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Increased stability of amyloid peptides
The developed illustrative biophysical model and the corresponding software make it possible to directly analyze the reactivity of amyloid peptides and molecules that bind to them.
Our method include:
-Study of hereditary forms of Alzheimer's disease,
- Study of the stability and kinetics of aggregation,
- Increased stability of amyloid peptides,
-determination of key amino acid residues,
-selection of antibodies to amyloids

Alzheimer's is the most common cause of dementia, a general term for memory loss and other cognitive abilities serious enough to interfere with daily life. Alzheimer's disease accounts for 60-80% of dementia cases.

The brain has 100 billion nerve cells (neurons). Each nerve cell connects with many others to form communication networks. Groups of nerve cells have special jobs. Some are involved in thinking, learning and remembering. Others help us see, hear and smell.

To do their work, brain cells operate like tiny factories. They receive supplies, generate energy, construct equipment and get rid of waste. Cells also process and store information and communicate with other cells. Keeping everything running requires coordination as well as large amounts of fuel and oxygen.

Scientists believe Alzheimer's disease prevents parts of a cell's factory from running well. They are not sure where the trouble starts. But just like a real factory, backups and breakdowns in one system cause problems in other areas. As damage spreads, cells lose their ability to do their jobs and, eventually die, causing irreversible changes in the brain.

Stabilization of amyloid peptides at the level of dimeric complexes

We have developed a method for increasing the stability of amyloid dimers in order to reduce their ability to enter into further biochemical reactions with the formation of toxic oligomers.

Numerical calculations are presented in the form of a graphical description, which clearly allows you to see an increase or decrease in the stability of dimeric amyloid complexes. Our proposed method allows us to vary the range of stability of dimeric complexes by substitutions of key amino acid residues.

chemical structure, chemical structure of water, chemical bonding and molecular structure class, chemical formula of water
Fig.2. Amino acid sequence of the ABeta(11–42) peptide, with
its missense mutations being indicated. AAR numbering is
given in two variants: for the peptide itself and relative to its
predecessor protein APP.
Since the method proposed in this work concerns a new insight into the stability of peptide amyloids, we will provide a fragment of such an amyloid peptide ABeta (11–42) that corresponds to the length of amino acid sequence of a three-dimensional structure from the PDB:2MXU database [5]. Figure 2 shows the amino acid sequence of ABeta (11–42) peptide in which missens mutations that lead to different biological and pathophysiological changes in the human body are indicated [23]. Figure 2. shows the amino acid sequence of AB (11–42) peptide indicating missense mutations (see table 1), which entail various biological and pathophysiological effects in the human body

If the dimeric complex is stable, then the formation of high molecular weight structures was much slower. This was due to the fact that the stable amyloid peptides were in no hurry to enter into chemical reactions with other amyloid peptides to achieve equilibrium. It remained to solve the question: how will we determine the stability of the dimeric complex?
Let me remind you that since 2015 our company has been dealing with the issues of the stability of protein compounds. We introduced a numerical stability criterion for protein dimers; this value is the condition number of the matrix of potential energy of pairwise electrostatic interaction between two proteins.

The higher this number, the more unstable the biological complex. We calculated the stability values for the known dimeric amyloid complexes taking into account mutations and obtained a numerical stability value for each dimer, which was presented in the form of a graph.

Higher values indicate a lower stability value and vice versa.

Our goal is the identification of dimeric amyloid complexes, which would be characterized as sufficiently stable dimeric complexes. One of the peptides in the dimer complex will be exactly the amyloid peptide, and the second companion we have to find. The second partner in the dimer complex will be an inhibitor based on the amino acid sequence.
Fig.3. Three-dimensional map of the potential energy of pairwise electrostatic interaction between two wild-type amyloid peptides (upper figure) and taking into account mutations (lower figure)
Next, we will consider the illustrative results obtained for the change in the stability of amyloid dimers taking into account various mutations in amyloid peptides.

The figures below show the numerical results obtained for various mutations and a brief description of their physical properties, which varied depending on changes in the resistance of amyloids at the level of dimeric complexes.

Part II

The following is the basic information on the effect of various mutations (mostly hereditary) on human physiology, as well as the physical properties of amyloids.
mut-mut peptides
Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-β (Aβ) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aβ.

First, due to the K16N mutation APP secretion is affected and a higher amount of Aβ peptides is being produced.

