Molecular Mechanism of Zinc-Dependent Oligomerization of Alzheimer's Amyloid-ß with Taiwan (D7H) Mutation.

Amyloid-ß (Aß) is a peptide formed by 39-43 amino acids, heterogenous by the length of its C-terminus. Aß constitutes a subnanomolar monomeric component of human biological fluids; however, in sporadic variants of Alzheimer's disease (AD), it forms soluble neurotoxic oligomers and accumulates as insoluble extracellular polymeric aggregates (amyloid plaques) in the brain tissues. The plaque formation is controlled by zinc ions; therefore, abnormal interactions between the ions and Aß seem to take part in the triggering of sporadic AD. The amyloid plaques contain various Aß isoforms, among which the most common is Aß with an isoaspartate in position 7 (isoD7). The spontaneous conversion of D7 to isoD7 is associated with Aß aging. Aß molecules with isoD7 (isoD7-Aß) easily undergo zinc-dependent oligomerization, and upon administration to transgenic animals (mice, nematodes) used for AD modeling, act as zinc-dependent seeds of the pathological aggregation of Aß. The formation of zinc-bound homo- and hetero-oligomers with the participation of isoD7-Aß is based on the rigidly structured segment 11-EVHH-14, located in the Aß metal binding domain (Aß16). Some hereditary variants of AD are associated with familial mutations within the domain. Among these, the most susceptible to zinc-dependent oligomerization is Aß with Taiwan (D7H) mutation (D7H-Aß). In this study, the D7H-Aß metal binding domain (D7H-Aß16) has been used as a model to establish the molecular mechanism of zinc-induced D7H-Aß oligomerization through turbidimetry, dynamic light scattering, isothermal titration calorimetry, mass spectrometry, and computer modelling. Additionally, the modeling data showed that a molecule of D7H-Aß, as well as isoD7-Aß in combination with two Aß molecules, renders a stable zinc-induced heterotrimer. The trimers are held together by intermolecular interfaces via zinc ions, with the primary interfaces formed by 11-EVHH-14 sites of the interacting trimer subunits. In summary, the obtained results confirm the role of the 11-EVHH-14 region as a structure and function determinant for the zinc-dependent oligomerization of all known Aß species (including various chemically modified isoforms and AD-associated mutants) and point at this region as a potent target for drugs aimed to stop amyloid plaque formation in both sporadic and hereditary variants of AD.

Natural Isoforms of Listeria monocytogenes Virulence Factor Inlb Differ in c-Met Binding Efficiency and Differently Affect Uptake and Survival Listeria in Macrophage.

Listeria monocytogenes virulence factor InlB specifically interacts with the receptors c-Met and gC1q-R. Both receptors are present in non-professional and professional phagocytes, including macrophages. Phylogenetically defined InlB isoforms differently support invasion into non-professional phagocytes. This work deals with the effects of InlB isoforms on L. monocytogenes uptake and intracellular proliferation in human macrophages. Three isoforms of the receptor binding domain (idInlB) were derived from phylogenetically distinct L. monocytogenes strains belonging to the highly virulent CC1 (idInlBCC1), medium-virulence CC7 (idInlBCC7), and low-virulence CC9 (idInlBCC9) clonal complexes. The constant dissociation increased in the order idInlBCC1 << idInlBCC7 < idInlBCC9 for interactions with c-Met, and idInlBCC1 ≈ idInlBCC7 < idInlBCC9 for interactions with gC1q-R. The comparison of uptake and intracellular proliferation of isogenic recombinant strains which expressed full-length InlBs revealed that the strain expressing idInlBCC1 proliferated in macrophages twice as efficiently as other strains. Macrophage pretreatment with idInlBCC1 followed by recombinant L. monocytogenes infection disturbed macrophage functions decreasing pathogen uptake and improving its intracellular multiplication. Similar pretreatment with idInlBCC7 decreased bacterial uptake but also impaired intracellular multiplication. The obtained results demonstrated that InlB impaired macrophage functions in an idInlB isoform-dependent manner. These data suggest a novel InlB function in L. monocytogenes virulence.

SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines.

Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.

Switching On/Off Amyloid Plaque Formation in Transgenic Animal Models of Alzheimer's Disease.

A hallmark of Alzheimer's disease (AD) are the proteinaceous aggregates formed by the amyloid-beta peptide (Aß) that is deposited inside the brain as amyloid plaques. The accumulation of aggregated Aß may initiate or enhance pathologic processes in AD. According to the amyloid hypothesis, any agent that has the capability to inhibit Aß aggregation and/or destroy amyloid plaques represents a potential disease-modifying drug. In 2023, a humanized IgG1 monoclonal antibody (lecanemab) against the Aß-soluble protofibrils was approved by the US FDA for AD therapy, thus providing compelling support to the amyloid hypothesis. To acquire a deeper insight on the in vivo Aß aggregation, various animal models, including aged herbivores and carnivores, non-human primates, transgenic rodents, fish and worms were widely exploited. This review is based on the recent data obtained using transgenic animal AD models and presents experimental verification of the critical role in Aß aggregation seeding of the interactions between zinc ions, Aß with the isomerized Asp7 (isoD7-Aß) and the α4ß2 nicotinic acetylcholine receptor.

