C-terminal Hydrophobic Region Research Articles

Salt-Induced Hydrophobic C-Terminal Region of α-Synuclein Triggers Its Fibrillation under the Mimic Physiologic Condition.

α-Synuclein (αS) causes Parkinson's disease due to the structural alteration into amyloid fibrils that form after the interaction with synaptic membranes in neurons. To understand the alternation mechanism, the effect of salt (NaCl) on the interaction of αS with synaptic mimic membrane was characterized at the molecular level because salt triggered the amyloid fibril formation. The membrane-bound conformation (or the initial conformation before fibrillation) showed that NaCl decreased the number of helical structures and Tyr residues interacting with the membrane surface compared to when NaCl was absent, implying an increase in solvent-exposed regions. The N-terminal region of αS interacted with the membrane, forming the helical structures regardless of NaCl, while the C-terminal region formed a random structure with weak membrane interaction, but NaCl inhibited the interaction of its hydrophobic area, suggesting that salt promoted amyloid fibril formations by exposing the hydrophobic C-terminal region, which can intermolecularly interact with free αS.

Synthesis and mechanistic study of Aβ42 C-terminus domain derived tetrapeptides that inhibit Alzheimer’s Aβ-aggregation-induced neurotoxicity

Amyloid plaque formation in the brain is mainly responsible for the onset of Alzheimer’s disease (AD). Structure-based peptides have gained importance in recent years, and rational design of the peptide sequences for the prevention of Aβ-aggregation and related toxicity is imperative. In this study, we investigate the structural modification of tetrapeptides derived from the hydrophobic C-terminal region of Aβ42 “VVIA-NH2” and its retro-sequence “AIVV-NH2.” A preliminary screening of synthesized peptides through an MTT cell viability assay followed by a ThT fluorescence assay revealed a peptide 13 (Ala-Ile-Aib-Val-NH2) that showed protection against Aβ-aggregation and associated neurotoxicity. The presence of the α-helix inducer “Aib” in peptide 13 manifested the conformational transition from cross-β-sheets to α-helical content in Aβ42. The absence of fibrils in electron microscopic analysis suggested the inhibitory potential of peptide 13. The HRMS, DLS, and ANS studies further confirmed the inhibitory activity of 13, and no cytotoxicity was observed. The structure-based peptide described herein is a promising amyloid-β inhibitor and provides a new lead for the development of AD therapeutics.

C-terminal region of Rv1039c (PPE15) protein of Mycobacterium tuberculosis targets host mitochondria to induce macrophage apoptosis.

Mycobacterium tuberculosis (Mtb) genome possesses a unique family called Proline-Glutamate/Proline-Proline-Glutamate (PE/PPE) gene family, exclusive to pathogenic mycobacterium. Some of these proteins are known to play role in virulence and immune response modulation, but many are still uncharacterized. This study investigated the role of C-terminal region of Rv1039c (PPE15) in inducing mitochondrial perturbations and macrophage apoptosis. Our in-silico studies revealed the disordered, coiled, and hydrophobic C-terminal region in Rv1039c has similarity with C-terminal of mitochondria-targeting pro-apoptotic host proteins. Wild type Rv1039c and C-terminal deleted Rv1039c (Rv1039c-/-Cterm) recombinant proteins were purified and their M. smegmatis knock-in strains were constructed which were used for in-vitro experiments. Confocal microscopy showed localization of Rv1039c to mitochondria of PMA-differentiated THP1 macrophages; and reduced mitochondrial membrane depolarization and production of mitochondrial superoxides were observed in response to Rv1039c-/-Cterm in comparison to full-length Rv1039c. The C-terminal region of Rv1039c was found to activate caspases 3, 7 and 9 along with upregulated expression of pro-apoptotic genes like Bax and Bim. Rv1039c-/-Cterm also reduced the Cytochrome-C release from the mitochondria and the expression of AnnexinV/PI positive and TUNEL positive cells as compared to Rv1039c. Additionally, Rv1039c was observed to upregulate the TLR4-NF-κB-TNF-α signalling whereas the same was downregulated in response to Rv1039c-/-Cterm. These findings suggested that the C-terminal region of Rv1039c is a molecular mimic of pro-apoptotic host proteins which induce mitochondria-dependent macrophage apoptosis and evoke host immune response. These observations enhance our understanding about the role of PE/PPE proteins at host-pathogen interface.