Second, Aβ peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells.
[Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers]
mut-wt peptides
Third, Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject.

Furthermore, Aβ42 K16N inhibits fibril formation of Aβ42 wild-type. Even more, Aβ42 K16N peptides are protected against clearance activity by the major Aβ-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease
[Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers]
Among the various hereditary mutants of amyloid β (Aβ) in familial Alzheimer's disease (AD), the A21G Flemish-type mutant has unique properties showing a low aggregation propensity.
[Conformational Effects of the A21G Flemish Mutation on the Aggregation of Amyloid β Peptide]

Minimal local change from A to G leads to significant conformational changes and wider free energy holes on the free energy surface as well as altered surface charges that lead to weaker affinity to the dipalmitoylphosphatidylcholine (DPPC) lipid bilayers. These results are consistent with experimental data that showed that A21G mutants of Aβ peptides have lower aggregation rates and membrane binding rates.
[The Effects of A21G Mutation on Transmembrane Amyloid Beta (11-40) Trimer: An In Silico Study]


H2O2 bound with the mutation site 22K residue of E22K and elicited more rapid aggregation of E22K than Aβ in vitro. Moreover, H2O2 acted with E22K synergistically to induce higher cellular toxicity than with Aβ. Notably, intrahippocampal infusion of E22K led to more severe plaque deposition, neuron death, and more rapid memory decline than Aβ-injected rats.
[Single Point Mutation from E22-to-K in Aβ Initiates Early-Onset Alzheimer's Disease by Binding with Catalase]

E22K-Aβ42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis.
[Neurotoxicity and Physicochemical Properties of Aβ Mutant Peptides from Cerebral Amyloid Angiopath]


This study presents in-vitro evidence that a heterozygous E22G pathogenic ("Arctic") mutation of Aβ40 can enhance misfolding of Aβ via cross-seeding from wild-type (WT) Aβ42 fibril. Thioflavin T (ThT) fluorescence analysis suggested that misfolding of E22G Aβ40 was enhanced by adding 5% (w/w) WT Aβ42 fibril as "seed", whereas WT Aβ40 was unaffected by Aβ42 fibril seed. 13C SSNMR analysis revealed that such cross-seeding prompted formation of E22G Aβ40 fibril that structurally mimics the seed Aβ42 fibril, suggesting unexpected cross talk of Aβ isoforms that potentially promotes early onset of AD. The SSNMR approach is likely applicable to elucidate structural details of heterogeneous amyloid fibrils produced in cross-seeding for amyloids linked to neurodegenerative diseases.[E22G Pathogenic Mutation of β-amyloid (Aβ) Enhances Misfolding of Aβ40 by Unexpected Prion-like Cross Talk between Aβ42 and Aβ40]

These studies revealed that the Arctic mutation accelerates both Aβ oligomerization and fibrillogenesis in vitro. In addition, Aβ40ARC was observed to affect both the morphology and the size distribution of Aβ protofibrils.

An increase in the ratio of AβWT/AβMUT(Arctic), therefore, may result in the accumulation of potential neurotoxic protofibrils and acceleration of disease progression in familial Alzheimer's disease mutation carriers.[Mixtures of Wild-type and a Pathogenic (E22G) Form of Aβ40 in Vitro Accumulate Protofibrils, Including Amyloid Pores]

the aggregation of E22G-Aβ42 and D23N-Aβ42 was similar to that of wild-type Aβ42[Neurotoxicity and Physicochemical Properties of Aβ Mutant Peptides from Cerebral Amyloid Angiopath]

E22Q-Aβ42 aggregated extensively, supporting the clinical evidence that Dutch and Italian patients are diagnosed as hereditary cerebral hemorrhage with amyloidosis.
[Neurotoxicity and Physicochemical Properties of Aβ Mutant Peptides from Cerebral Amyloid Angiopath]

Part III

The calculated values of the resistance of amyloid peptides for the mutations described above

Fig.4. Graph of values for stability indicator lg(cond(w)) that were obtained by considering the interaction of wild form wtABeta and interaction of mutant peptide forms mutABeta from Table 1.

The stability of biological complex is plotted in Fig. 3. The mutation names and the way the structures of a higher order are formed are indicated in the plots. A large arrow between the plots indicates the direction for values characteristic of the formation of structures with a higher molecular weight such as oligomers, protofibrils, and fibrils. A thick line at a level of 5.53 arbitrarily separates structures that tend to (the region below the line). The "FAB" designation on the plot corresponds to the stability value upo interaction of the solanezumab region with an amyloid peptide (a structure in the PDB:4XXD database) .

