Probing the Dissociation Pathway of a Kinetically Labile Transthyretin Mutant.

Aggregation of transthyretin (TTR) is associated with devastating amyloid diseases. Amyloidosis begins with the dissociation of the native homotetramer (a dimer of dimers) to form a monomeric intermediate that assembles into pathogenic aggregates. This process is accelerated in vitro at low pH, but the process by which TTR dissociates and reassembles at neutral pH remains poorly characterized due to the low population of intermediates. Here, we use 19F-nuclear magnetic resonance (NMR) and a highly sensitive trifluoromethyl probe to determine the relative populations of the species formed by the dissociation of a destabilized variant, A25T. The A25T mutation perturbs both the strong dimer and weak dimer-dimer interfaces. A tetramer ⇌ dimer ⇌ monomer (TDM) equilibrium model is proposed to account for concentration- and temperature-dependent population changes. Thermodynamic and kinetic parameters and activation energetics for dissociation of the native A25T tetramer, as well as a destabilized alternative tetramer (T*) with a mispacked F87 side chain, were extracted by van't Hoff and 19F-NMR line shape analysis, saturation transfer, and transition state theory. Chemical shifts for the dimer and T* species are degenerate for 19F and methyl probes close to the strong dimer interface, implicating interfacial perturbation as a common structural feature of these destabilized species. All-atom molecular dynamics simulations further suggest more frequent F87 ring flipping on the nanosecond time scale in the A25T dimer than in the native A25T tetramer. Our integrated approach offers quantitative insights into the energy landscape of the dissociation pathway of TTR at neutral pH.

Characterization of the G-quadruplexes in the transthyretin gene and its role in silencing transthyretin mRNA transcription.

Transthyretin Amyloidosis arises from the misfolding of monomers or oligomers of the normal transthyretin protein. Our investigation revealed that certain guanine-rich regions within the 5' UTR sequence of the transthyretin gene possess the ability to form G2-quadruplex structures, as determined through analysis with QGRS mapper. We demonstrated that small molecule ligands, including TMPyP4, Braco-19, NMM, and TO, have a significant impact on the stabilization of transthyretin G-quadruplexes. The objective of this study was to confirm the effect of ligands on transthyretin gene transcription through the stabilization of G-quadruplexes. To comprehend the interaction between ligands and transthyretin G-quadruplexes, a range of analytical techniques were employed, includingUV titration, fluorescence titration assays, circular dichroism, quantitative RT-PCR and cytotoxicity tests. The results revealed the presence of four putative G2-quadruplex sequences, which formed stable anti-parallel, parallel, and hybrid G2-quadruplex structures. Notably, Ttrg 3 (5'-GGAAGGAAGGGAGGGAGGG-3') exhibited the highest stability to form G-quadruplex. Furthermore, TmPyP4, Braco-19, NMM and TO were found to stabilize the parallel topology of Ttrg 3. After 48 h of incubation, the RT-PCR experiments revealed a significant reduction in transthyretin mRNA transcription in HepG2 cells when treated with 20 µM TmPyP4 and Braco-19, without inducing apoptosis. Our findings suggested that ligand-mediated stabilization of G-quadruplexes within the 5'-UTR can effectively silence transthyretin expression, highlighting the potential of G-quadruplex as a novel therapeutic target for Transthyretin Amyloidosis. This study might shed valuable lights for the development of innovative therapeutic approach against Transthyretin Amyloidosis.

A transthyretin monomer intermediate undergoes local unfolding and transient interaction with oligomers in a kinetically concerted aggregation pathway.

Transthyretin (TTR) amyloidosis is associated with tissue deposition of TTR aggregates. TTR aggregation is initiated by dissociation of the native tetramer to form a monomeric intermediate, which locally unfolds and assembles into soluble oligomers and higher-order aggregates. However, a detailed mechanistic understanding requires kinetic and structural characterization of the low population intermediates formed. Here, we show that the monomeric intermediate exchanges with an ensemble of oligomers on the millisecond timescale. This transient and reversible exchange causes broadening of the 19F resonance of a trifluoromethyl probe coupled to the monomeric intermediate at S85C. We show the 19F linewidth and R2 relaxation rate increase with increasing concentration of the oligomer. Furthermore, introduction of 19F probes at additional TTR sites yielded distinct 19F chemical shifts for the TTR tetramer and monomer when the trifluoromethyl probe was attached at S100C, located near the same subunit interface as S85C, but not with probes attached at S46C or E63C, which are distant from any interfaces. The 19F probe at E63C shows that part of the DE loop, which is solvent accessible in the tetramer, becomes more buried in the NMR-visible oligomers. Finally, using backbone amides as probes, we show that parts of the EF helix and H-strand become highly flexible in the otherwise structured monomeric intermediate at acidic pH. We further find that TTR aggregation can be reversed by increasing pH. Taken together, this work provides insights into location-dependent conformational changes in the reversible early steps of a kinetically concerted TTR aggregation pathway.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Variation of tafamidis plasma levels during the treatment of TTR amyloidosis patients with Glu89Gln mutation.