Deciphering the influence of Y12L and N17H substitutions on the conformation and oligomerization of human calcitonin.

The abnormal aggregation of human calcitonin (hCT) hormone peptides impairs their physiological function, leading to harmful immune responses and cytotoxicity, which limits their clinical utility. Interestingly, a representative hCT analog incorporating Y12L and N17H substitutions (DM-hCT) has shown reduced aggregation tendencies while maintaining bioactivity. But the molecular mechanism of Y12L and N17H substitutions on the conformational dynamics of hCT remains unclear. Here, we systematically investigated the folding and self-assembly dynamics of hCT and DM-hCT using atomistic discrete molecular dynamics (DMD) simulations. Our findings revealed that hCT monomers predominantly adopted unstructured conformations with dynamic helices. Oligomerization of hCT resulted in the formation of β-sheet-rich aggregates and β-barrel intermediates. The Y12L and N17H substitutions enhanced helical conformations and suppressed β-sheet formation in both monomers and oligomers. These substitutions stabilized the dynamic helices and disrupted aromatic interactions responsible for β-sheet formation at residue 12. Notably, DM-hCT assemblies still exhibited β-sheets in phenylalanine-rich and C-terminal hydrophobic regions, suggesting that future optimizations should focus on these areas. Our simulations provide insights into the molecular mechanisms underlying hCT aggregation and the amyloid-resistant effects of Y12L and N17H substitutions. These findings have valuable implications for the development of clinical hCT analogs.

Multi-Target Actions of Flavonoid Derivatives from Mesua ferrea Linn Flower against Alzheimer’s disease Pathogenesis

OBJECTIVE Kaempferol-3-O-rhamnoside (compound 1) and quercetin- 3-O-rhamnoside (compound 2), two flavonoids isolated from Mesua ferrea L. flowers, were examined for their activities related Alzheimer’s disease (AD) pathogenesis including antioxidant, acetylcholinesterase (AChE) inhibition, anti-beta amyloid (Ab) aggregation and neuroprotection. METHODS The two flavonoids were isolated from M. ferrea L. flowers using the column chromatography technique. Both compounds were evaluated for their effects on AD pathogenesis, including antioxidant action by ABTS assay, AChE inhibition by Ellman’s method, and anti-Ab aggregation by thioflavin T (ThT) assay and neuroprotection by cell base assay. To explain the mechanism of AChE inhibition and anti-Ab aggregation, binding interactions between the test compounds and AChE and Ab were studied in-silico. RESULTS Compounds 1 and 2 showed an ability to scavenge ABTS radicals, with IC50 values of 424.57±2.97 and 308.67±9.90 µM, respectively, and to inhibit AChE function with IC50 values of 769.23±6.23 and 520.64±5.94, respectively. ThT assay indicated that both compounds inhibited Ab aggregation with IC50 values of 406.43±9.95 and 300.69 ±1.18 µM, respectively. The neuroprotection study revealed that the two flavonoids could reduce human neuroblastoma (SH-SY5Y) cell death induced by H2O2. The in-silico study showed that both compounds bound AChE at catalytic anionic and peripheral anionic sites. In addition, the test compounds prevented Ab aggregation by interacting at the central hydrophobic core, the C-terminal hydrophobic region, and the important residues of Ile41. CONCLUSIONS Together, the results showed that kaempferol-3-O- rhamnoside and quercetin-3-O- rhamnoside exhibit multiple mechanisms of action that are involved in the pathogenesis of AD including antioxidant, AChE inhibition, anti-Ab aggregation, and neuroprotection. KEYWORDS flavonoid rhamnosides, Alzheimer’s disease, oxidation, beta amyloid, acetylcholinesterase, molecular docking

Dissecting the Molecular Mechanisms of the Co-Aggregation of Aβ40 and Aβ42 Peptides: A REMD Simulation Study.