On the strength of experimental data on missense mutations and their biological effect, we draw a separation line at a level of 5.53 that separates mutations in peptides leading to enhanced formation of higherorder structures from mutations in ABeta peptides exhibiting a reduced capacity to form high-molecular weight structures.

Two vertical arrows pointing away from the line drawn at 5.53 indicate directions in the regions characterized by the lower (arrow up) or higher (arrow down) stability of dimer complexes. The present grading was obtained and tested for a three-dimensional complex from the PDB:2MXU database [12].
We find that secondary nucleation proceeds at a much higher rate for D23N, E22G, E22Q, and E22K compared with WT at low and physiological monomer concentrations such that generation of toxic oligomers from monomers of these mutants at the surface of fibrils may become even more severe than for WT [On the role of sidechain size and charge in the
aggregation of A 42 with familial mutations]

Part IV

Determination of a combination of modified amyloid peptides characterized by the greatest thermodynamic stability.
The purpose of this work is the analytical identification of the most stable combinations of modified amyloid peptides. In this case, the first part of the work will be devoted to the analysis and processing of the available experimental base, and the second part will be a logical continuation of the search for stable dimeric complexes characterized by thermodynamic stability and a longer aggregation time.
The first part will compare numerical and experimental data on the example of mutant hereditary forms of Alzheimer's disease characterized by mutations in the APP protein. Each such hereditary mutation is characterized by its biochemical and physiological characteristics.

All calculations presented in this paper will be performed using the 2MXU or 5KK3 structure database PDB. The calculations will use amyloid peptides from the 11th to the 42nd amino acid residue, while the dimers will consist of either two wild-type amyloid peptides [wtABeta(11-42)]2, or one modified amyloid peptide, in which various amino acid residues will be replaced [mutABeta(11-42)] and one wild-type amyloid peptide [wtABeta(11-42)] to form a dimer [mutABeta(11-42)]-[wtABeta(11-42)] or involving two identically modified amyloid peptides , to form the [mutABeta(11-42)]2 dimer.
Fig.5. Comparison of experimental and calculated values that characterize the stability of amyloid peptides and their ability to enter into biochemical processes (self-aggregation), depending on the substitutions performed. Upper graph a) was obtained from experimental work[*] at a concentration of 1.1 μM. The time of half completion, t1/2, for the serine mutants in comparison with the WT Aβ42 in 20 mM sodium phosphate and 200 μM EDTA at pH 8.0 is shown in the upper graph. Below them are the corresponding calculated stability data for dimeric complexes taking into account the performed substitutions. At the same time, for clarity of viewing and comparison, the reciprocal value 1/lg(cond(W)) is given. Thus, we see a relationship between the stability of dimeric modified amyloid peptides and the half-life of their self-aggregation.

We explain it this way: the more stable the dimeric complex is, the slower it will enter into new biochemical processes.

In this work, we pay special attention to the peptide with four Ser substitutions, since it shows the least tendency to aggregation, with the longest half-life. The lower graph is the calculated value; the higher the value of the inverse of 1\lg(cond(W)) lies, the more stable the biological complex. This is a dimeric complex with four substitutions of amino acid residues for Serine (Ser).
[*]- Proc Natl Acad Sci U S A 2020 Oct 13;117(41):25272-25283. doi: 10.1073/pnas.2002956117. Epub 2020 Oct 1.The role of fibril structure and surface hydrophobicity in secondary nucleation of amyloid fibrils

The table summarizes the main studied vectors with the name of the vector and the substitutions made.

Fig.6. Results of Numerical Calculations Performed for Replacement of Amyloid Peptides in the Dimer ABeta(11-42) when replacing amino acid residues in one of the vectors a) and simultaneously replacing In two vectors b)

Analysis of the graphs allows us to conclude that single substitutions of the 16th amino acid residue do not lead to significant changes in the value of lg(cond(W)) as compared to the triple substitution of the 16th, 21st, and 30th amino acid residues.

Let us dwell in more detail on triple substitutions in amyloid peptides.

