit exchange of TTR with FT2·TTR can be analyzed and quantified using a semi-native polyacrylamide gel electrophoresis technique. In addition, we biophysically characterized two FT2·TTR variants derived from wild-type and the amyloidogenic variant Val122Ile TTR, both of which are associated with cardiac amyloid deposition late in life. The FT2·TTR variants have similar amyloidogenic potential and similar thermodynamic and kinetic stabilities compared to those of their nontagged counterparts. We utilized the methodology to study the potential of the small molecule SOM0226, a repurposed drug under clinical development for the prevention and treatment of the TTR amyloidoses, to stabilize TTR. The results enabled us to characterize the binding energetics of SOM0226 to TTR. The described technique is well-suited to study the quaternary structure of other human aggregation-prone proteins under physiological conditions.
Subject(s)
Amyloid/chemistry , Amyloidosis, Familial/genetics , Models, Molecular , Mutant Proteins/chemistry , Prealbumin/chemistry , Amino Acid Substitution , Amyloid/metabolism , Amyloidosis, Familial/metabolism , Benzophenones/pharmacology , Benzoxazoles/pharmacology , Binding Sites , Drugs, Investigational/pharmacology , Fluorescent Dyes/chemistry , Humans , Kinetics , Mutant Proteins/metabolism , Nitrophenols/pharmacology , Nootropic Agents/pharmacology , Prealbumin/genetics , Prealbumin/metabolism , Protein Aggregation, Pathological , Protein Stability/drug effects , Protein Structure, Quaternary , Protein Unfolding/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Thermodynamics , TolcaponeABSTRACT
Increased neuronal synthesis of transthyretin (TTR) may favorably impact on Alzheimer's disease (AD) because TTR has been shown to inhibit Aß aggregation and detoxify cell-damaging conformers. The mechanism whereby hippocampal and cortical neurons from AD patients and APP23 AD model mice produce more TTR is unknown. We now show that TTR expression in SH-SY5Y human neuroblastoma cells, primary hippocampal neurons and the hippocampus of APP23 mice, is significantly enhanced by heat shock factor 1 (HSF1). Chromatin immunoprecipitation (ChIP) assays demonstrated occupation of TTR promoter heat shock elements by HSF1 in APP23 hippocampi, primary murine hippocampal neurons, and SH-SY5Y cells, but not in mouse liver, cultured human hepatoma (HepG2) cells, or AC16 cultured human cardiomyocytes. Treating SH-SY5Y human neuroblastoma cells with heat shock or the HSF1 stimulator celastrol increased TTR transcription in parallel with that of HSP40, HSP70, and HSP90. With both treatments, ChIP showed increased occupancy of heat shock elements in the TTR promoter by HSF1. In vivo celastrol increased the HSF1 ChIP signal in hippocampus but not in liver. Transfection of a human HSF1 construct into SH-SY5Y cells increased TTR transcription and protein production, which could be blocked by shHSF1 antisense. The effect is neuron specific. In cultured HepG2 cells, HSF1 was either suppressive or had no effect on TTR expression confirming the differential effects of HSF1 on TTR transcription in different cell types.