The aggregation of amyloid-β protein (Aβ) into oligomers and amyloid fibrils is closely related to Alzheimer's disease (AD). Aβ40 and Aβ42, as two most prominent isoforms of Aβ peptides, can cross-interact with each other and form co-aggregates, which affect the progression of the disease. However, the molecular determinants underlying Aβ40 and Aβ42 cross-interaction and the structural details of their co-oligomers remain elusive. Herein, we performed all-atom explicit-solvent replica exchange molecular dynamics simulations on Aβ40-Aβ42 heterogeneous and Aβ40/Aβ42 homogeneous dimer systems to dissect the co-aggregation mechanisms of the two isoforms. Our results show that the interpeptide main-chain interaction of Aβ40-Aβ42 is stronger than that of Aβ40-Aβ40 and Aβ42-Aβ42. The positions of hotspot residues in heterodimers and homodimers display high similarity, implying similar molecular recognition sites for both cross-interaction and self-interaction. Contact maps of Aβ40-Aβ42 heterodimers reveal that residue pairs crucial for cross-interaction are mostly located in the C-terminal hydrophobic regions of Aβ40 and Aβ42 peptides. Conformational analysis shows that Aβ40 and Aβ42 monomers can co-assemble into β-sheet-rich heterodimers with shorter β-sheets than those in homodimers, which is decremental to monomer addition. Similar molecular recognition sites and β-sheet distribution of Aβ40 and Aβ42 peptides are observed in heterodimers and homodimers, which may provide the molecular basis for the two isoforms' co-aggregation and cross-seeding. Our work dissects the co-aggregation mechanisms of Aβ40 and Aβ42 peptides at the atomic level, which will help for in-depth understanding of the cross-talk between the two Aβ isoforms and the pathogenesis of AD.

Higher-order structure formation using refined monomer structures of lipid raft markers, Stomatin, Prohibitin, Flotillin, and HflK/C-related proteins.

Currently, information on the higher-order structure of Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH)-domain proteins is limited. Briefly, the coordinate information (Refined PH1511.pdb) of the stomatin ortholog, PH1511 monomer, was obtained using the artificial intelligence, ColabFold: AlphaFold2. Thereafter, the 24mer homo-oligomer structure of PH1511 was constructed using the superposing method, with HflK/C and FtsH (KCF complex) as templates. The 9mer-12mer homo-oligomer structures of PH1511 were also constructed using the ab initio docking method, with the GalaxyHomomer server for artificiality elimination. The features and functional validity of the higher-order structures were discussed. The coordinate information (Refined PH1510.pdb) of the membrane protease PH1510 monomer, which specifically cleaves the C-terminal hydrophobic region of PH1511, was obtained. Thereafter, the PH1510 12mer structure was constructed by superposing 12 molecules of the Refined PH1510.pdb monomer onto a 1510-C prism-like 12mer structure formed along the crystallographic threefold helical axis. The 12mer PH1510 (prism) structure revealed the spatial arrangement of membrane-spanning regions between the 1510-N and 1510-C domains within the membrane tube complex. Based on these refined 3D homo-oligomeric structures, the substrate recognition mechanism of the membrane protease was investigated. These refined 3D homo-oligomer structures are provided via PDB files as Supplementary data and can be used for further reference.

Effects of Familial Alzheimer's Disease Mutations on the Folding Free Energy and Dipole-Dipole Interactions of the Amyloid β-Peptide.