PURPOSE: The transthyretin kinetic stabilizer tafamidis, used as a first-line therapy of amyloidosis patients, binds selectively to the transthyretin protein structure and thus prevents its dissociation. The limited information regarding tafamidis application in Glu89Gln amyloidosis patients imposed our research team to determine and evaluate its individual mean plasma levels and their biological variation. METHODS: The present cohort study investigated Bulgarian amyloidosis patients, grouped by gender, age, and therapy duration. A total of sixty patients aged 40-75 years and therapy duration up to 9 years were included. A precise and accurate high-performance liquid chromatography method with ultraviolet detection was used for plasma concentration measurement. RESULTS: Mean plasma concentrations were 5.13 ± 2.64 µmol/L and showed low intra-individual (18.50%) and high inter-individual variability (51.43%). No significant difference was observed between tafamidis plasma levels and therapy duration with p = 0.5941 (p < 0.05 considered significant), but a significant positive correlation was found between plasma concentration, gender, and age with obtained results about p-value 0.0001 and 0.0235, respectively. CONCLUSION: The summary results of the study showed differences that may be based on some specific clinical features of the Glu89Gln mutation.

Binding of common organic UV-filters to the thyroid hormone transport protein transthyretin using in vitro and in silico studies: Potential implications in health.

Several anthropogenic contaminants have been identified as competing with the thyroid hormone thyroxine (T4) for binding to transport proteins as transthyretin (TTR). This binding can potentially create toxicity mechanisms posing a threat to human health. Many organic UV filters (UVFs) and paraben preservatives (PBs), widely used in personal care products, are chemicals of emerging concern due to their adverse effects as potential thyroid-disrupting compounds. Recently, organic UVFs have been found in paired maternal and fetal samples and PBs have been detected in placenta, which opens the possibility of the involvement of TTR in the transfer of these chemicals across physiological barriers. We aimed to investigate a discrete set of organic UVFs and PBs to identify novel TTR binders. The binding affinities of target UVFs towards TTR were evaluated using in vitro T4 competitive binding assays. The ligand-TTR affinities were determined by isothermal titration calorimetry (ITC) and compared with known TTR ligands. In parallel, computational studies were used to predict the 3-D structures of the binding modes of these chemicals to TTR. Some organic UVFs, compounds 2,2',4,4'-tetrahydroxybenzophenone (BP2, Kd = 0.43 µM); 2,4-dihydroxybenzophenone (BP1, Kd = 0.60 µM); 4,4'-dihydroxybenzophenone (4DHB, Kd = 0.83 µM), and 4-hydroxybenzophenone (4HB, Kd = 0.93 µM), were found to display a high affinity to TTR, being BP2 the strongest TTR binder (ΔH = -14.93 Kcal/mol). Finally, a correlation was found between the experimental ITC data and the TTR-ligand docking scores obtained by computational studies. The approach integrating in vitro assays and in silico methods constituted a useful tool to find TTR binders among common organic UVFs. Further studies on the involvement of the transporter protein TTR in assisting the transplacental transfer of these chemicals across physiological barriers and the long-term consequences of prenatal exposure to them should be pursued.

The Regulatory Mechanism of Transthyretin Irreversible Aggregation through Liquid-to-Solid Phase Transition.

Transthyretin (TTR) aggregation and amyloid formation are associated with several ATTR diseases, such as senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP). However, the mechanism that triggers the initial pathologic aggregation process of TTR remains largely elusive. Lately, increasing evidence has suggested that many proteins associated with neurodegenerative diseases undergo liquid-liquid phase separation (LLPS) and subsequent liquid-to-solid phase transition before the formation of amyloid fibrils. Here, we demonstrate that electrostatic interactions mediate LLPS of TTR, followed by a liquid-solid phase transition, and eventually the formation of amyloid fibrils under a mildly acidic pH in vitro. Furthermore, pathogenic mutations (V30M, R34T, and K35T) of TTR and heparin promote the process of phase transition and facilitate the formation of fibrillar aggregates. In addition, S-cysteinylation, which is a kind of post-translational modification of TTR, reduces the kinetic stability of TTR and increases the propensity for aggregation, while another modification, S-sulfonation, stabilizes the TTR tetramer and reduces the aggregation rate. Once TTR was S-cysteinylated or S-sulfonated, they dramatically underwent the process of phase transition, providing a foundation for post-translational modifications that could modulate TTR LLPS in the context of pathological interactions. These novel findings reveal molecular insights into the mechanism of TTR from initial LLPS and subsequent liquid-to-solid phase transition to amyloid fibrils, providing a new dimension for ATTR therapy.