Impact of Troponin in Cardiomyopathy Development Caused by Mutations in Tropomyosin.

Tropomyosin (Tpm) mutations cause inherited cardiac diseases such as hypertrophic and dilated cardiomyopathies. We applied various approaches to investigate the role of cardiac troponin (Tn) and especially the troponin T (TnT) in the pathogenic effects of Tpm cardiomyopathy-associated mutations M8R, K15N, A277V, M281T, and I284V located in the overlap junction of neighboring Tpm dimers. Using co-sedimentation assay and viscosity measurements, we showed that TnT1 (fragment of TnT) stabilizes the overlap junction of Tpm WT and all Tpm mutants studied except Tpm M8R. However, isothermal titration calorimetry (ITC) indicated that TnT1 binds Tpm WT and all Tpm mutants similarly. By using ITC, we measured the direct KD of the Tpm overlap region, N-end, and C-end binding to TnT1. The ITC data revealed that the Tpm C-end binds to TnT1 independently from the N-end, while N-end does not bind. Therefore, we suppose that Tpm M8R binds to TnT1 without forming the overlap junction. We also demonstrated the possible role of Tn isoform composition in the cardiomyopathy development caused by M8R mutation. TnT1 dose-dependently reduced the velocity of F-actin-Tpm filaments containing Tpm WT, Tpm A277V, and Tpm M281T mutants in an in vitro motility assay. All mutations impaired the calcium regulation of the actin-myosin interaction. The M281T and I284V mutations increased the calcium sensitivity, while the K15N and A277V mutations reduced it. The Tpm M8R, M281T, and I284V mutations under-inhibited the velocity at low calcium concentrations. Our results demonstrate that Tpm mutations likely implement their pathogenic effects through Tpm interaction with Tn, cardiac myosin, or other protein partners.

Bacterial hepatocyte growth factor receptor agonist stimulates hepatocyte proliferation and accelerates liver regeneration in a partial hepatectomy rat model.

Hepatocyte growth factor (HGF) is central to liver regeneration. The Internalin B (InlB) protein is a virulence factor produced by the pathogenic bacterium Listeria monocytogenes. InlB is known to mimic HGF activity by interacting with the HGF receptor (HGFR) and activating HGFR-controlled signaling pathways. We expressed and purified the HGFR-binding InlB domain, InlB321/15, cloned from the fully virulent clinical L. monocytogenes strain. HGFR and Erk1/2 phosphorylation was determined using Western blotting. The capacity of InlB321/15 to bind HGFR was measured using microscale thermophoresis. Liver regeneration was studied in a model of 70% partial hepatectomy (70%PHx) in male Wistar rats. The nuclear grade parameters were quantified using manual (percentage of binuclear hepatocytes), automated (nuclear diameters), or combined (Ki67 proliferation index) scoring methods. Purified InlB321/15 stimulated HGFR and Erk1/2 phosphorylation and accelerated the proliferation of HepG2 cells. InlB321/15 bound HGFR with Kd = 7.4 ± 1.3 nM. InlB321/15 injected intravenously on the second, fourth, and sixth days after surgery recovered the liver mass and improved the nuclear grade parameters. Seven days post 70% PHx, the liver weight indexes were 2.9 and 2.0%, the hepatocyte proliferation indexes were 19.8 and 0.6%, and the percentages of binucleated hepatocytes were 6.7 and 4.0%, in the InlB321/15-treated and control animals, respectively. Obtained data demonstrated that InlB321/15 improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

Amyloid-ß peptides slightly affect lifespan or antimicrobial peptide gene expression in Drosophila melanogaster.

BACKGROUND: Beta-amyloid peptide (Aß) is the key protein in the pathogenesis of Alzheimer's disease, the most common age-related neurodegenerative disorder in humans. Aß peptide induced pathological phenotypes in different model organisms include neurodegeneration and lifespan decrease. However, recent experimental evidence suggests that Aß may utilize oligomerization and fibrillization to function as an antimicrobial peptide (AMP), and protect the host from infections. We used the power of Drosophila model to study mechanisms underlying a dual role for Aß peptides. RESULTS: We investigated the effects of Drosophila treatment with three Aß42 peptide isoforms, which differ in their ability to form oligomers and aggregates on the lifespan, locomotor activity and AMP genes expression. Aß42 slightly decreased female's median lifespan (by 4.5%), but the effect was not related to the toxicity of peptide isoform. The lifespan and relative levels of AMP gene expression in male flies as well as locomotor activity in both sexes were largely unaffected by Aß42 peptide treatment. Regardless of the effects on lifespan, Aß42 peptide treatment induced decrease in AMP genes expression in females, but the effects were not robust. CONCLUSIONS: The results demonstrate that chronic treatment with Aß42 peptides does not drastically affect fly aging or immunity.