Familial Alzheimer's disease (FAD) mutations of the amyloid β-peptide (Aβ) are known to lead to early onset and more aggressive Alzheimer's disease. FAD mutations such as "Iowa" (D23N), "Arctic" (E22G), "Italian" (E22K), and "Dutch" (E22Q) have been shown to accelerate Aβ aggregation relative to the wild-type (WT). The mechanism by which these mutations facilitate increased aggregation is unknown, but each mutation results in a change in the net charge of the peptide. Previous studies have used nonpolarizable force fields to study Aβ, providing some insight into how this protein unfolds. However, nonpolarizable force fields have fixed charges that lack the ability to redistribute in response to changes in local electric fields. Here, we performed polarizable molecular dynamics simulations on the full-length Aβ42 of WT and FAD mutations and calculated folding free energies of the Aβ15-27 fragment via umbrella sampling. By studying both the full-length Aβ42 and a fragment containing mutations and the central hydrophobic cluster (residues 17-21), we were able to systematically study how these FAD mutations impact secondary and tertiary structure and the thermodynamics of folding. Electrostatic interactions, including those between permanent and induced dipoles, affected side-chain properties, salt bridges, and solvent interactions. The FAD mutations resulted in shifts in the electronic structure and solvent accessibility at the central hydrophobic cluster and the hydrophobic C-terminal region. Using umbrella sampling, we found that the folding of the WT and E22 mutants is enthalpically driven, whereas the D23N mutant is entropically driven, arising from a different unfolding pathway and peptide-bond dipole response. Together, the unbiased, full-length, and umbrella sampling simulations of fragments reveal that the FAD mutations perturb nearby residues and others in hydrophobic regions to potentially alter solubility. These results highlight the role electronic polarizability plays in amyloid misfolding and the role of heterogeneous microenvironments that arise as conformational change takes place.

Syntaxin 17, an ancient SNARE paralog, plays different and conserved roles in different organisms.

Mammalian syntaxin 17 (Stx17) has several roles in processes other than membrane fusion, including in mitochondrial division, autophagosome formation and lipid droplet expansion. In contrast to conventional syntaxins, Stx17 has a long C-terminal hydrophobic region with a hairpin-like structure flanked by a basic amino acid-enriched C-terminal tail. Although Stx17 is one of the six ancient SNAREs and is present in diverse eukaryotic organisms, it has been lost in multiple lineages during evolution. In the present study, we compared the localization and function of fly and nematode Stx17s expressed in HeLa cells with those of human Stx17. We found that fly Stx17 predominantly localizes to the cytosol and mediates autophagy, but not mitochondrial division. Nematode Stx17, on the other hand, is predominantly present in mitochondria and facilitates mitochondrial division, but is irrelevant to autophagy. These differences are likely due to different structures in the C-terminal tail. Non-participation of fly Stx17 and nematode Stx17 in mitochondrial division and autophagy, respectively, was demonstrated in individual organisms. Our results provide an insight into the evolution of Stx17 in metazoa. This article has an associated First Person interview with the first author of the paper.

Multi-Target Actions of Acridones from Atalantia monophylla towards Alzheimer's Pathogenesis and Their Pharmacokinetic Properties.

Ten acridones isolated from Atalantia monophylla were evaluated for effects on Alzheimer’s disease pathogenesis including antioxidant effects, acetylcholinesterase (AChE) inhibition, prevention of beta-amyloid (Aβ) aggregation and neuroprotection. To understand the mechanism, the type of AChE inhibition was investigated in vitro and binding interactions between acridones and AChE or Aβ were explored in silico. Drug-likeness and ADMET parameters were predicted in silico using SwissADME and pKCSM programs, respectively. All acridones showed favorable drug-likeness and possessed multifunctional activities targeting AChE function, Aβ aggregation and oxidation. All acridones inhibited AChE in a mixed-type manner and bound AChE at both catalytic anionic and peripheral anionic sites. In silico analysis showed that acridones interfered with Aβ aggregation by interacting at the central hydrophobic core, C-terminal hydrophobic region, and the key residues 41 and 42. Citrusinine II showed potent multifunctional action with the best ADMET profile and could alleviate neuronal cell damage induced by hydrogen peroxide and Aβ1-42 toxicity.

Evaluating the Multifunctionality of a New Modulator of Zinc-Induced Aβ Aggregation Using a Novel Computational Approach.

