Rational design of a conformation-specific antibody for the quantification of Aβ oligomers.
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ABSTRACT:
SUBMITTER: Aprile FA
PROVIDER: S-EPMC7306997 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced exon skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and exon skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable exon skipping. We report substantially different levels of exon skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted skipping of human dystrophin exons for therapeutic outcomes.
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Project description:Aggregation of amyloid-β (Aβ) that leads to the formation of plaques in Alzheimer's disease (AD) occurs through the stepwise formation of oligomers and fibrils. An earlier onset of aggregation is obtained upon intracerebral injection of Aβ-containing brain homogenate into human APP transgenic mice that follows a prion-like seeding mechanism. Immunoprecipitation of these brain extracts with anti-Aβ oligomer antibodies or passive immunization of the recipient animals abrogated the observed seeding activity, although induced Aβ deposition was still evident. Here, we establish that, together with Aβ monomers, Aβ oligomers trigger the initial phase of Aβ seeding and that the depletion of oligomeric Aβ delays the aggregation process, leading to a transient reduction of seed-induced Aβ deposits. This work extends the current knowledge about the role of Aβ oligomers beyond its cytotoxic nature by pointing to a role in the initiation of Aβ aggregation in vivo. We conclude that Aβ oligomers are important for the early initiation phase of the seeding process.
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Project description:BackgroundThe form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.ResultsMOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunoreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.ConclusionsBoth intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.
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Project description:Aβ is the toxic amyloid polypeptide responsible for Alzheimer's disease (AD). Prevention and elimination of the Aβ misfolded aggregates are the promising therapeutic strategies for the AD treatments. Gammabody, the Aβ-Specific Single-domain (VH) antibody, recognizes Aβ aggregates with high affinity and specificity and reduces their toxicities. Employing the molecular dynamics simulations, we studied diverse gammabody-Aβ recognition complexes to get insights into their structural and dynamic properties and gammabody-Aβ recognitions. Among many heterogeneous binding modes, we focused on two gammabody-Aβ recognition scenarios: recognition through Aβ β-sheet backbone and on sidechain surface. We found that the gammabody primarily uses the complementarity-determining region 3 (CDR3) loop with the grafted Aβ sequence to interact with the Aβ fibril, while CDR1/CDR2 loops have very little contact. The gammabody-Aβ complexes with backbone binding mode are more stable, explaining the gammabody's specificity towards the C-terminal Aβ sequence.
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Project description:As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.
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Project description:BackgroundPartially acetylated chito-oligosaccharides (paCOS) have a variety of potential applications in different fields, but to harness their benefits, pure paCOS or well-defined paCOS mixtures are essential. For example, if one could produce fully acetylated (A4) and fully deacetylated (D4) tetramers in abundance, all possible variants of tetrameric paCOS could be generated reliably from them. A promising approach for generating defined paCOS is by enzymatic depolymerization of chitosan polymers using chitosanases, since these enzymes' subsite specificities directly influence the composition of the paCOS produced; however, enzymatic production of e.g. D4 is challenging because the substrate is generally hydrolyzed further by most chitosanases. To overcome this, chitosanases could potentially be engineered so that upon hydrolyzing chitosan, they are unable to hydrolyze certain substrates, leaving well-defined oligomers intact in the hydrolysate.ResultsFor this purpose, we performed rational protein engineering on the extensively studied GH 8 chitosanase CSN from Bacillus sp. MN. By specifically targeting residues with a predicted function in substrate binding, we created new muteins incapable of efficiently hydrolyzing the fully deacetylated tetramer D4, and we were able to demonstrate efficient large-scale production of D4 with an altered version of CSN. Furthermore, we were able to uncover differences in the substrate positioning and subsite specificities of the muteins, which result in altered paCOS mixtures produced from partially acetylated chitosan polymers, with possibly altered bioactivities.ConclusionThe value of protein engineering as a tool for the more efficient production of pure oligomers and potentially bioactive paCOS mixtures was demonstrated by the results and the suitability of specific muteins for the large-scale production of strictly defined, pure paCOS in a batch process was shown using the example of D4.
| S-EPMC6419424 | biostudies-literature
Project description:The assembly of the β-amyloid peptide (Aβ) to form oligomers and fibrils is closely associated with the pathogenesis and progression of Alzheimer's disease. Aβ is a shape-shifting peptide capable of adopting many conformations and folds within the multitude of oligomers and fibrils the peptide forms. These properties have precluded detailed structural elucidation and biological characterization of homogeneous, well-defined Aβ oligomers. In this paper, we compare the structural, biophysical, and biological characteristics of two different covalently stabilized isomorphic trimers derived from the central and C-terminal regions Aβ. X-ray crystallography reveals the structures of the trimers and shows that each trimer forms a ball-shaped dodecamer. Solution-phase and cell-based studies demonstrate that the two trimers exhibit markedly different assembly and biological properties. One trimer forms small soluble oligomers that enter cells through endocytosis and activate capase-3/7-mediated apoptosis, while the other trimer forms large insoluble aggregates that accumulate on the outer plasma membrane and elicit cellular toxicity through an apoptosis-independent mechanism. The two trimers also exhibit different effects on the aggregation, toxicity, and cellular interaction of full-length Aβ, with one trimer showing a greater propensity to interact with Aβ than the other. The studies described in this paper indicate that the two trimers share structural, biophysical, and biological characteristics with oligomers of full-length Aβ. The varying structural, assembly, and biological characteristics of the two trimers provide a working model for how different Aβ trimers can assemble and lead to different biological effects, which may help shed light on the differences among Aβ oligomers.
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Project description:RNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Due to the extremely high proportion of ribosomal RNA (rRNA) in total RNA, sequencing libraries usually incorporate messenger RNA (mRNA) enrichment. Although polyadenylate (poly(A)) tail selection is widely used, many applications require alternate approaches such as rRNA depletion. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the length of target RNAs, has emerged as an easy, cost-effective alternative to commercial rRNA depletion kits. Here, we present a streamlined RNaseH-mediated rRNA depletion method that uses shorter antisense oligos that only sparsely tile the target RNA, in a digestion reaction of only 5 minutes. We wrote a Web tool, Oligo-ASST, that simplifies oligo design to favor target regions with optimal thermodynamic properties, and additionally allows users to design common oligo pools that can simultaneously target divergent RNAs in their regions of higher sequence similarity. We demonstrate the efficacy of these oligos by building rRNA-depleted sequencing libraries for Xenopus laevis as well as zebrafish, which expresses two distinct versions of the 28S, 18S, 5.8S, and 5S rRNAs during embryogenesis. These libraries efficiently deplete rRNA to <5% of total reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.
2020-06-28 | GSE152902 | GEO