Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

Formation of toxic fibrils of Alzheimer's amyloid beta-protein-(1-40) by monosialoganglioside GM1, a neuronal membrane component

A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid beta-protein (Abeta) in fibrillar form on neuronal cells. However, the role of Abeta fibrils in neuronal dysfunction is highly controversial. This study demonstrates that monosialoganglioside GM1 (GM1) released from damaged neurons catalyzes the formation of Abeta fibrils, the toxicity and the cell affinity of which are much stronger than those of Abeta fibrils formed in phosphate-buffered saline. Abeta-(1-40) was incubated with equimolar GM1 at 37 degrees C. After a lag period of 6-12 h, amyloid fibrils were formed, as confirmed by circular dichroism, thioflavin-T fluorescence, size-exclusion chromatography, and transmission electron microscopy. The fibrils showed significant cytotoxicity against PC12 cells differentiated with nerve growth factor. Trisialoganglioside GT1b also facilitated the fibrillization, although the effect was weaker than that of GM1. Our study suggests an exacerbation mechanism of AD and an importance of polymorphisms in Abeta fibrils during the pathogenesis of the disease.     

The amyloid fibrils of the constant domain of immunoglobulin light chain

Light chain-associated (AL) amyloidosis is characterized by dominant fibril deposition of the variable domain (VL) of an immunoglobulin light chain, and thus its constant domain (CL) has been considered not to be amyloidogenic. We examined the in vitro fibril formation of the isolated CL in comparison with β₂-microglobulin (β2-m), an immunoglobulin domain-like amyloidogenic protein responsible for dialysis-related amyloidosis. Two methods useful for β2-m at neutral pH also induced amyloid fibrils of CL, which were monitored by thioflavin-T binding and electron microscopy (EM). These results suggest that CL plays an important role, more than previously assumed, in the development of AL-amyloidosis.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of “functional amyloids” expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.     

Synergistic Amyloid Switch Triggered by Early Heterotypic Oligomerization of Intrinsically Disordered α-Synuclein and Tau

Amyloidogenic intrinsically disordered proteins, α-synuclein and tau are linked to Parkinson's disease and Alzheimer's disease, respectively. A body of evidence suggests that α-synuclein and tau, both present in the presynaptic nerve terminals, co-aggregate in many neurological ailments. The molecular mechanism of α-synuclein-tau hetero-assembly is poorly understood. Here we show that amyloid formation is synergistically facilitated by heterotypic association mediated by binding-induced misfolding of both α-synuclein and tau K18. We demonstrate that the intermolecular association is largely driven by the electrostatic interaction between the negatively charged C-terminal segment of α-synuclein and the positively charged tau K18 fragment. This heterotypic association results in rapid formation of oligomers that readily mature into hetero-fibrils with a much shorter lag phase compared to the individual proteins. These findings suggested that the critical intermolecular interaction between α-synuclein and tau can promote facile amyloid formation that can potentially lead to efficient sequestration of otherwise long-lived lethal oligomeric intermediates into innocuous fibrils. We next show that a well-known familial Parkinson's disease mutant (A30P) that is known to aggregate slowly via accumulation of highly toxic oligomeric species during the long lag phase converts into amyloid fibrils significantly faster in the presence of tau K18. The early intermolecular interaction profoundly accelerates the fibrillation rate of A30P α-synuclein and impels the disease mutant to behave similar to wild-type α-synuclein in the presence of tau. Our findings suggest a mechanistic underpinning of bypassing toxicity and suggest a general strategy by which detrimental amyloidogenic precursors are efficiently sequestered into more benign amyloid fibrils.     

C-terminal truncation exacerbates the aggregation and cytotoxicity of α-Synuclein: A vicious cycle in Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disease which usually associates with neuroinflammation. The main pathological characteristics of PD are dopaminergic neurons death and the presence of Lewy bodies which are composed of aggregated α-synuclein (α-Syn). Truncated forms of α-Syn are found in the brain of PD patients, and account for 10–30% of total synuclein in Lewy bodies. Caspase-1, which plays an important role in neuroinflammation, cleaves full-length α-Syn (α-Syn FL) to generate a C-terminus 19-residues truncated α-Syn (α-Syn121). However, the role of truncated α-Syn in the onset and/or pathogenesis of PD is unclear. Here, we used α-Syn121 as a model to explore its aggregation, membrane disruption and cytotoxicity properties. Compared with α-Syn FL, α-Syn121 aggregated at an accelerated rate, and formed amorphous aggregates rich in random coil structures rather than β-sheet-rich linear fibrils formed by α-Syn FL. Importantly, higher cytotoxicity with lower membrane disruption capacity was found for α-Syn121 aggregates. Furthermore, α-Syn121 aggregates could activate the apoptosis signaling pathway and stimulate the caspase-1-mediated cleavage of α-Syn FL to generate α-Syn121, which as a result leading to increased levels of endogenous α-Syn121 and intracellular S129 phosphorylated α-Syn inclusions. Together, our data suggests a hidden vicious cycle in PD that α-Syn121 rapidly forms amorphous aggregates, which activate caspase-1 to cleave α-Syn FL and generate more α-Syn121, and this cycle may contribute to the onset and/or pathogenesis of PD.     