Subject(s)
Amyloid beta-Protein Precursor/genetics , DNA-Binding Proteins/pharmacology , Neuroblastoma/metabolism , Prealbumin/metabolism , Transcription Factors/pharmacology , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/metabolism , Animals , Carcinoma, Hepatocellular/pathology , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Heat Shock Transcription Factors , Heat-Shock Proteins/pharmacology , Hippocampus/pathology , Humans , Liver Neoplasms/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuroblastoma/pathology , Neurons/drug effects , Neurons/metabolism , Oligonucleotides, Antisense/pharmacology , Prealbumin/geneticsABSTRACT
The misassembly of soluble proteins into toxic aggregates, including amyloid fibrils, underlies a large number of human degenerative diseases. Cardiac amyloidoses, which are most commonly caused by aggregation of Ig light chains or transthyretin (TTR) in the cardiac interstitium and conducting system, represent an important and often underdiagnosed cause of heart failure. Two types of TTR-associated amyloid cardiomyopathies are clinically important. The Val122Ile (V122I) mutation, which alters the kinetic stability of TTR and affects 3% to 4% of African American subjects, can lead to development of familial amyloid cardiomyopathy. In addition, aggregation of WT TTR in individuals older than age 65 y causes senile systemic amyloidosis. TTR-mediated amyloid cardiomyopathies are chronic and progressive conditions that lead to arrhythmias, biventricular heart failure, and death. As no Food and Drug Administration-approved drugs are currently available for treatment of these diseases, the development of therapeutic agents that prevent TTR-mediated cardiotoxicity is desired. Here, we report the development of AG10, a potent and selective kinetic stabilizer of TTR. AG10 prevents dissociation of V122I-TTR in serum samples obtained from patients with familial amyloid cardiomyopathy. In contrast to other TTR stabilizers currently in clinical trials, AG10 stabilizes V122I- and WT-TTR equally well and also exceeds their efficacy to stabilize WT and mutant TTR in whole serum. Crystallographic studies of AG10 bound to V122I-TTR give valuable insights into how AG10 achieves such effective kinetic stabilization of TTR, which will also aid in designing better TTR stabilizers. The oral bioavailability of AG10, combined with additional desirable drug-like features, makes it a very promising candidate to treat TTR amyloid cardiomyopathy.
Subject(s)
Amyloid/antagonists & inhibitors , Amyloidosis/prevention & control , Benzoates/therapeutic use , Cardiomyopathies/prevention & control , Prealbumin/metabolism , Pyrazoles/therapeutic use , Amyloid/genetics , Amyloid/metabolism , Amyloidosis/genetics , Amyloidosis/metabolism , Animals , Area Under Curve , Benzoates/chemistry , Benzoates/pharmacokinetics , Benzoxazoles/metabolism , Benzoxazoles/pharmacokinetics , Benzoxazoles/pharmacology , Cardiomyopathies/genetics , Cardiomyopathies/metabolism , Cell Line , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , HeLa Cells , Humans , MCF-7 Cells , Mice , Mice, Inbred ICR , Models, Molecular , Molecular Structure , Mutation , Prealbumin/chemistry , Prealbumin/genetics , Protein Binding , Protein Stability/drug effects , Protein Structure, Tertiary , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Rats , Rats, WistarABSTRACT
The transthyretin amyloidoses are diseases of protein misfolding characterized by the extracellular deposition of fibrils and other aggregates of the homotetrameric protein transthyretin (TTR) in peripheral nerves, heart, and other tissues. Age is the major risk factor for the development of these diseases. We hypothesized that an age-associated increase in the level of protein oxidation could be involved in the onset of the senile forms of the TTR amyloidoses. To test this hypothesis, we have produced and characterized relevant age-related oxidative modifications of the wild type (WT) and the Val122Ile (V122I) TTR variant, both involved in cardiac TTR deposition in the elderly. Our studies show that methionine/cysteine-oxidized TTR and carbonylated TTR from either the WT or the V122I variant are thermodynamically less stable than their nonoxidized counterparts. Moreover, carbonylated WT and carbonylated V122I TTR have a stronger propensity to form aggregates and fibrils than WT and V122I TTR, respectively, at physiologically attainable pH values. It is well-known that TTR tetramer dissociation, the limiting step for aggregation and amyloid fibril formation, can be prevented by small molecules that bind the TTR tetramer interface. Here, we report that carbonylated WT TTR is less amenable to resveratrol-mediated tetramer stabilization than WT TTR. All the oxidized forms of TTR tested are cytotoxic to a human cardiomyocyte cell line known to be a target for cardiac-specific TTR variants. Overall, these studies demonstrate that age-related oxidative modifications of TTR can contribute to the onset of the senile forms of the TTR amyloidoses.