Toxic Misfolded Transthyretin Oligomers with Different Molecular Conformations Formed through Distinct Oligomerization Pathways.

Protein aggregation is initiated by structural changes from native polypeptides to cytotoxic oligomers, which form cross-ß structured amyloid. Identification and characterization of oligomeric intermediates are critically important for understanding not only the molecular mechanism of aggregation but also the cytotoxic nature of amyloid oligomers. Preparation of misfolded oligomers for structural characterization is, however, challenging because of their transient, heterogeneous nature. Here, we report two distinct misfolded transthyretin (TTR) oligomers formed through different oligomerization pathways. A pathogenic TTR variant with a strong aggregation propensity (L55P) was used to prepare misfolded oligomers at physiological pH. Our mechanistic studies showed that the full-length TTR initially forms small oligomers, which self-assemble into short protofibrils at later stages. Enzymatic cleavage of the CD loop was also used to induce the formation of N-terminally truncated oligomers, which was detected in ex vivo cardiac TTR aggregates extracted from the tissues of patients. Structural characterization of the oligomers using solid-state nuclear magnetic resonance and circular dichroism revealed that the two TTR misfolded oligomers have distinct molecular conformations. In addition, the proteolytically cleaved TTR oligomers exhibit a higher surface hydrophobicity, suggesting the presence of distinct oligomerization pathways for TTR oligomer formation. Cytotoxicity assays also revealed that the cytotoxicity of cleaved oligomers is stronger than that of the full-length TTR oligomers, indicating that hydrophobicity might be an important property of toxic oligomers. These comparative biophysical analyses suggest that the toxic cleaved TTR oligomers formed through a different misfoling pathway may adopt distinct structural features that produce higher surface hydrophobicity, leading to the stronger cytotoxic activities.

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer-dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

Analysis of amyloidogenic transthyretin mutations using continuum solvent free energy calculations.

Many proteins can undergo pathological conformational changes that result in the formation of amyloidogenic fibril structures. Various neurodegenerative diseases are associated with such pathological fibril formation of specific proteins. Transthyretin (TTR) is a tetrameric globular transport protein in the blood plasma that can dissociate, unfold, and form long and stable fibrils. Many TTR mutations are known that promote (TTR) amyloidosis and cause severe diseases. TTR amyloidosis has been studied extensively using biochemical methods and structures of various mutations in the globular form have been characterized. Recently, also the structure of a TTR fibril has been determined. In an effort to better understand why some mutations increase or decrease the tendency of amyloid formation, we have applied a combined molecular dynamics and continuum solvent approach to calculate the energetic influence of residue changes in the globular versus fibril form. For 29 out of 36 tested TTR single residue mutations, the approach correctly predicts the increased or decreased tendency for amyloidosis allowing us also to elucidate the origins of the tendency. We find that indeed the destabilization of the globular monomer or changes in dimer and tetramer stability due to mutation has a dominant influence on the amyloidogenic tendency. The continuum solvent model predicts a significantly more favorable mean energy per residue of the fibril form compared to the globular form. This effect is only slightly modulated by single-point mutations preserving the energetic preference for fibril formation upon protein unfolding. It explains why no correlation between experimental amyloidosis and calculated change in fibril stability was observed.

Atomic Mechanisms of Transthyretin Tetramer Dissociation Studied by Molecular Dynamics Simulations.

