Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3.

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ In ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we "reverse engineered" PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.

Structural discordance in HIV-1 Vpu from brain isolate alarms amyloid fibril forming behavior- a computational perspective.

HIV-1 being the most widespread type worldwide, its accounts for almost 95% of all infections including HIV associated dementia (HAD) that triggers neurological dysfunction and neurodegeneration in patients. The common features associated with HAD and other neurodegenerative diseases are accumulation of amyloid plaques, neuronal loss and deterioration of cognitive abilities, amongst which amyloid fibrillation is considered to be a hallmark. The success of effective therapeutics lies in the understanding of mechanisms leading to neurotoxicity. Few viral proteins like gp-120 are known to be involved in aggregation and enhancement of viral infectivity while comprehending the neurotoxic role of some other proteins is still underway. In the current study, amyloidogenic potential of HIV-1 Vpu protein from brain isolate is investigated through computational approaches. The aggregation propensity of brain derived HIV-1 Vpu was assessed by several amyloid prediction servers that projected the region 4-35 to be amyloidogenic. The protein structure was modeled and subjected to 70 ns molecular dynamics (MD) simulation to investigate the transformation of α-helical conformation of the predicted aggregate region into ß-sheet, proposing the protein's ability to initiate fibril formation that is central to amyloidogenic proteins. The structural features of brain derived HIV-1 Vpu were consistent with the in silico amyloid prediction results that depicts the conformational change in the region 8-28 of which residues Ala8, Ile9, Val10, Ala19, Ile20 and Val21 constitutes ß-sheet formation. The α-helix/ß-sheet discordance of the predicted region was reflected in the simulation study highlighting the possible structural transition associated with HIV-1 Vpu protein of brain isolate.

Characterization of Non-amyloidogenic G101S Transthyretin.

Transthyretin (TTR) is a tetrameric beta-sheet-rich protein that is important in the plasma transport of thyroxine and retinol. Mutations in the TTR gene cause TTR tetramer protein to dissociate to monomer, which is the rate-limiting step in familial amyloid polyneuropathy. Amyloidogenicity of individual TTR variants depends on the types of mutation that induce significant changes in biophysical, biochemical and/or biological properties. G101S TTR variant was previously identified in a Japanese male without amyloidotic symptom, and was considered as a non-amyloidogenic TTR variant. However, little is known about G101S TTR. Here, we found slight but possibly important biophysical differences between wild-type (WT) and G101S TTR. G101S TTR had slower rate of tetramer dissociation and lower propensity for amyloid fibril formation, especially at mild low pH (4.2 and 4.5), and was likely to have strong hydrophobic interaction among TTR monomers, suggesting relatively higher stability of G101S TTR compared with WT TTR. Cycloheximide (CHX)-based assay in HEK293 cells revealed that intracellular G101S TTR expression level was lower, but extracellular expression was higher than WT TTR, implying enhanced secretion efficiency of G101S TTR protein compared with WT TTR. Moreover, we found that STT3B-dependent posttranslational N-glycosylation at N98 residue occurred in G101S TTR but not in other TTR variants, possibly due to amino acid alterations that increase N-glycosylation preference or accelerate rigid structure formation susceptible to N-glycosylation. Taken together, our study characterizes G101S TTR as a stable and N-glycosylable TTR, which may be linked to its non-amyloidogenic characteristic.

Complete characterization of the mutation landscape reveals the effect on amylin stability and amyloidogenicity.

Type-II diabetes is believed to be partially aggravated by the emergence of toxic amylin protein deposits in the extracellular space of the pancreas ß-cells. Amylin, the regulatory hormone that is co-secreted with insulin, has been observed to misfold into toxic structures. Pramlintide, an FDA approved injectable amylin analog mutated at positions 25, 28, and 29 was therefore developed to create a more stable, soluble, less-aggregating, and equipotent peptide that is used as an adjunctive therapy for diabetes. However, because Pramlintide is not ideal, researchers have been exploring other amylin analogs as therapeutic replacements. In this work, we assist the finding of optimal analogs by computationally revealing the mutational landscape of amylin. We computed the structure energies of all possible single-point mutations and studied the effect they have on amylin stability and amyloidogenicity. Each of the 37 amylin residues was mutated in silico into the 19 canonical amino acids and an energy function computing the Lennard-Jones, Coulomb and solvation energy was used to analyze changes in stability. The mutation landscape identified amylin's conserved stable regions, residues that can be tweaked to further stabilize structure, regions that are susceptible to mutations, and mutations that are amyloidogenic. We used the single-point mutational landscape data to generate estimations for higher-order multiple-point mutational landscapes and discovered millions of three-point mutations that are more stable and less amyloidogenic than Pramlintide. The landscapes provided an explanation for the effect of the S20G and Q10R mutations on the onset of diabetes of the Chinese and Maori populations, respectively.