Kinetics of the thermal inactivation and aggregate formation of rabbit muscle pyruvate kinase in the presence of trehalose

In a previous study we found that 30-40% dimethylsulfoxide induces the active conformation of rabbit muscle pyruvate kinase. Because dimethylsulfoxide is known to perturb structure and function of many proteins, we have explored the effect of trehalose on the kinetics of thermal inactivation and stability of pyruvate kinase; this is because trehalose, in contrast to dimethyl sulfoxide, is totally excluded from the hydration shell of proteins. The results show that 600 mM trehalose inhibits the activity of pyruvate kinase by about 20% at 25 °C, however, trehalose protects pyruvate kinase from thermal inactivation at 60 °C, increases the Tm_app of unfolding by 7.2 °C, induces a more compact state, and stabilizes its tetrameric structure. The inactivation process is irreversible due to the formation of protein aggregates. Trehalose diminishes the rate of formation of intermediates with propensity to aggregate, but does not affect the extent of aggregation. Remarkably, trehalose affects the aggregation process by inducing aggregates with amyloid-like characteristics.     

Binding Modes of Thioflavin-T to the Single-Layer β-Sheet of the Peptide Self-Assembly Mimics

Although the amyloid dye thioflavin-T (ThT) is among the most widely used tools in the study of amyloid fibrils, the mechanism by which ThT binds to fibrils and other β-rich peptide self-assemblies remains elusive. The development of the water-soluble peptide self-assembly mimic (PSAM) system has provided a set of ideal model proteins for experimentally exploring the properties and minimal dye-binding requirements of amyloid fibrils. PSAMs consist of a single-layer β-sheet (SLB) capped by two globular domains, which capture the flat, extended β-sheet features common among fibril-like surfaces. Recently, a PSAM that binds to ThT with amyloid-like affinity (low micromolar K_d) has been designed, and its crystal structure in the absence of bound ThT was determined. This PSAM thus provides a unique opportunity to examine the interactions of ThT with a β-rich structure. Here, we present molecular dynamics simulations of the binding of ThT to this PSAM β-sheet. We show that the primary binding site for ThT is along a shallow groove formed by adjacent Tyr and Leu residues on the β-sheet surface. These simulations provide an atomic-scale rationale for this PSAM's experimentally determined dye-binding properties. Together, our results suggest that an aromatic-hydrophobic groove spanning across four consecutive β-strands represents a minimal ThT binding site on amyloid fibrils. Grooves formed by aromatic-hydrophobic residues on amyloid fibril surfaces may therefore offer a generic mode of recognition for amyloid dyes.     

A Generic Mechanism of β2-Microglobulin Amyloid Assembly at Neutral pH Involving a Specific Proline Switch

Although numerous measurements of amyloid assembly of different proteins under distinct conditions in vitro have been performed, the molecular mechanisms underlying the specific self-association of proteins into amyloid fibrils remain obscure. Elucidating the nature of the events that initiate amyloid formation remains a particularly difficult challenge because of the heterogeneity and transient nature of the species involved. Here, we have used site-directed mutagenesis to create five proline to glycine variants in the naturally amyloidogenic protein β₂-microglobulin (β₂m). One of these variants, P5G, allowed us to isolate and characterise an intermediate containing a non-native trans Pro32 backbone conformation, a feature that is known to be required for amyloid elongation at neutral pH. By analysing oligomerisation and amyloid formation using analytical size-exclusion chromatography, multi-angle static light-scattering, analytical ultracentrifugation, circular dichroism and thioflavin T fluorescence we reveal a pathway for β₂m amyloid assembly at pH 7.5 that does not require the addition of metal ions, detergents, co-solvents or other co-factors that have been used to facilitate amyloid formation at physiological pH and temperature. Assembly is shown to involve the transient formation of a non-native monomer containing a trans P32 backbone conformation. This is followed by the formation of dimeric species and higher molecular mass oligomers that accumulate before the development of amyloid fibrils. On the basis of these results, we propose a generic mechanism for β₂m fibrillogenesis at neutral pH that is consistent with the wide range of published studies of this protein. In this mechanism, amyloid formation is initiated by a specific cis to trans proline switch, the rate of which we show to be controlled by the amino acid sequence proximal to P32 and to the applied solution conditions.     

In silico and in vitro characterization of anti-amyloidogenic activity of vitamin K3 analogues for Alzheimer's disease

Background

Aggregation of amyloid-beta (Aβ) has been proposed as the main cause of Alzheimer's disease (AD). Vitamin K deficiency has been linked to the pathogenesis of AD. Therefore, 15 synthesized vitamin K3 (VK3) analogues were studied for their anti-amyloidogenic activity.

Methods

Biological and spectroscopic assays were used to characterize the effect of VK3 analogues on amyloidogenic properties of Aβ, such as aggregation, free radical formation, and cell viability. Molecular dynamics simulation was used to calculate the binding affinity and mode of VK3 analogue binding to Aβ.

Results

Both numerical and experimental results showed that several VK3 analogues, including VK3-6, VK3-8, VK3-9, VK3-10, and VK3-224 could effectively inhibit Aβ aggregation and conformational conversion. The calculated inhibition constants were in the μM range for VK3-10, VK3-6, and VK3-9 which was similar to the IC₅₀ of curcumin. Cell viability assays indicated that VK3-9 could effectively reduce free radicals and had a protective effect on cytotoxicity induced by Aβ.

Conclusions

The results clearly demonstrated that VK3 analogues could effectively inhibit Aβ aggregation and protect cells against Aβ induced toxicity. Modified VK3 analogues can possibly be developed as effective anti-amyloidogenic drugs for the treatment of AD.

General significance

VK3 analogues effectively inhibit Aβ aggregation and are highly potent as anti-amyloidogenic drugs for therapeutic treatment of AD.