The high concentration of zinc metal ions in Aβ aggregations is one of the most cited hallmarks of Alzheimer's disease (AD), and several substantial pieces of evidence emphasize the key role of zinc metal ions in the pathogenesis of AD. In this study, while designing a multifunctional peptide for simultaneous targeting Aβ aggregation and chelating the zinc metal ion, a novel and comprehensive approach is introduced for evaluating the multifunctionality of a multifunctional drugs based on computational methods. The multifunctional peptide consists of inhibitor and chelator domains, which are included in the C-terminal hydrophobic region of Aβ, and the first four amino acids of human albumin. The ability of the multifunctional peptide in zinc ion chelation has been investigated using molecular dynamics (MD) simulations of the peptide-zinc interaction for 300 ns, and Bennett's acceptance ratio (BAR) method has been used to accurately calculate the chelation free energy. Data analysis demonstrates that the peptide chelating domain can be stably linked to the zinc ion. Besides, the introduced method used for evaluating chelation and calculating the free energy of peptide binding to zinc ions was successfully validated by comparison with previous experimental and theoretical published data. The results indicate that the multifunctional peptide, coordinating with the zinc metal ion, can be effective in Aβ inhibition by preserving the native helical structure of the Aβ42 monomer as well as disrupting the β-sheet structure of Aβ42 aggregates. Detailed assessments of the Aβ42-peptide interactions elucidate that the inhibition of Aβ is achieved by considerable hydrophobic interactions and hydrogen bonding between the multifunctional peptide and the hydrophobic Aβ regions, along with interfering in stable bridges formed inside the Aβ aggregate.

Mechanistic Insights into the Co-Aggregation of Aβ and hIAPP: An All-Atom Molecular Dynamic Study.

Patients with Alzheimer's disease (AD) have a high risk of developing Type II diabetes (T2D). The co-aggregation of the two disease-related proteins, Aβ and hIAPP, has been proposed as a potential molecular mechanism. However, the detailed Aβ-hIAPP interactions and structural characteristics of co-aggregates are mostly unknown at atomic level. Here, we explore the conformational ensembles of the Aβ-hIAPP heterodimer and Aβ or hIAPP homodimer by performing all-atom explicit-solvent replica exchange molecular dynamic simulations. Our simulations show that the interaction propensity of Aβ-hIAPP in the heterodimer is comparable with that of Aβ-Aβ/hIAPP-hIAPP in the homodimer. Similar hot spot residues of Aβ/hIAPP in the homodimer and heterodimer are identified, indicating that both Aβ and hIAPP have similar molecular recognition sites for self-aggregation and co-aggregation. Aβ in the heterodimer possesses three high β-sheet probability regions: the N-terminal region E3-H6, the central hydrophobic core region K16-E22, and the C-terminal hydrophobic region I31-A41, which is highly similar to Aβ in the homodimer. More importantly, in the heterodimer, the regions E3-H6, F19-E22, and I31-M35 of Aβ and the amyloid core region N20-T30 of hIAPP display higher β-sheet probability than they do in homodimer, implying their crucial roles in the formation of β-sheet-rich co-aggregates. Our study sheds light on the co-aggregation of Aβ and hIAPP at an atomic level, which will be helpful for an in-depth understanding of the molecular mechanism for epidemiological correlation of AD and T2D.

Role of Chain Extension in the Ability of Peptide Oligomers to Damage the Lipid Membrane Studied by the l- to d-Amino Acid Substitutions of hIAPP18-27.

Exploration of the relation between the structural feature of oligomers and the ability of oligomers to damage the membrane has been an important subject in the study of the cytotoxic mechanism of amyloid proteins. In this work, we selected the hIAPP18-27 fragment as a model peptide and modified it by an alternating substitution of a d-amino acid for an l-amino acid in the hydrophilic N-terminal region, the hydrophobic C-terminal region, and the entire sequence. We prepared the oligomers using these peptides and investigated the effects of chain extension in different regions of the peptide on the ability of the oligomers to damage the membrane composed of POPC/POPG 4:1. We examined the morphology, structure, surface hydrophobicity, and packing compactness of the oligomers and monitored the changes in the structure and aggregation of the peptides upon interaction with the membrane. We found that the surface hydrophobicity and the disruptive ability of the oligomers are increased by an alternating l- and d-amino acid arrangement in the hydrophobic region of the peptide, while the packing compactness of the oligomers is increased and the disruptive ability of the oligomers decreased by an alternating l- and d-amino acid arrangement only in the hydrophilic region. The extension of the hydrophobic chain plays a significant role in the disruptive ability of the oligomers. Our results suggest that a positive relation between the surface hydrophobicity and the disruptive ability could be established only for the oligomers in which the peptide chains are flexible and loosely packed.