The dissociation of the transthyretin (TTR) tetramer into a monomer is closely related to various TTR amyloidoses in humans. While the tetramer dissociation has been reported to be the rate-limiting step for TTR aggregation, few details are known about the mechanism. Here, molecular dynamics (MD) simulations were performed by combining conventional MD and biased metadynamics to investigate the mechanism for the wild-type (WT) and mutant (T119M) structures. Both were found to have a great deal in common. Conventional MD simulations reveal that interfacial hydrophobic interactions contribute significantly to stabilize the tetramer. Interfacial residues including L17, V20, L110, and V121 with close contacts form a hydrophobic channel. Metadynamics simulations indicate that the mouth opening of the hydrophobic channel is the first and the most difficult step for dissociation. Interactions of V20 between opposing dimers lock four monomers into the tetramer, and disruption of the interactions is found to be involved in the final step. During the dissociation, an increasing extent of solvation was observed by calculating the radial distribution functions of water around interfacial hydrophobic residues, suggesting that water plays a role in driving the tetramer dissociation. Moreover, compared to T119, residue M119 has a longer side chain that extends into the hydrophobic channel, making solvation more difficult, consistent with a higher energy barrier for dissociation of the T119M tetramer. This result provides a good explanation for the protective role of the T119M mutation. Overall, this study can provide atomic-level insights to better understand the pathogenesis of TTR amyloidosis and guide rational drug design in the future.

The role of transthyretin in cell biology: impact on human pathophysiology.

Transthyretin (TTR) is an extracellular protein mainly produced in the liver and choroid plexus, with a well-stablished role in the transport of thyroxin and retinol throughout the body and brain. TTR is prone to aggregation, as both wild-type and mutated forms of the protein can lead to the accumulation of amyloid deposits, resulting in a disease called TTR amyloidosis. Recently, novel activities for TTR in cell biology have emerged, ranging from neuronal health preservation in both central and peripheral nervous systems, to cellular fate determination, regulation of proliferation and metabolism. Here, we review the novel literature regarding TTR new cellular effects. We pinpoint TTR as major player on brain health and nerve biology, activities that might impact on nervous systems pathologies, and assign a new link between TTR and angiogenesis and cancer. We also explore the molecular mechanisms underlying TTR activities at the cellular level, and suggest that these might go beyond its most acknowledged carrier functions and include interaction with receptors and activation of intracellular signaling pathways.

Thermodynamic Stability and Aggregation Kinetics of EF Helix and EF Loop Variants of Transthyretin.

Misfolding and aggregation of transthyretin (TTR) are linked to amyloid disease. Amyloidosis occurs when the TTR homotetramer dissociates into aggregation-prone monomers that self-assemble into amyloid. In familial transthyretin amyloidosis, hereditary amino acid substitutions destabilize TTR and promote aggregation. In this work, we used 19F nuclear magnetic resonance (NMR) to determine the effect of mutations in the EF helix (Y78F, K80D, K80E, and A81T) and EF loop (G83R and I84S) on the aggregation kinetics and stability of the TTR tetramer and monomer. The EF region acts as a scaffold that stabilizes interactions in both the strong and weak dimer interfaces of the tetramer and is the site of a cluster of pathogenic mutations. K80D and K80E are non-natural mutants that destabilize the EF helix and yield an equilibrium mixture of tetramer and monomer at neutral pH, providing a unique opportunity to determine the thermodynamic parameters for tetramer assembly under nondenaturing conditions. Of the pathogenic mutants studied, only A81T formed appreciable monomer at neutral pH. Real-time 19F NMR measurements showed that the pathogenic Y78F mutation accelerates aggregation by destabilizing both the tetrameric and monomeric species. The pathogenic mutations A81T, G83R, and I84S destabilize the monomer and increase its aggregation rate by disrupting a Schellman helix C-capping motif. These studies provide new insights into the mechanism by which relatively subtle mutations that affect tetramer or monomer stability promote entry of TTR into the dissociation-aggregation pathway.

Comparative study of the stabilities of synthetic in vitro and natural ex vivo transthyretin amyloid fibrils.

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36, -8.10, and -10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods of structural comparison between ex vivo fibrils and fibrils generated in vitro Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology.

The inhibition of cellular toxicity of amyloid-ß by dissociated transthyretin.

The protective effect of transthyretin (TTR) on cellular toxicity of ß-amyloid (Aß) has been previously reported. TTR is a tetrameric carrier of thyroxine in blood and cerebrospinal fluid, the pathogenic aggregation of which causes systemic amyloidosis. However, studies have documented a protective effect of TTR against cellular toxicity of pathogenic Aß, a protein associated with Alzheimer's disease. TTR binds Aß, alters its aggregation, and inhibits its toxicity both in vitro and in vivo In this study, we investigate whether the amyloidogenic ability of TTR and its antiamyloid inhibitory effect are associated. Using protein aggregation and cytotoxicity assays, we found that the dissociation of the TTR tetramer, required for its amyloid pathogenesis, is also necessary to prevent cellular toxicity from Aß oligomers. These findings suggest that the Aß-binding site of TTR may be hidden in its tetrameric form. Aided by computational docking and peptide screening, we identified a TTR segment that is capable of altering Aß aggregation and toxicity, mimicking TTR cellular protection. EM, immune detection analysis, and assessment of aggregation and cytotoxicity revealed that the TTR segment inhibits Aß oligomer formation and also promotes the formation of nontoxic, nonamyloid amorphous aggregates, which are more sensitive to protease digestion. Finally, this segment also inhibits seeding of Aß catalyzed by Aß fibrils extracted from the brain of an Alzheimer's patient. Together, these findings suggest that mimicking the inhibitory effect of TTR with peptide-based therapeutics represents an additional avenue to explore for the treatment of Alzheimer's disease.