Relative Quantitation of Beta-Amyloid Peptide Isomers with Simultaneous Isomerization of Multiple Aspartic Acid Residues by Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry can be used for rapid quantitation of peptides with various post-translational modifications (PTM), even if they do not shift the mass of the native peptide. Previously, it was shown that MALDI-TOF MS can be used for quantitation of isoD7 beta-amyloid 1-42 peptide. On the basis of the differences in the collision-induced dissociation fragmentation pattern of native Aß, isoD7 Aß, isoD23 Aß, and isoD7_23 peptide (a di-isomerized peptide with both isomerization of D7 and D23 residues), we developed a MALDI-TOF-based method for simultaneous quantitation of all of these isoforms. Using multivariate regression for analysis of fragment MS data, the method allows the determination of the molar fractions of all of these isoforms with up to 16% error for mixtures with 2 pmol total amount of the beta-amyloid peptide.

Discrimination between G/C Binding Sites by Olivomycin A Is Determined by Kinetics of the Drug-DNA Interaction.

Olivomycin A (OA) exerts its cytotoxic potency due to binding to the minor groove of the G/C-rich DNA and interfering with replication and transcription. Screening of the complete set of tetranucleotide G/C sites by electrophoretic mobility gel shift assay (EMSA) revealed that the sites containing central GC or GG dinucleotides were able to bind OA, whereas the sites with the central CG dinucleotide were not. However, studies of equilibrium OA binding in solution by fluorescence, circular dichroism and isothermal titration calorimetry failed to confirm the sequence preference of OA, indicating instead a similar type of complex and comparable affinity of OA to all G/C binding sites. This discrepancy was resolved by kinetics analysis of the drug-DNA interaction: the dissociation rate significantly differed between SGCS, SGGS and SCGS sites (S stands for G or C), thereby explaining the disintegration of the complexes during EMSA. The functional relevance of the revealed differential kinetics of OA-DNA interaction was demonstrated in an in vitro transcription assay. These findings emphasize the crucial role of kinetics in the mechanism of OA action and provide an important approach to the screening of new drug candidates.

The English (H6R) Mutation of the Alzheimer's Disease Amyloid-ß Peptide Modulates Its Zinc-Induced Aggregation.

The coordination of zinc ions by histidine residues of amyloid-beta peptide (Aß) plays a critical role in the zinc-induced Aß aggregation implicated in Alzheimer's disease (AD) pathogenesis. The histidine to arginine substitution at position 6 of the Aß sequence (H6R, English mutation) leads to an early onset of AD. Herein, we studied the effects of zinc ions on the aggregation of the Aß42 peptide and its isoform carrying the H6R mutation (H6R-Aß42) by circular dichroism spectroscopy, dynamic light scattering, turbidimetric and sedimentation methods, and bis-ANS and thioflavin T fluorescence assays. Zinc ions triggered the occurrence of amorphous aggregates for both Aß42 and H6R-Aß42 peptides but with distinct optical properties. The structural difference of the formed Aß42 and H6R-Aß42 zinc-induced amorphous aggregates was also supported by the results of the bis-ANS assay. Moreover, while the Aß42 peptide demonstrated an increase in the random coil and ß-sheet content upon complexing with zinc ions, the H6R-Aß42 peptide showed no appreciable structural changes under the same conditions. These observations were ascribed to the impact of H6R mutation on a mode of zinc/peptide binding. The presented findings further advance the understanding of the pathological role of the H6R mutation and the role of H6 residue in the zinc-induced Aß aggregation.

Aberrant interactions between amyloid-beta and alpha5 laminins as possible driver of neuronal disfunction in Alzheimer's disease.