As can be seen from the figure, when a dimer is formed by one wild-type vector and one vector with a replacement of amino acid residues, the numerical values of lg(cond(W)) (in the range of 5.48)
They are in a higher range of values than the obtained numerical results for dimers formed by modified peptides lg(cond(W)) is in the range of 5.44 (Fig. B)

Fig.7. The results of numerical calculations performed when replacing amino acid residues in amyloid peptides during the formation of the dimeric complex ABet(11-42). The values of lg(cond(W)) are shown in fig.a) and the value of (delta)H in fig. B).
We will analyze graphs a) and b) simultaneously

Fig.10. The results of numerical calculations obtained for two different X-ray structures 2MXU a)-b) and 5KK3 c) with identical substitutions of the 21st, 30th and 42nd amino acid residues in one vector a) and in two vectors simultaneously b)-c) for all three graphs.
Numerical calculations for the last three vectors: wt-wt, K16N-wt, K16N-K16N are given as reference points for a visual comparison of the change in the value of lg(cond(W)).
The numbers in the margins of each graph indicate the average value of the minimum values of lg(cond(W)) obtained for each graph.
Fig.12. Results of numerical calculations performed for dimer complexes of amyloid peptides for two X-ray structures 2MXU a) and 5KK3 b)-c)
At the same time, in Fig. a) and b) identical substitutions were made in one of the vectors, namely, 2 amino acid residues 21 and 30 were replaced.
The numerical results presented in Fig.c) are subsequent substitutions in one of the vectors of two amino acid residues 21 and 30.

Selection of flexible chains of Gosuranemab for a peptide mimic of a tau protein

Gosuranemab, a humanized monoclonal antibody directed against N-terminal tau that is currently being investigated as a treatment for AD. Binding experiments showed that gosuranemab exhibited high affinity for tau monomer, tau fibrils, and insoluble tau from different tauopathies.
Interaction of amyloid peptides with various proteins
Toll-Like Receptors
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. They are single-pass membrane-spanning receptors usually expressed on sentinel cells such as macrophages and dendritic cells, that recognize structurally conserved molecules derived from microbes.
One family of receptors involved in the immune response to Aβ amyloid are the Toll-like receptors (TLRs) a class of pattern recognition receptors that recognize conserved microbial structures (Kawasaki and Kawai, 2014). TLRs are type I integral membrane proteins which recognize ligands with their leucine-rich repeat (LRR)-containing ectodomains. RNA sequencing revealed that the expression of six TLR genes (1,2,4,5,6,8) is upregulated in the temporal cortex of AD patients when compared to control brains, likely resulting from increased microglial activation (Chakrabarty et al., 2018). A direct interaction was identified between Aβ fibrils and CD14, a TLR co-receptor previously shown to associated with the inflammatory response to fibrillar Aβ (Fassbender et al., 2004; Reed-Geaghan et al., 2009). This interaction was shown to facilitate the internalization of Aβ fibrils by microglia, at lower concentrations than that required for cell activation (Liu et al., 2005). This suggests that CD14 could be involved in the phagocytosis of Aβ fibrils at low concentrations, but increased Aβ levels in AD results in cellular activation. Consistent with a role in Aβ uptake, TLR4 deficiency in AD mouse models results in increased fibrillar and soluble Aβ deposition (Tahara et al., 2006). Conversely, stimulation of the murine microglial cell line BV-2 with TLR2 and TLR4 ligands significantly increased the internalization of Aβ in vitro, further implicating TLR receptors in Aβ uptake and clearance (Tahara et al., 2006; Song et al., 2011).
scavenger receptors (SRs)
which are highly expressed by microglia (Christie et al., 1996; Wilkinson and El Khoury, 2012). It was found initially that class A SRs, characterized by an extracellular collagen-like domain, are involved in the binding to Aβ fibrils to microglial cells (El Khoury et al., 1996). It was then shown that coincubation of microglia with SR ligands such as acetyl-low density lipoprotein (Ac-LDL) reduced Aβ uptake, and CHO cells transfected with class A, or class B SR's showed enhanced Aβ uptake, suggesting that SRs are important in the uptake and clearance of Aβ (Paresce et al., 1996). Further investigation using microglia that are deficient in SR-A1 confirmed the role of SR-A1 and also SR-B1 in binding Aβ fibrils, consistent with a role in the clearance of Aβ amyloid (Husemann et al., 2001). CD36 is a class B scavenger receptor identified to form a receptor complex with the α6β1-integrin and the integrin-associated protein CD47 in microglia. This complex was shown to mediate the binding of Aβ fibrils to microglial cells and the subsequent activation of intracellular signaling pathways (Bamberger et al., 2003). While initial studies reported that Aβ fibril binding to this complex is largely involved in the activation of an inflammatory response, it was also reported that the interaction of Aβ fibrils with this complex is involved in the phagocytic uptake of fibrils by microglia (Coraci et al., 2002; Moore et al., 2002; Bamberger et al., 2003; Koenigsknecht and Landreth, 2004).
The TREM2 gene provides instructions for making a protein called triggering receptor expressed on myeloid cells 2. As its name suggests, this protein is made in myeloid cells, which are cells produced in bone marrow.