A low amyloidogenic E61K transthyretin mutation may cause familial amyloid polyneuropathy.

Patients with transthyretin (TTR)-type familial amyloid polyneuropathy (FAP) typically exhibit sensory dominant polyneuropathy and autonomic neuropathy. However, the molecular pathogenesis of the neuropathy remains unclear. In this study, we characterize the features of FAP TTR the substitution of lysine for glutamic acid at position 61 (E61K). This FAP was late-onset, with sensory dominant polyneuropathy, autonomic neuropathy, and cardiac amyloidosis. Interestingly, no amyloid deposits were found in the endoneurium of the four nerve specimens examined. Therefore, we examined the amyloidogenic properties of E61K TTR in vitro. Recombinant wild-type TTR, the substitution of methionine for valine at position 30 (V30M) TTR, and E61K TTR proteins were incubated at 37°C for 72 hr, and amyloid fibril formation was assessed using the thioflavin-T binding assay. Amyloid fibril formation by E61K TTR was less than that by V30M TTR, and similar to that by wild-type TTR. E61K TTR did not have an inhibitory effect on neurite outgrowth from adult rat dorsal root ganglion (DRG) neurons, but V30M TTR did. Furthermore, we studied the sural nerve of our patient by terminal deoxynucleotidyl transferase dUTP nick end labeling and electron microscopy. A number of apoptotic cells were observed in the endoneurium of the nerve by transferase dUTP nick end labeling. Chromatin condensation was confirmed in the nucleus of non-myelinating Schwann cells by electron microscopy. These findings suggest that E61K TTR is low amyloidogenic, in vitro and in vivo. However, TTR aggregates and amyloid fibrils in the DRG may cause sensory impairments in FAP because the DRG has no blood-nerve barrier. Moreover, Schwann cell apoptosis may contribute to the neurodegeneration.

Transthyretin affects the proliferation and migration of human retinal microvascular endothelial cells in hyperglycemia via hnRNPA2B1.

Transthyretin (TTR) has been proved to repress neovascularization in diabetic retinopathy environment by regulating the molecules in and downstream of the STAT-4/miR-223-3p/FBXW7 signal pathway; however, the details of its direct targets are still not well understood. The interaction between TTR and a target in nucleus of human retinal microvascular endothelial cells (hRECs), heterogeneous nuclear ribonucleoprotein (hnRNP) A2B1, was screened by immunoprecipitation (IP) and mass spectrum (MS), and it was further confirmed by co-immunoprecipitation (co-IP). Regarding ZDOCK analysis using Discovery Studio, the interface and potential binding sites between TTR and hnRNPA2B1 were simulated; mutants were designed in these regions and five soluble ones were recombinantly expressed and prepared; the interaction between TTR and hnRNPA2B1 were disrupted by several mutated residues. In addition, for several mutated TTRs, the inhibition activities against the proliferation, migration and tube formation of hRECs were absent in vitro. Following the disruption of TTR-hnRNPA2B1, the molecules in and downstream of STAT-4/miR-223-3p/FBXW7 signal pathway, including STAT-4, miR-223-3p, FBXW7 p-Akt and Notch1 could not be regulated by TTR mutants; therefore, a TTR-hnRNPA2B1/STAT-4/miR-223-3p/FBXW7 was proposed. In conclusion, this work suggested that TTR should play a physiological role in diabetic environment by the direct binding with hnRNPA2B1, and it provided a theoretical basis for clinical diagnosis, therapy and further application.

Confirmation of intersubunit connectivity and topology of designed protein complexes by native MS.

Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein-protein interactions to generate specific protein assemblies. In this work, we designed protein-protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches.

Kinetic analysis of the multistep aggregation pathway of human transthyretin.

Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a 19F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct 19F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time 19F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119M) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119M remains resistant to tetramer dissociation and aggregation.

Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition.

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutant TTR gene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.