Subject(s)
Amyloid/chemistry , Amyloid/metabolism , Amyloidosis/metabolism , Prealbumin/chemistry , Prealbumin/metabolism , Age Factors , Aging , Amyloid/genetics , Amyloid/toxicity , Amyloidosis/epidemiology , Amyloidosis/genetics , Amyloidosis/pathology , Benzothiazoles , Cell Line , Humans , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Oxidation-Reduction , Point Mutation , Prealbumin/genetics , Prealbumin/toxicity , Protein Carbonylation , Protein Multimerization/drug effects , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/toxicity , Resveratrol , Stilbenes/pharmacology , Thiazoles/metabolismABSTRACT
Pursuit of the actinomycete pyrrolobenzodiazepine natural product sibiromycin as a chemotherapeutic agent has been limited by its cardiotoxicity. Among pyrrolobenzodiazepines, cardiotoxicity is associated with hydroxylation at position 9. Deletion of the methyltransferase gene sibL abolishes the production of sibiromycin. Supplementation of growth media with 4-methylanthranilic acid can substitute for its native 3-hydroxy congener. Cultures grown in this fashion yielded 9-deoxysibiromycin. In this study, we characterize the structure and biological activity of sibiromycin and 9-deoxysibiromycin methyl carbinolamines. Preliminary in vitro evidence suggests that 9-deoxysibiromycin exhibits reduced cardiotoxicity while gaining antitumor activity. These results strongly support further exploration of the production and evaluation of monomeric and dimeric glycosylated pyrrolobenzodiazepine analogues of sibiromycin.
Subject(s)
Actinomycetales/metabolism , Aminoglycosides/chemistry , Aminoglycosides/metabolism , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/metabolism , Actinomycetales/chemistry , Actinomycetales/enzymology , Actinomycetales/genetics , Aminoglycosides/genetics , Aminoglycosides/pharmacology , Antibiotics, Antineoplastic/pharmacology , Cardiotoxins/chemistry , Cardiotoxins/genetics , Cardiotoxins/metabolism , Cardiotoxins/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Gene Deletion , Humans , Methyltransferases/genetics , Neoplasms/drug therapy , ortho-Aminobenzoates/metabolismABSTRACT
Transthyretin (TTR), a systemic amyloid precursor in the human TTR amyloidoses, interacts with ß-amyloid (Aß) in vitro, inhibits Aß fibril formation, and suppresses the Alzheimer's disease (AD) phenotype in APP23 mice bearing a human APP gene containing the Swedish autosomal dominant AD mutation. In the present study, we show that TTR is a neuronal product upregulated in AD. Immunohistochemical analysis reveals that, in contrast to brains from non-demented age-matched individuals and control mice, the majority of hippocampal neurons from human AD and all those from the APP23 mouse brains contain TTR. Quantitative PCR for TTR mRNA and Western blot analysis show that primary neurons from APP23 mice transcribe TTR mRNA, and the cells synthesize and secrete TTR protein. TTR mRNA abundance is greatly increased in cultured cortical and hippocampal embryonic neurons and cortical lysates from adult APP23 mice. Antibodies specific for TTR and Aß pulled down TTR/Aß complexes from cerebral cortical extracts of APP23 mice and some human AD patients but not from control brains. In complementary tissue culture experiments, recombinant human TTR suppressed the cytotoxicity of soluble Aß aggregates added to mouse neurons and differentiated human SH-SY5Y neuroblastoma cells. The findings that production of Aß, its precursor, or its related peptides induces neuronal TTR transcription and synthesis and the presence of Aß/TTR complexes in vivo suggest that increased TTR production coupled with interaction between TTR and Aß and/or its related peptides may play a role in natural resistance to human AD.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Brain/pathology , Neurons/metabolism , Prealbumin/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Analysis of Variance , Animals , Cell Line, Tumor , Disease Models, Animal , Female , Gene Expression Regulation/genetics , Humans , Lipids/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neuroblastoma/pathology , Prealbumin/deficiency , Prealbumin/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Transfection/methodsABSTRACT
A valine-to-isoleucine mutation at position 122 of the serum protein transthyretin (TTR), found in 3 to 4% of African Americans, alters its stability, leading to amyloidogenesis and cardiomyopathy. In addition, 10 to 15% of individuals older than 65 years develop senile systemic amyloidosis and cardiac TTR deposits because of wild-type TTR amyloidogenesis. Although several drugs are in development, no approved therapies for TTR amyloid cardiomyopathy are yet available, so the identification of additional compounds that prevent amyloid-mediated cardiotoxicity is needed. To this aim, we developed a fluorescence polarization-based high-throughput screen and used it to identify several new chemical scaffolds that target TTR. These compounds were potent kinetic stabilizers of TTR and prevented TTR tetramer dissociation, partial unfolding, and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR. High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. These compounds effectively inhibited the proteotoxicity of V122I-TTR toward human cardiomyocytes. Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is a well-studied inhibitor of TTR aggregation, and may be promising leads for the treatment or prevention of TTR-mediated cardiomyopathy.
