Monomeric Fragment Research Articles

Single-Domain Antibodies as Therapeutic and Imaging Agents for the Treatment of CNS Diseases.

Antibodies have become one of the most successful therapeutics for a number of oncology and inflammatory diseases. So far, central nervous system (CNS) indications have missed out on the antibody revolution, while they remain ‘hidden’ behind several hard to breach barriers. Among the various antibody modalities, single-domain antibodies (sdAbs) may hold the ‘key’ to unlocking the access of antibody therapies to CNS diseases. The unique structural features of sdAbs make them the smallest monomeric antibody fragments suitable for molecular targeting. These features are of particular importance when developing antibodies as modular building blocks for engineering CNS-targeting therapeutics and imaging agents. In this review, we first introduce the characteristic properties of sdAbs compared to traditional antibodies. We then present recent advances in the development of sdAbs as potential therapeutics across brain barriers, including their use for the delivery of biologics across the blood–brain and blood–cerebrospinal fluid (CSF) barriers, treatment of neurodegenerative diseases and molecular imaging of brain targets.

Spatial and temporal organization of RecA in the Escherichia coli DNA-damage response

The RecA protein orchestrates the cellular response to DNA damage via its multiple roles in the bacterial SOS response. Lack of tools that provide unambiguous access to the various RecA states within the cell have prevented understanding of the spatial and temporal changes in RecA structure/function that underlie control of the damage response. Here, we develop a monomeric C-terminal fragment of the λ repressor as a novel fluorescent probe that specifically interacts with RecA filaments on single-stranded DNA (RecA*). Single-molecule imaging techniques in live cells demonstrate that RecA is largely sequestered in storage structures during normal metabolism. Upon DNA damage, the storage structures dissolve and the cytosolic pool of RecA rapidly nucleates to form early SOS-signaling complexes, maturing into DNA-bound RecA bundles at later time points. Both before and after SOS induction, RecA* largely appears at locations distal from replisomes. Upon completion of repair, RecA storage structures reform.

On the synthesis of polyphenylene sulfide and copolymers based on it by the reaction of nonequilibrium polycondensation

Polyphenylene sulfide is a semicrystalline engineering thermoplastic, in the world market of superconstructive materials is presented in two modifications: the first is a cross-linked material, the second is a linear polymer with a highly ordered supramolecular structure.Polyphenylene sulfides, synthesized according to the classical procedure in N-methyl-2-pyrrolidone, are characterized by low molecular weight characteristics due to the occurrence of side reactions. The main yield of the target product does not exceed 45%, even with an increase in the synthesis time of up to 120 hours.Polymers with higher viscosity characteristics can be obtained by heat treatment of synthesized polyphenylene sulfide at 240-260 °С. Copolymers with new monomeric fragments, such as sulfonic, ketone, amide, imide, etc., have been synthesized, which lead to amorphous polymers. The copolymers obtained are well soluble at room temperature and have a higher yield and a molecular weight. The introduction of cyclic groups into the macromolecule of polyphenylene sulfide results in an increase in heat resistance (500 °C) and solubility. Over the past 10-15 years, special attention has been paid to the synthesis of macrocyclic oligomers, which are used subsequently for the preparation of high molecular copolymers by a ring-opening polymerization reaction. This technique allows to obtain polymers in a short time and without the formation of by-products.During the synthesis of copolymers, extensive knowledge of the effect of the chemical structure on the performance properties of materials was obtained, and new methods were developed that reduced the possibility of percolation reactions. Thus, the synthesis of new polyheteroarylenes becomes topical, the review reflects the results of research over the past few years on the development on their basis of materials with unique valuable properties.

Oligomers as Triggers for Responsive Liquid Crystals.

We report an investigation of the influence of aqueous solutions of amphiphilic oligomers on the ordering of micrometer-thick films of thermotropic liquid crystals (LCs), thus addressing the gap in knowledge arising from previous studies of the interactions of monomeric and polymeric amphiphiles with LCs. Specifically, we synthesized amphiphilic oligomers (with decyl hydrophobic and pentaethylene glycol hydrophilic domains) in monomer, dimer, and trimer forms, and incubated aqueous solutions of the oligomers against nematic films of 4'-pentyl-4-biphenylcarbonitrile (5CB). All amphiphilic oligomers caused sequential surface-driven orientational (planar to homeotropic) and then bulk phase transitions (nematic to isotropic) with dynamics depending strongly on the degree of oligomerization. The dynamics of the orientational transitions accelerated from monomer to trimer, consistent with the effects of an increase in adsorption free energy. The mechanism underlying the orientational transition, however, involved a decrease in anchoring energy and not change in the easy axis of the LC. In contrast, the rate of the phase transition induced by absorption of oligomers into the LC decreased from monomer to trimer, suggesting that constraints on configurational degrees of freedom influence the absorption free energies of the oligomers. Interestingly, the oligomer-induced transition from the nematic to isotropic phase of 5CB was observed to nucleate at the aqueous-5CB interface, consistent with surface-induced disorder underlying the above-reported decrease in anchoring energy caused by the oligomers. Finally, we provided proof-of-concept experiments of the triggering of LCs using a trimeric amphiphile that is photocleaved by UV illumination into monomeric fragments. Overall, our results provide insight into the rational design of oligomers that can be used as triggers to create responsive LCs.