Inactive dimeric structure of the protease domain of stomatin operon partner protein.

The N-terminal region of the stomatin operon partner protein (STOPP) PH1510 (1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii is a serine protease with a catalytic Ser-Lys dyad (Ser97 and Lys138) and specifically cleaves the C-terminal hydrophobic region of the p-stomatin PH1511. In a form of human hemolytic anemia known as hereditary stomatocytosis, stomatin is deficient in the erythrocyte membrane owing to mis-trafficking. Stomatin is thought to act as an oligomeric scaffolding protein to support cell membranes. The cleavage of stomatin by STOPP might be involved in a regulatory system. Several crystal structures of 1510-N have previously been determined: the wild type, the K138A mutant and its complex with a substrate peptide. Here, the crystal structure of the S97A mutant of 1510-N (1510-N S97A) was determined at 2.25 Å resolution. The structure contained two 1510-N S97A molecules in the asymmetric unit. On the superposition of one monomer of the 1510-N S97A and wild-type dimers, the S97A Cα atom of the other monomer of 1510-N S97A deviated by 23 Å from that of the wild type. This result indicates that 1510-N can greatly change the form of its dimer. Because of crystallographic symmetry in space group P65, a sixfold helical structure is constructed using the 1510-N dimer as a basic unit. This helical structure may be common to STOPP structures.

The C-terminal region of the Plasmodium yoelii microgamete surface antigen PyMiGS induces potent anti-malarial transmission-blocking immunity in mice

Malaria transmission-blocking vaccines (TBVs) aim to inhibit parasite fertilization or further development within the mosquito midgut. Because TBV-immunized individuals reduce the transmission of malaria parasites to mosquito vectors, TBVs could serve as a promising strategy to eliminate malaria. We previously reported that a male specific protein, PyMiGS (Plasmodium yoelii microgamete surface protein), is localized to the surface of microgametes and anti-PyMiGS antibodies have strong transmission-blocking activity. In this study we determine a region of PyMiGS that contains epitopes inducing potent transmission-blocking antibodies. PyMiGS excluding the N-terminal signal sequence and C-terminal hydrophobic region (PyMiGS-full) was divided into five overlapping regions, named I through V, and corresponding truncated recombinant proteins were produced. Anti-region V antibody, affinity-purified from anti-PyMiGS-full rabbit antiserum, significantly reduced the number of oocysts in a mosquito membrane-feeding assay. Antibodies from mice immunized with PyMiGS-V recognized the microgamete surface and showed higher transmission-blocking efficacy than antibodies obtained by PyMiGS-full immunization. These results indicate that the major epitopes for transmission-blocking antibodies are within region V at the C-terminal region of PyMiGS. Therefore, region V of MiGS could be a promising pre-fertilization TBV candidate antigen.

Tau/Aβ chimera peptides: Evaluating the dual function of metal coordination and membrane interaction in one sequence

Several abnormal events may concur as major risk factors for Alzheimer's disease (AD) pathogenesis. For instance, dysregulation of brain's metal homeostasis and amyloid-mediated membrane damage are established toxic mechanisms causing neuronal death. In this study, we assess the amyloidogenic propensity and membrane-damage effects, either in the presence or in absence of metal ions, of two newly synthesized bifunctional peptides. These were designed to comprise a metal chelating N-terminus region derived from Tau protein namely the Tau9–16 (EVMEDHAG) or Tau26–33 (QGGYTMHQ) sequences, merged with the C-terminal hydrophobic region analogous to the Amyloid beta (Aβ) 16–20 aminoacid sequence KLVFF (KL). Comparative circular dichroism or fluorescence experiments were carried out to look at the peptide conformation, fibril formation and membrane affinity of Tau9–16KL and Tau26–33KL peptides. We found that Tau9–16KL and Tau26–33KL perturb the fibrillogenic process of Aβ1–40. Furthermore Cu(II) and, to a lower extent, Zn(II) induced conformational changes Tau26–33KL both in water and in membrane-mimicking environment. By contrast, due to a different metal coordination mode we observed for Tau9–16KL an unstructured peptide conformation in all the experimental conditions. Unlike aqueous solution, a certain propensity to form amyloid structures at the lipid membrane interface clearly emerged for both the peptides. However, the two peptides exhibit a different capability to elicit membrane damage depending on the presence or absence of metal ions. Tau9–16KL and Tau26–33KL can be used as peptide-based molecular systems able to interfere with the metal dependent Aβ/Tau cross-seeded generation of membrane active amyloid species.