Subject(s)
Amyloidosis/metabolism , Cardiomyopathies/metabolism , Prealbumin/metabolism , Amyloidosis/prevention & control , Benzophenones/pharmacology , Cell Line , Cell Line, Tumor , Fluorescence Polarization , Humans , Molecular Structure , Protein Binding/drug effects , Protein Multimerization/drug effectsABSTRACT
The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.
Subject(s)
Amyloidosis/metabolism , Antioxidants/chemistry , Antioxidants/pharmacology , Myocytes, Cardiac/drug effects , Prealbumin/antagonists & inhibitors , Stilbenes/chemistry , Stilbenes/pharmacology , Cell Line , Humans , Myocytes, Cardiac/metabolism , Prealbumin/metabolism , Prealbumin/toxicity , ResveratrolABSTRACT
Glycosaminoglycans (GAGs), which are found in association with all extracellular amyloid deposits in humans, are known to accelerate the aggregation of various amyloidogenic proteins in vitro. However, the precise molecular mechanism(s) by which GAGs accelerate amyloidogenesis remains elusive. Herein, we show that sulfated GAGs, especially heparin, accelerate transthyretin (TTR) amyloidogenesis by quaternary structural conversion. The clustering of sulfate groups on heparin and its polymeric nature are essential features for accelerating TTR amyloidogenesis. Heparin does not influence TTR tetramer stability or TTR dissociation kinetics, nor does it alter the folded monomer-misfolded monomer equilibrium directly. Instead, heparin accelerates the conversion of preformed TTR oligomers into larger aggregates. The more rapid disappearance of monomeric TTR in the presence of heparin likely reflects the fact that the monomer-misfolded amyloidogenic monomer-oligomer-TTR fibril equilibria are all linked, a hypothesis that is strongly supported by the light scattering data. TTR aggregates prepared in the presence of heparin exhibit a higher resistance to trypsin and proteinase K proteolysis and a lower exposure of hydrophobic side chains comprising hydrophobic clusters, suggesting an active role for heparin in amyloidogenesis. Our data suggest that heparin accelerates TTR aggregation by a scaffold-based mechanism, in which the sulfate groups comprising GAGs interact primarily with TTR oligomers through electrostatic interactions, concentrating and orienting the oligomers, facilitating the formation of higher molecular weight aggregates. This model raises the possibility that GAGs may play a protective role in human amyloid diseases by interacting with proteotoxic oligomers and promoting their association into less toxic amyloid fibrils.