Recombinant IgG1 Fc hexamers block cytotoxicity and pathological changes in experimental in vitro and rat models of neuromyelitis optica

Intravenous human immunoglobulin G (IVIG) may have therapeutic benefit in neuromyelitis optica spectrum disorders (herein called NMO), in part because of the anti-inflammatory properties of the IgG Fc region. Here, we evaluated recombinant Fc hexamers consisting of the IgM μ-tailpiece fused with the Fc region of human IgG1. In vitro, the Fc hexamers prevented cytotoxicity in aquaporin-4 (AQP4) expressing cells and in rat spinal cord slice cultures exposed to NMO anti-AQP4 autoantibody (AQP4-IgG) and complement, with >500-fold greater potency than IVIG or monomeric Fc fragments. Fc hexamers at low concentration also prevented antibody-dependent cellular cytotoxicity produced by AQP4-IgG and natural killer cells. Serum from rats administered a single intravenous dose of Fc hexamers at 50 mg/kg taken at 8 h did not produce complement-dependent cytotoxicity when added to AQP4-IgG-treated AQP4-expressing cell cultures. In an experimental rat model of NMO produced by intracerebral injection of AQP4-IgG, Fc hexamers at 50 mg/kg administered before and at 12 h after AQP4-IgG fully prevented astrocyte injury, complement activation, inflammation and demyelination. These results support the potential therapeutic utility of recombinant IgG1 Fc hexamers in AQP4-IgG seropositive NMO.

Crystal Structure of the Polymer Copper(II) Complex with 1-Phenyl-3-Methyl-4-Formyl-5-Hydroxypyrazole Hetarylhydrazone

A new coordination polymer of Cu(II) based on 1-phenyl-3-methyl-4-formyl-5-hydroxypyrazole 4,6-dimethylpyrimidylhydrazone is synthesized. The polymer is studied by X-ray diffraction analysis (CIF file CCDC no. 1565772). It is shown that the polymer is formed due to the coordination of the nitrogen atom of the pyrazolone fragment to the copper(II) ion of the adjacent monomeric fragment.

Molecular-Level Processing of Si-(B)-C Materials with Tailored Nano/Microstructures.

The design of Si-(B)-C materials is investigated, with detailed insight into the precursor chemistry and processing, the precursor-to-ceramic transformation, and the ceramic microstructural evolution at high temperatures. In the early stage of the process, the reaction between allylhydridopolycarbosilane (AHPCS) and borane dimethyl sulfide is achieved. This is investigated in detail through solid-state NMR and FTIR spectroscopy and elemental analyses for Si/B ratios ranging from 200 to 30. Boron-based bridges linking AHPCS monomeric fragments act as crosslinking units, extending the processability range of AHPCS and suppressing the distillation of oligomeric fragments during the low-temperature pyrolysis regime. Polymers with low boron contents display appropriate requirements for facile processing in solution, leading to the design of monoliths with hierarchical porosity, significant pore volume, and high specific surface area after pyrolysis. Polymers with high boron contents are more appropriate for the preparation of dense ceramics through direct solid shaping and pyrolysis. We provide a comprehensive study of the thermal decomposition mechanisms, and a subsequent detailed study of the high-temperature behavior of the ceramics produced at 1000 °C. The nanostructure and microstructure of the final SiC-based ceramics are intimately linked to the boron content of the polymers. B4 C/C/SiC nanocomposites can be obtained from the polymer with the highest boron content.

A Bimolecular Fluorescence Complementation Tool for Identification of Protein-Protein Interactions in Candida albicans.