An updated AL-Base reveals ranked enrichment of immunoglobulin light chain variable genes in AL amyloidosis.

Background: Each monoclonal antibody light chain associated with AL amyloidosis has a unique sequence. Defining how these sequences lead to amyloid deposition could facilitate faster diagnosis and lead to new treatments. Methods: Light chain sequences are collected in the Boston University AL-Base repository. Monoclonal sequences from AL amyloidosis, multiple myeloma and the healthy polyclonal immune repertoire were compared to identify differences in precursor gene use, mutation frequency and physicochemical properties. Results: AL-Base now contains 2,193 monoclonal light chain sequences from plasma cell dyscrasias. Sixteen germline precursor genes were enriched in AL amyloidosis, relative to multiple myeloma and the polyclonal repertoire. Two genes, IGKV1-16 and IGLV1-36, were infrequently observed but highly enriched in AL amyloidosis. The number of mutations varied widely between light chains. AL-associated κ light chains harbored significantly more mutations compared to multiple myeloma and polyclonal sequences, whereas AL-associated λ light chains had fewer mutations. Machine learning tools designed to predict amyloid propensity were less accurate for new sequences than their original training data. Conclusions: Rarely-observed light chain variable genes may carry a high risk of AL amyloidosis. New approaches are needed to define sequence-associated risk factors for AL amyloidosis. AL-Base is a foundational resource for such studies.

The effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin.

It is widely accepted that the formation of amyloid fibrils is one of the natural properties of proteins. The amyloid formation process is associated with a variety of factors, among which the hydrophobic residues play a critical role. In this study, insulin was used as a model to investigate the effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin. Porcine insulin was digested with trypsin to obtain desoctapeptide-(B23-B30) insulin (DOI), whose hydrophilic C-terminal of B-chain was removed and hydrophobic core was exposed. The results showed that DOI, of which the ordered structure (predominantly α-helix) was markedly decreased, was more prone to aggregate than intact insulin. As to the secondary structure of amyloid fibrils, DOI fibrils were similar to insulin fibrils formed under acidic condition, whereas under neutral condition, insulin formed less polymerized aggregates by showing decreased ß-sheet contents in fibrils. Further investigation on membrane damage and hemolysis showed that DOI fibrils induced significantly less membrane damage and less hemolysis of erythrocytes compared with those of insulin fibrils. In conclusion, exposing the hydrophobic core of insulin can induce the increase of amyloidogenicity and formation of higher-order polymerized fibrils, which is less toxic to membranes.

Biophysical characterization as a tool to predict amyloidogenic and toxic properties of amyloid-ß42 peptides.

Amyloid-ß42 (Aß42) peptides are central to the amyloid pathology in Alzheimer's disease (AD). As biological mimetics, properties of synthetic Aß peptides usually vary between vendors and batches, thus impacting the reproducibility of experimental studies. Here, we tested recombinantly expressed Aß42 (Asp1 to Ala42) against synthetic Aß42 from different suppliers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), circular dichroism (CD) spectroscopy, thioflavin T aggregation, surface plasmon resonance, and MTT cell viability assays. Overall, our recombinant Aß42 provided a reproducible mimetic of desired properties. Across experimental approaches, the combined detection of Aß42 dimers and random coil to ß-sheet transition only correlated with aggregation-prone and cytotoxic peptides. Conclusively, combining MALDI-MS with CD appears to provide a rapid, reliable means to predict the 'bioactivity' of Aß42.

Hexameric Aggregation Nucleation Core Sequences and Diversity of Pathogenic Tau Strains.