Subject(s)
Glycosaminoglycans/chemistry , Prealbumin/chemistry , Amyloidogenic Proteins/chemistry , Amyloidogenic Proteins/metabolism , Circular Dichroism , Heparin/metabolism , Kinetics , Models, Molecular , Molecular Weight , Prealbumin/metabolism , Protein Folding , Protein Structure, QuaternaryABSTRACT
Transthyretin aggregation-associated proteotoxicity appears to cause several human amyloid diseases. Rate-limiting tetramer dissociation and monomer misfolding of transthyretin (TTR) occur before its aggregation into cross-beta-sheet amyloid fibrils. Small molecule binding to and preferential stabilization of the tetrameric state of TTR over the dissociative transition state raises the kinetic barrier for dissociation, imposing kinetic stabilization on TTR and preventing aggregation. This is an effective strategy to halt neurodegeneration associated with polyneuropathy, according to recent placebo-controlled clinical trial results. In three recent papers, we systematically ranked possibilities for the three substructures composing a typical TTR kinetic stabilizer, using fibril inhibition potency and plasma TTR binding selectivity data. Herein, we have successfully employed a substructure combination strategy to use these data to develop potent and selective TTR kinetic stabilizers that rescue cells from the cytotoxic effects of TTR amyloidogenesis. Of the 92 stilbene and dihydrostilbene analogues synthesized, nearly all potently inhibit TTR fibril formation. Seventeen of these exhibit a binding stoichiometry of >1.5 of a maximum of 2 to plasma TTR, while displaying minimal binding to the thyroid hormone receptor (<20%). Six analogues were definitively categorized as kinetic stabilizers by evaluating dissociation time-courses. High-resolution TTR.(kinetic stabilizer)(2) crystal structures (1.31-1.70 A) confirmed the anticipated binding orientation of the 3,5-dibromo-4-hydroxyphenyl substructure and revealed a strong preference of the isosteric 3,5-dibromo-4-aminophenyl substructure to bind to the inner thyroxine binding pocket of TTR.
Subject(s)
Amyloid/antagonists & inhibitors , Amyloidosis/prevention & control , Prealbumin/antagonists & inhibitors , Prealbumin/metabolism , Stilbenes/chemistry , Stilbenes/pharmacology , Amyloid/metabolism , Amyloidosis/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Humans , Models, Molecular , Plasma/metabolism , Prealbumin/chemistry , Protein Binding , Protein Folding , Protein Stability , Receptors, Thyroid Hormone/metabolismABSTRACT
A small molecule that could bind selectively to and then react chemoselectively with a non-enzyme protein in a complex biological fluid, such as blood, could have numerous practical applications. Herein, we report a family of designed stilbenes that selectively and covalently modify the prominent plasma protein transthyretin in preference to more than 4,000 other human plasma proteins. They react chemoselectively with only one of eight lysine e-amino groups within transthyretin. The crystal structure confirms the expected binding orientation of the stilbene substructure and the anticipated conjugating amide bond. These covalent transthyretin kinetic stabilizers exhibit superior amyloid inhibition potency compared to their noncovalent counterparts, and they prevent cytotoxicity associated with amyloidogenesis. Though there are a few prodrugs that, upon metabolic activation, react with a cysteine residue inactivating a specific non-enzyme, we are unaware of designed small molecules that react with one lysine e-amine within a specific non-enzyme protein in a complex biological fluid.
Subject(s)
Lysine/chemistry , Prealbumin/chemistry , Stilbenes/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Crystallography, X-Ray , Drug Design , Humans , Kinetics , Lysine/genetics , Lysine/metabolism , Models, Molecular , Prealbumin/genetics , Prealbumin/metabolism , Protein Multimerization , Protein Structure, Tertiary , Stilbenes/pharmacologyABSTRACT
Transthyretin (TTR) (formerly, thyroxine binding prealbumin) is an evolutionarily conserved serum and cerebrospinal fluid protein that transports holo-retinol-binding protein and thyroxine. Its serum concentration has been widely used to assess clinical nutritional status. It is also well known that wild-type transthyretin and approximately 100 different mutants give rise to a variety of forms of systemic amyloid deposition. It has been suspected and recently established that TTR can suppress the Alzheimer's disease phenotype in transgenic animal models of cerebral Abeta deposition. Thus, while TTR is a systemic amyloid precursor, in the brain it seems to have an anti-amyloidogenic effect. TTR is found in other organs as a result of local synthesis or transport, suggesting that it may have other, as yet undiscovered, functions. It is possible that its capacity to bind many classes of compounds allows it to serve as an endogenous detoxifier of molecules with potential pathologic effects.