Investigation of protein-protein interactions (PPI) in Candida albicans is essential for understanding the regulation of the signal transduction network that triggers its pathogenic lifestyle. Unique features of C. albicans, such as its alternative codon usage and incomplete meiosis, have enforced the optimization of standard genetic methods as well as development of novel approaches. Since the existing methods for detection of PPI are limited for direct visualization of the interacting complex in vivo, we have established a bimolecular fluorescence complementation (BiFC) assay in C. albicans, a powerful technique for studying PPI. We have developed an optimized set of plasmids that allows for N- and C-terminal tagging of proteins with split yeast-enhanced monomeric Venus fragments, so that all eight combinations of fusion orientations can be analyzed. With the use of our BiFC assay we demonstrate three interaction complexes in vivo, which were also confirmed by two-hybrid analysis. Our Candida-optimized BiFC assay represents a useful molecular tool for PPI studies and shows great promise in expanding our knowledge of molecular mechanisms of protein functions.

NOVEL METHOD FOR OLIGOMERIC Aβ DETECTION REVEALS INTRACELLULAR ACCUMULATION OF Aβ UPON LOW-DOSE TREATMENT WITH A GAMMA-SECRETASE INHIBITOR

As the focus for Aβ-induced Alzheimer's disease (AD) pathology has shifted towards the oligomeric forms, there is a need for specific and sensitive detection of oligomeric Aβ (oAβ). Additionally, clinical trials using γ-secretase inhibitors have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors (GSI) may increase Aβ production. We hypothesised that low-dose treatment with the GSI DAPT could cause intracellular accumulation of oAβ and developed a novel method to monitor this process. A novel method for specific detection of oligomeric Aβ (oAβ) was developed using Duolink techology, where an Aβ N-terminal specific antibody was conjugated to Duolink PLUS and MINUS probes, respectively. The method was optimized using oAβ-fed SH-SY5Y cells and was thereafter used to study intracellular accumulation of oAβ in iPSC-derived human cortical neurons exposed to low doses of DAPT. Secreted levels of Aβ were also investigated upon DAPT treatment using mass spectrometry and immunocytochemical methods. Duolink probe-labelled 82E1 antibody successfully detected oAβ in cultured SH-SY5Y cells. The method was sensitive (lower limit of detection <125 nM) (Figure 1) and specific for the oligomeric form (no cross-reactivity with monomeric N-terminal Aβ fragments). Furthermore, low-dose treatment (2 and 20 nM) with DAPT increased the secretion of several Aβ peptides from iPSC derived human cortical neurons. This was accompanied by intracellular accumulation oAβ (Figure 2).

CpFe(CO)2TePh as a ligand for organometallic dihalides Fe(CO)4I2, (p-Cymene)RuI2, and Me5C5RhI2: Synthesis and molecular structures

CpFe(CO)2TePh (I) can substitute one carbonyl group in Fe(CO)4I2 providing Fe(CO)3I2(μ-TePh)Fe(CO)2Cp (II) or play role of ligand to monomeric fragments [(p-Cymene)RuI2] and [Сp*RhI2] (p-Cymene = (η6-1-isopropyl-4-methylbenzene); Сp* = η5-pentamethylcyclopentadienyl) provides the complexes (p-Cymene)RuI2(μ-TePh)Fe(CO)2Cp (III) and Cp*RhCl2(μ-TePh)Fe(CO)2Cp (IV), respectively. The single-crystal X-ray diffraction of complexes II–IV (CIF files CCDC nos. 1038124 (II), 1038127 (III), 1038125 (IV)) revealed the shortening of М–Те bonds and the presence of intramolecular I…Te contacts.

IgG Fc Fragment as a Scaffold for Development of Targeted Therapeutics.

Monoclonal antibodies and Fc fusion proteins have been successful therapeutics in the field of cancer and immune disease. As their pharmacological activity is dependent on the Fc fragment governing their effector functions and long in vivo half-life, the extensive engineering of the Fc for altered binding of its natural ligands that enable these properties has delivered molecules optimized for their therapeutic effect. Recently, the IgG1 Fc region itself and its subunits monomeric Fc fragment, CH2 and monomeric CH3 domain, have been engineered into scaffolds with favorable biophysical properties and a high potential for de novo antigen recognition. A dimeric Fc fragment with an antigen binding site has proven suitable for evaluation in animal models and has already entered human trials. Such modified constant domains can easily be incorporated into an antibody or fused with antibody domains of a second specificity. The small size of the Fc and its subunits that enhances their tissue penetration, as well as the unique topology of their binding sites that allows novel modes of contact with the antigen, are attractive features that prompt their development into promising candidate therapeutics.

Calcium-dependent binding of Myc to calmodulin.