It has been widely accepted that laminins are involved in pathogenesis of Alzheimer's disease (AD). Amyloid plaques in AD patients are associated with immunostaining using antibodies raised against laminin-111, and laminin-111 has been shown to prevent aggregation of amyloid peptides. Although numerous articles describe small peptides from laminin-111 that are capable to disaggregate amyloid buildups and reduce neurotoxicity in in vitro and in vivo models, there is no approved laminin-111-based therapeutic approaches for treatment of AD. Also, it has been shown that immunoreactivity to laminin-111 appears late in development of cerebral amyloidosis. Based on the published data, we hypothesize that aberrant interaction between amyloid-beta and α5-laminins such as laminin-511 prevents the necessary laminin signaling into neurons leading to neurodegeneration and contributing to the early development of AD. Laminin-511 is the key extracellular protein that protects neurons from anoikis, inhibits excitoxicity and provides signaling that stabilizes dendritic spines and synapses in the developed brain. Absence of the signaling from laminin-511 leads to behavioral defects in mice. Laminin-511 and hippocampal neurons are in direct contact and accumulation of amyloid-beta that has been shown to avidly bind laminin-511 may physically decouple the interaction between α5-laminins and the neuronal membrane receptors inhibiting the signaling. Under this hypothesis, protein domains and peptides from laminin α5 chain may have a therapeutic potential in treatment of AD and the appearance of laminin-111 in the amyloid plaques is simply a consequence of the disease.

Williams-Beuren syndrome-related methyltransferase WBSCR27: cofactor binding and cleavage.

Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.

Beta-amyloid induces apoptosis of neuronal cells by inhibition of the Arg/N-end rule pathway proteolytic activity.

Alzheimer's disease (AD) is accompanied by the dysfunction of intracellular protein homeostasis systems, in particular the ubiquitin-proteasome system (UPS). Beta-amyloid peptide (Aß), which is involved in the processes of neurodegeneration in AD, is a substrate of this system, however its effect on UPS activity is still poorly explored. Here we found that Aß peptides inhibited the proteolytic activity of the antiapoptotic Arg/N-end rule pathway that is a part of UPS. We identified arginyltransferase Ate1 as a specific component of the Arg/N-end rule pathway targeted by Aßs. Aß bearing the familial English H6R mutation, known to cause early-onset AD, had an even greater inhibitory effect on protein degradation through the Arg/N-end rule pathway than intact Aß. This effect was associated with a significant decrease in Ate1-1 and Ate1-3 catalytic activity. We also found that the loss of Ate1 in neuroblastoma Neuro-2a cells eliminated the apoptosis-inducing effects of Aß peptides. Together, our results show that the apoptotic effect of Aß peptides is linked to their impairment of Ate1 catalytic activity leading to suppression of the Arg/N-end rule pathway proteolytic activity and ultimately cell death.

Interplay of histidine residues of the Alzheimer's disease Aß peptide governs its Zn-induced oligomerization.

Conformational changes of Aß peptide result in its transformation from native monomeric state to the toxic soluble dimers, oligomers and insoluble aggregates that are hallmarks of Alzheimer's disease (AD). Interactions of zinc ions with Aß are mediated by the N-terminal Aß(1-16) domain and appear to play a key role in AD progression. There is a range of results indicating that these interactions trigger the Aß plaque formation. We have determined structure and functional characteristics of the metal binding domains derived from several Aß variants and found that their zinc-induced oligomerization is governed by conformational changes in the minimal zinc binding site 6HDSGYEVHH14. The residue H6 and segment 11EVHH14, which are part of this site are crucial for formation of the two zinc-mediated interaction interfaces in Aß. These structural determinants can be considered as promising targets for rational design of the AD-modifying drugs aimed at blocking pathological Aß aggregation.

Zinc-induced heterodimer formation between metal-binding domains of intact and naturally modified amyloid-beta species: implication to amyloid seeding in Alzheimer's disease?

Zinc ions and modified amyloid-beta peptides (Aß) play a critical role in the pathological aggregation of endogenous Aß in Alzheimer's disease (AD). Zinc-induced Aß oligomerization is mediated by the metal-binding domain (MBD) which includes N-terminal residues 1-16 (Aß1-16). Earlier, it has been shown that Aß1-16 as well as some of its naturally occurring variants undergoes zinc-induced homodimerization via the interface in which zinc ion is coordinated by Glu11 and His14 of the interacting subunits. In this study using surface plasmon resonance technique, we have found that in the presence of zinc ions Aß1-16 forms heterodimers with MBDs of two Aß species linked to AD: Aß containing isoAsp7 (isoAß) and Aß containing phosphorylated Ser8 (pS8-Aß). The heterodimers appear to possess the same interface as the homodimers. Simulation of 200 ns molecular dynamic trajectories in two constructed models of dimers ([Aß1-16/Zn/Aß1-16] and [isoAß1-16/Zn/Aß1-16]), has shown that conformational flexibility of the N-terminal fragments of the dimer subunits is controlled by the structure of corresponding sites 6-8. The data suggest that isoAß and pS8-Aß can be involved in the AD pathogenesis by means of their zinc-dependent interactions with endogenous Aß resulting in the formation of heterodimeric seeds for amyloid aggregation.