Subject(s)
Prealbumin/physiology , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Eye/metabolism , Humans , Mice , Pancreas/metabolism , Plasma/metabolism , Prealbumin/genetics , Prealbumin/metabolismABSTRACT
Cells that have evolved to produce large quantities of secreted proteins to serve the integrated functions of complex multicellular organisms are equipped to compensate for protein misfolding. Hepatocytes and plasma cells have well developed chaperone and proteasome systems to ensure that secreted proteins transit the cell efficiently. The number of neurodegenerative disorders associated with protein misfolding suggests that neurons are particularly sensitive to the pathogenic effects of aggregates of misfolded molecules because those systems are less well developed in this lineage. Aggregates of the amyloidogenic (Abeta(1-42)) peptide play a major role in the pathogenesis of Alzheimer's disease (AD), although the precise mechanism is unclear. In genetic studies examining protein-protein interactions that could constitute native mechanisms of neuroprotection in vivo, overexpression of a WT human transthyretin (TTR) transgene was ameliorative in the APP23 transgenic murine model of human AD. Targeted silencing of the endogenous TTR gene accelerated the development of the neuropathologic phenotype. Intraneuronal TTR was seen in the brains of normal humans and mice and in AD patients and APP23 mice. The APP23 brains showed colocalization of extracellular TTR with Abeta in plaques. Using surface plasmon resonance we obtained in vitro evidence of direct protein-protein interaction between TTR and Abeta aggregates. These findings suggest that TTR is protective because of its capacity to bind toxic or pretoxic Abeta aggregates in both the intracellular and extracellular environment in a chaperone-like manner. The interaction may represent a unique normal host defense mechanism, enhancement of which could be therapeutically useful.
Subject(s)
Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Amyloid beta-Peptides/toxicity , Behavior, Animal/drug effects , Prealbumin/therapeutic use , Animals , Biochemical Phenomena , Biochemistry , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Transgenic , Receptors, Albumin/genetics , Receptors, Albumin/metabolismABSTRACT
The transthyretin amyloidoses appear to be caused by rate-limiting tetramer dissociation and partial monomer unfolding of the human serum protein transthyretin, resulting in aggregation and extracellular deposition of amorphous aggregates and amyloid fibrils. Mice transgenic for few copies of amyloid-prone human transthyretin variants, including the aggressive L55P mutant, failed to develop deposits. Silencing the murine transthyretin gene in the presence of the L55P human gene resulted in enhanced tissue deposition. To test the hypothesis that the murine protein interacted with human transthyretin, preventing the dissociation and partial unfolding required for amyloidogenesis, we produced recombinant murine transthyretin and human/murine transthyretin heterotetramers and compared their structures and biophysical properties to recombinant human transthyretin. We found no significant differences between the crystal structures of murine and human homotetramers. Murine transthyretin is not amyloidogenic because the native homotetramer is kinetically stable under physiologic conditions and cannot dissociate into partially unfolded monomers, the misfolding and aggregation precursor. Heterotetramers composed of murine and human subunits are also kinetically stable. These observations explain the lack of transthyretin deposition in transgenics carrying a low copy number of human transthyretin genes. The incorporation of mouse subunits into tetramers otherwise composed of human amyloid-prone transthyretin subunits imposes kinetic stability, preventing dissociation and subsequent amyloidogenesis.
Subject(s)
Amyloid/chemistry , Prealbumin/chemistry , Protein Folding , Amyloid/genetics , Amyloid/metabolism , Animals , Crystallography, X-Ray , Humans , Kinetics , Mice , Prealbumin/genetics , Prealbumin/metabolism , Protein Structure, Quaternary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Species SpecificityABSTRACT
Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.