The bHLH-LZ (basic region/helix-loop-helix/leucine zipper) oncoprotein Myc and the bHLH-LZ protein Max form a binary transcription factor complex controlling fundamental cellular processes. Deregulated Myc expression leads to neoplastic transformation and is a hallmark of most human cancers. The dynamics of Myc transcription factor activity are post-translationally coordinated by defined protein-protein interactions. Here, we present evidence for a second messenger controlled physical interaction between the Ca2+ sensor calmodulin (CaM) and all Myc variants (v-Myc, c-Myc, N-Myc, and L-Myc). The predominantly cytoplasmic Myc:CaM interaction is Ca2+-dependent, and the binding site maps to the conserved bHLH domain of Myc. Ca2+-loaded CaM binds the monomeric and intrinsically disordered Myc protein with high affinity, whereas Myc:Max heterodimers show less, and Max homodimers no affinity for CaM. NMR spectroscopic analyses using alternating mixtures of 15N-labeled and unlabeled preparations of CaM and a monomeric Myc fragment containing the bHLH-LZ domain corroborate the biochemical results on the Myc:CaM interaction and confirm the interaction site mapping. In electrophoretic mobility shift assays, addition of CaM does not affect high-affinity DNA-binding of Myc:Max heterodimers. However, cell-based reporter analyses and cell transformation assays suggest that increasing CaM levels enhance Myc transcriptional and oncogenic activities. Our results point to a possible involvement of Ca2+ sensing CaM in the fine-tuning of Myc function.

LCu(μ-X)2CuL compounds: An induced cuprophilic interaction

A trio of dimeric copper(I) complexes of the formula [(Ph3P)Cu(μ-X)2Cu(PPh3)] (X=OC6F5, 1, OC4F9, 2, OCPh(CF3)2, 3) were prepared and characterized by X-ray crystallography, elemental analysis, and NMR spectroscopy (1H, 13C, 19F, 31P), as was the monomeric [(cy3P)Cu(OC4F9)] (cy=cyclohexyl, 4). Solution conductivity studies demonstrate that all three new dinuclear compounds, as well as the known [(Ph3P)Cu(μ-OC4H9)2Cu(PPh3)], 2-H, are neutral species in solution and do not rearrange into ion pairs. The fold angle (β) varies among these four dimers and the Cu(I)⋯Cu(I) distance in the structure of 2, 2.8315(5)Å, is cuprophilic. The Cu(I)⋯Cu(I) distances for 1, 2-H, and 3 are 3.0533(5), 2.890(2), and 3.0169(6)Å respectively. Density functional theory (DFT) calculations were performed on 1, 2, 3, and 2-H, as well as the hypothetical 1-H, and several related models. Five PMe3 models, 1(Me), 1-H(Me), 2(Me), 2-H(Me), and 3(Me) were also studied as well as five monomers [(Me3P)CuX] 1-mon, 1-H(mon), 2-mon, 2-H(mon), and 3-mon to understand the electronic reasons for folding in this group of compounds. A Natural Energy Decomposition Analysis (NEDA) indicates that electrostatic stabilizations are the dominant factor governing the strength of interaction between monomeric fragments in 1(Me)–3(Me). NBO analysis reveals that 1(Me) and 1-H(Me) do not display any cuprophilic interactions. The folding angle observed in 2(Me), 2-H(Me), and 3(Me), which is correlated with an increased delocalization from the oxygen 2pz lone pairs, brings the metal centers into sufficient propinquity to have weak Cu⋯Cu orbital interactions. Weak luminescence behavior at room-temperature is also consistent with these assignments.

Iridium Complexes of the Conformationally Rigid IBioxMe4 Ligand: Hydride Complexes and Dehydrogenation of Cyclooctene

A method for accessing the formally 14 VE iridium(III) hydride fragment {Ir(IBioxMe4)2(H)2}+ (2), containing the conformationally rigid NHC ligand IBioxMe4, is reported. Hydrogenation of trans-[Ir(IBioxMe4)2(COE)Cl] (1) in the presence of excess Na[BArF4] leads to the formation of dimeric [{Ir(IBioxMe4)2(H)2}2Cl][BArF4] (3), which is structurally fluxional in solution and acts as a reservoir of monomeric 2 in the presence of excess halogen ion abstractor. Stable dihydride complexes trans-[Ir(IBioxMe4)2(2,2′-bipyridine)(H)2][BArF4] (4) and [Ir(IBioxMe4)3(H)2][BArF4] (5) were subsequently isolated through in situ trapping of 2 using 2,2′-bipyridine and IBioxMe4, respectively, and fully characterized. Using mixtures of 3 and Na[BArF4] as a latent source of 2, the reactive monomeric fragment’s reactivity was explored with excess ethylene and cyclooctene, and trans-[Ir(IBioxMe4)2(C2H4)2][BArF4] (6) and cis-[Ir(IBioxMe4)2(COD)][BArF4] (7) were isolated, respectively, through sacrificial hydrogenation of the alkenes. Complex 6 is notable for the adoption of a very unusual orthogonal arrangement of the trans-ethylene ligands in the solid state, which has been analyzed computationally using energy and charge decomposition (EDA-NOCV). The formation of 7 via transfer dehydrogenation of COE highlights the ability to partner IBioxMe4 with reactive metal centers capable of C–H bond activation, without intramolecular activation. Reaction of 7 with CO slowly formed trans-[Ir(IBioxMe4)2(CO)2][BArF4] (8), but the equivalent reaction with bis-ethylene 6 was an order of magnitude faster, quantifying the strong coordination of COD in 7.