Triggering receptor expressed on myeloid cells 2 also known as TREM-2 is a protein that in humans is encoded by the TREM2 gene.The TREM-2 protein is expressed primarily in immune cells across many different tissues. In the brain, this receptor is found in microglia,which are the central nervous system's immune response system. In the liver, TREM2 is expressed in a multitude of cells, notably in macrophages that respond to insults to the tissue.In the bowel and intestine, TREM2 is found on dendritic cells. In these and other tissues, TREM2 is increasingly becoming an important receptor in different inflammatory diseases, and may have future potential as a therapeutic target.

The deletion of TREM2 in primary microglia was shown to significantly reduce the phagocytosis of aggregated Aβ1–42 (Kleinberger et al., 2014). Similarly, TREM2 deficiency reduced the efficacy of antibody-targeted Aβ phagocytosis by microglia (Xiang et al., 2016). There is evidence for direct interactions between TREM2 and Aβ1–42 fibrils, although no difference in binding affinity was identified for TREM2 R47H and R62H variants that are associated with an increased risk of AD (Lessard et al., 2018). However, the internalization of monomeric Aβ was reduced with the expression of these TREM2 AD variants (Lessard et al., 2018). In another study, TREM2 was found to bind to Aβ oligomers with a similar affinity to previously described Aβ receptors, CD36 and receptor for advanced glycation end products (RAGE), and this interaction was compromised by R47H and R62H TREM2 mutations (Zhao et al., 2018). In this study, TREM2 deficiency had little effect on Aβ uptake but led to significantly reduced Aβ degradation once internalized by microglia (Zhao et al., 2018). In TREM2 knock out mice injected with Aβ oligomers, there was reduced microglial migration to the site of injection and reduced Aβ clearance (Zhao et al., 2018). A recent study found that loss of TREM2 function led to an acceleration in early amyloidogenesis, accompanied by a reduction in microglial recruitment as previously described, again suggesting that TREM2 has a role in microglial clearance of Aβ (Parhizkar et al., 2019). Together this evidence suggests that Aβ is a ligand for TREM2, and that TREM2 has a role to play in both Aβ clearance and Aβ-stimulated microglial activation.
LC3-associated endocytosis
LANDO in microglia is a critical regulator of immune-mediated aggregate removal and microglial activation in a murine model of AD. Mice lacking LANDO but not canonical autophagy in the myeloid compartment or specifically in microglia have a robust increase in pro-inflammatory cytokine production in the hippocampus and increased levels of neurotoxic β-amyloid. This inflammation and β-amyloid deposition were associated with reactive microgliosis and tau hyperphosphorylation. LANDO-deficient AD mice displayed accelerated neurodegeneration, impaired neuronal signaling, and memory deficits. Our data support a protective role for LANDO in microglia in neurodegenerative pathologies resulting from β-amyloid deposition.
Evidence from this study suggests that LANDO facilitates recycling of the Ab receptors CD36, TLR4 and TREM2, thus allowing cycles of Ab endocytosis to continue,
promoting Ab uptake and clearance (Heckmann et al., 2019).

The autophagy proteins ATG5 and Rubicon were found to be protective against Ab deposition, with their absence leading to increased pathology. The expression of autophagy proteins declines with age, which may be related to the development of
Ab pathology in AD (Rubinsztein et al., 2011). It is important to note that macroautophagy has previously been implicated in the secretion of Ab into the extracellular space where it forms plaques in AD (Nilsson et al., 2013). When autophagyrelated gene 7 (ATG7) was conditionally knocked out in excitatory neurons of APP transgenic mice, extracellular Ab plaque pathology was significantly decreased, and Ab instead accumulated intracellularly (Nilsson et al., 2013). Thus, a
reduction in expression of proteins involved in macroautophagy could affect both Ab secretion and clearance.

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