Identification of a Novel Multifunctional Ligand for Simultaneous Inhibition of Amyloid-Beta (Aβ42) and Chelation of Zinc Metal Ion

Zinc binding to β-amyloid structure could promote amyloid-β aggregation, as well as reactive oxygen species (ROS) production, as suggested in many experimental and theoretical studies. Therefore, the introduction of multifunctional drugs capable of chelating zinc metal ion and inhibiting Aβ aggregation is a promising strategy in the development of AD treatment. The present study has evaluated the efficacy of a new bifunctional peptide drug using molecular docking and molecular dynamics (MD) simulations. This drug comprises two different domains, an inhibitor domain, obtained from the C-terminal hydrophobic region of Aβ, and a Zn2+ chelating domain, derived from rapeseed meal, merge with a linker. The multifunctionality of the ligand was evaluated using a comprehensive set of MD simulations spanning up to 3.2 μs including Aβ relaxation, ligand-Zn2+ bilateral interaction, and, more importantly, ligand-Zn2+-Aβ42 trilateral interactions. Analysis of the results strongly indicated that the bifunctional ligand can chelate zinc metal ion and avoid Aβ aggregation simultaneously. The present study illustrated that the proposed ligand has considerable hydrophobic interactions and hydrogen bonding with monomeric Aβ in the presence of zinc metal ion. Therefore, in light of these considerable interactions and contacts, the α-helical structure of Aβ has been enhanced, while the β-sheet formation is prevented and the α-helix native structure is protected. Furthermore, the analysis of interactions between Aβ and ligand-zinc complex revealed that the zinc metal ion is coordinated to Met13, the ending residue of the ligand and merely one residue in Aβ. The results have proven the previous experimental and theoretical findings in the literature about Aβ interactions with zinc metal ion and also Aβ interactions with the first domain of the proposed ligand. Moreover, the current research has evaluated the chelation using MD simulation and linear interaction energy (LIE) methods, and the result has been satisfactorily verified with previous experimental and theoretical (DFT) studies.

Impact of K16A and K28A mutation on the structure and dynamics of amyloid-β42 peptide in Alzheimer’s disease: key insights from molecular dynamics simulations

The aggregation of amyloid-β42 (Aβ42) peptide into toxic oligomers and fibrils is a key step in the Alzheimer disease pathogenesis. The recent studies highlighted that lysine residues (K16 and K28) play a critical role in the Aβ42 self-assembly and are the target of entities like molecular tweezer, CLR01. The studies reveal that lysine to alanine mutation significantly affect Aβ oligomerization, toxicity and aggregation process. However, the molecular mechanism behind reduced Aβ toxicity on K16A and K28A mutation remain elusive. In this regard, molecular dynamics (MD) simulations were performed in the present study to get insights into the effect of K16A and K28A mutation in Aβ42 self-assembly. The MD simulations highlighted that K16A and K28A mutation in the aggregation-prone region, i.e., central hydrophobic core (KLVFF, 16–20) and bend region (D23–K28), cause major structural changes in the Aβ42 monomer. The secondary structure analysis highlight that modulation of aggregation in K16A and K28A is linked to the increase in the overall helix content and a concomitant decrease in the β–sheet content of Aβ42 monomer. The short-range tertiary contacts between central hydrophobic core and C-terminal region were relatively reduced in K16A and K28A as compare to wild type (wt) Aβ42. The mechanistic insights from the study will be beneficial for the design and development of novel inhibitors that will bind and block the interactions, mediated by lysine residues specifically, critical for the Aβ42 self-assembly in Alzheimer disease. The molecular mechanism behind modulation of amyloid-β42 (Aβ42) self-assembly on K16A and K28A mutation has been investigated using molecular dynamics (MD) simulations. MD simulations reveal that reduced aggregation in K16A and K28A is linked to the increase in the overall helix content and a concomitant decrease in the β–sheet content, particularly at the C–terminal region, of Aβ42.Communicated by Ramaswamy H. Sarma