Tau aggregation associates with multiple neurodegenerative diseases including Alzheimer's disease and rare tauopathies such as Pick's disease, progressive supranuclear palsy, and corticobasal degeneration. The molecular and structural basis of tau aggregation and related diverse misfolded tau strains are not fully understood. To further understand tau-protein aggregation mechanisms, we performed systematic truncation mutagenesis and mapped key segments of tau proteins that contribute to tau aggregation, where it was determined that microtubule binding domains R2 and R3 play critical roles. We validated that R2- or R3-related hexameric PHF6 and PHF6* peptide sequences are necessary sequences that render tau amyloidogenicity. We also determined that the consensus VQI peptide sequence is not sufficient for amyloidogenicity. We further proposed single- and dual-nucleation core-based strain classifications based on recent cryo-EM structures. We analyzed the structural environment of the hexameric peptide sequences in diverse tau strains in tauopathies that, in part, explains why the VQI consensus core sequence is not sufficient to induce tau aggregation. Our experimental work and complementary structural analysis highlighted the indispensible roles of the hexameric core sequences, and shed light on how the interaction environment of these core sequences contributes to diverse pathogenic tau-strains formation in various tauopathy brains.

Comparison of the aggregation of homologous ß2-microglobulin variants reveals protein solubility as a key determinant of amyloid formation.

The mouse and human ß2-microglobulin protein orthologs are 70% identical in sequence and share 88% sequence similarity. These proteins are predicted by various algorithms to have similar aggregation and amyloid propensities. However, whilst human ß2m (hß2m) forms amyloid-like fibrils in denaturing conditions (e.g. pH2.5) in the absence of NaCl, mouse ß2m (mß2m) requires the addition of 0.3M NaCl to cause fibrillation. Here, the factors which give rise to this difference in amyloid propensity are investigated. We utilise structural and mutational analyses, fibril growth kinetics and solubility measurements under a range of pH and salt conditions, to determine why these two proteins have different amyloid propensities. The results show that, although other factors influence the fibril growth kinetics, a striking difference in the solubility of the proteins is a key determinant of the different amyloidogenicity of hß2m and mß2m. The relationship between protein solubility and lag time of amyloid formation is not captured by current aggregation or amyloid prediction algorithms, indicating a need to better understand the role of solubility on the lag time of amyloid formation. The results demonstrate the key contribution of protein solubility in determining amyloid propensity and lag time of amyloid formation, highlighting how small differences in protein sequence can have dramatic effects on amyloid formation.

Diethylpyrocarbonate modification reveals HisB5 as an important modulator of insulin amyloid formation.

More than 30 amyloid proteins are reported to be associated with amyloidosis diseases. Studies have implicated histidine may be critically involved in amyloid formation. Here, we used diethylpyrocarbonate (DEPC) modification to obtain a His(B5) mono-ethyloxyformylated insulin (DMI-B(5)). The secondary structure, amyloidogenicity, metal ion interaction, and cytotoxicity of DMI-B(5) and insulin were compared. DMI-B(5) was less prone to aggregation in acidic condition but easier to aggregate at neutral pH. DEPC modification resulted in attenuated inhibitory effect of Zn(2+) on aggregation, whereas DMI-B(5) fibrils induced more severe erythrocytes haemolysis compared to insulin fibrils. This study not only provides a fast new approach for studying the impact of imidazole ring in amyloid formation, but also reveals the critical modulating role of histidine imidazole ring on the amyloidogenicity of insulin.

C-terminal unfolding of an amyloidogenic ß2-microglobulin fragment: ΔN6ß2-microglobulin.

OBJECTIVES: A ß2-microglobulin (ß2m) fragment that lacks the first six amino acids, i.e., ΔN6ß2-microglobulin (ΔN6ß2m), is an endogenous, proteolytically derived, amyloidogenic fragment of ß2m, the precursor protein in Aß2M amyloidosis (dialysis-related amyloidosis). As reports suggest the importance of C-terminal unfolding for the amyloidogenicity of ß2m, in this study we aimed to investigate conformational characteristics of ΔN6ß2m related to amyloidogenicity. We also measured the concentration of an amyloidogenic intermediate of ß2m with C-terminal unfolding (ß2m92-99) in serum samples from 10 patients undergoing hemodialysis (HD). METHODS: We utilized capillary electrophoretic analysis, surface plasmon resonance and enzyme-linked immunosorbent assay. RESULTS AND CONCLUSIONS: We confirmed the normal core structure of ΔN6ß2m with a commercial monoclonal anti-ß2m antibody. In addition, using the specific monoclonal antibody for the C-terminal peptide, i.e. mAb 92-99, we confirmed unfolding in the C-terminal region of ΔN6ß2m. On the basis of these findings, we established an ELISA to measure ß2m92-99 using ΔN6ß2m as a standard molecule in circulation. However, we did not detect ß2m92-99 in serum from 10 HD patients, despite the absence of uremic inhibitors in the serum.