The Norepinephrine Inhibits Alzheimer's Amyloid-β Peptide Aggregation by Binding to the C-Terminal Hydrophobic Region

The abnormal self-assembly of amyloid-β (Aβ) peptides into toxic prefibrillar aggregates is associated with the pathogenesis of Alzheimer's disease (AD). The inhibition of β-sheet-rich oligomers formation is considered as the primary therapeutic strategy for AD. Previous experimental studies reported that norepinephrine (NE), one of the neurotransmitters, is able to inhibit Aβ aggregation and disaggregate the preformed fibrils. However, the underlying inhibitory and disruptive mechanisms remain elusive. In this work, we performed extensive replica-exchange molecular dynamic (REMD) simulations on the conformational ensemble of Aβ1-42 dimer with and without NE molecules. Our results show that in the absence of NE molecules, Aβ1-42 dimer transiently adopts a β-hairpin involving two β-strands of residues 15QKLVFFA21 and residues 33GLMVGGVV40, whose β-strand regions strongly resemble those of the Aβ fibril structure (β1: residues Q15-A21, β2: residues I30-G37) reported in an electron paramagnetic resonance spectroscopy study. NE molecules greatly reduce the interpeptide β-sheet content and suppress the formation of the above-mentioned β-hairpin, leading to a more disordered coil-rich Aβ dimer. Four dominant binding sites are identified via contact probability and binding energy analyses, among which the C-terminal I31-V36 hydrophobic region is the most favorable one. Our data reveal that hydrophobic, aromatic stacking, cation-π and hydrogen-bonding interactions synergistically contribute to the NE-induced conformational shift. This work reveals the molecular mechanism of NE molecules against Aβ1-42 aggregation and provides valuable information for the development of new drug candidates against AD.

Specific regions of the SulA protein recognized and degraded by the ATP-dependent ClpYQ (HslUV) protease in Escherichia coli

In Escherichia coli, ClpYQ (HslUV) is a two-component ATP-dependent protease, in which ClpQ is the peptidase subunit and ClpY is the ATPase and unfoldase. ClpY functions to recognize protein substrates, and denature and translocate the unfolded polypeptides into the proteolytic site of ClpQ for degradation. However, it is not clear how the natural substrates are recognized by the ClpYQ protease and the mechanism by which the substrates are selected, unfolded and translocated by ClpY into the interior site of ClpQ hexamers. Both Lon and ClpYQ proteases can degrade SulA, a cell division inhibitor, in bacterial cells. In this study, using yeast two-hybrid and in vivo degradation analyses, we first demonstrated that the C-terminal internal hydrophobic region (139th∼149th aa) of SulA is necessary for binding and degradation by ClpYQ. A conserved region, GFIMRP, between 142th and 147th residues of SulA, were identified among various Gram-negative bacteria. By using MBP-SulA(F143Y) (phenylalanine substituted with tyrosine) as a substrate, our results showed that this conserved residue of SulA is necessary for recognition and degradation by ClpYQ. Supporting these data, MBP-SulA(F143Y), MBP-SulA(F143N) (phenylalanine substituted with asparagine) led to a longer half-life with ClpYQ protease in vivo. In contrast, MBP-SulA(F143D) and MBP-SulA(F143S) both have shorter half-lives. Therefore, in the E. coli ClpYQ protease complex, ClpY recognizes the C-terminal region of SulA, and F143 of SulA plays an important role for the recognition and degradation by ClpYQ protease.