Characterization and optimization of a novel vaccine for protection against Lyme borreliosis

Lyme borreliosis (LB) is the most common vector-borne disease in the northern hemisphere and there is no vaccine available for disease prevention. The majority of LB cases in Europe are caused by four different Borrelia species expressing six different OspA serotypes, whereas in the US only one of these serotypes is present. Immunization with the outer surface protein A (OspA) can prevent infection and the C-terminal part of OspA is sufficient for protection against infection transmitted by Ixodes ticks. Here we show that the order of the stabilized monomeric OspA fragments making up the heterodimers in our LB vaccine does not influence the induced immunogenicity and protection. Using bioinformatics analysis (surface electrostatics), we have designed an improved version of an LB vaccine which has an increased immunogenicity for OspA serotype 3 and an optimized expression and purification profile. The OspA heterodimers were highly purified with low amounts of endotoxin, host cell proteins and host cell DNA. All three proteins were at least 85% triacylated which ensured high immunogenicity. The LB vaccine presented here was designed, produced and characterized to a level which warrants further development as a second generation human LB vaccine.

A monomeric fab fragment capable of inducing CD3 conformational change increases T cell receptor reactivity to poorly immunogenic antigens (TUM2P.1020)

Abstract TCR interactions with agonist pMHCs induce a conformational change in the tails of the associated CD3 complex (CD3Δc) that contributes to efficient TCR/CD3 triggering and productive T cell responses. In contrast, TCR engagement by antagonistic pMHCs does not induce either CD3Δc or full TCR/CD3 signaling in mature T cells. Here, we have used a monovalent Fab fragment (Mono-Fab) specific for the murine CD3 complex to provide induction of CD3Δc to mature T cells engaging pMHC ligands. This Mono-Fab does not interfere with TCR capacity to bind pMHC and fails to elicit signaling in the absence of TCR/MHC interactions. Using OT-I Tg T cells and a collection of altered peptide ligands derived of the agonist peptide OVA, we show that addition of CD3Δc to OT-I T cells using Mono-Fab enables T cell responses to antagonist ligands that fail to induce CD3Δc on their own. Moreover, using a mouse model of metastatic melanoma, we show that Mono-Fab promotes in vivo T cell dependent anti-melanoma responses that are antigen-specific. This Mono-Fab can also be combined with established therapies targeting signals 2 and 3 involved in T cell activation for a synergistic effect. Our data suggests that in-trans CD3Δc provision decreases the activation threshold of T cells. We propose that targeting the CD3 complex for the induction of CD3Δc could be exploited to achieve efficient TCR/CD3 triggering by poorly immunogenic antigens in the context of vaccination against cancer and infectious diseases.

Conversion of technical lignins to functional materials with retained polymeric properties

A significant amount of technical lignins is produced in the pulp and paper industries. However, most technical lignins are burned for thermal recycling and a few percent are used as materials, such as lignosulfonate as a dispersant. Native lignin has a highly complex structure and is susceptible to structural variations depending on the pulping process, thus hindering the effective utilization of lignins. The procedures used to convert lignins into functional materials include depolymerization to monomeric fragments followed by re-building to functional materials, and chemical modifications to generate functional polymers with retained polymeric properties. In this paper, the latter is disserted. The characteristics of technical lignins, which include kraft lignin, lignosulfonate, and organosolv lignin, and their conversion to functional materials such as polyesters, polyethers, polyurethanes, etc. and the applications of lignin-based materials in some fields are discussed.

Peptide Immunotherapy; Short but Long Lasting?

Allergen immunotherapy (AIT) with whole allergens or allergen extracts has been in use for more than one hundred years. It is clinically efficacious and disease-modifying. However, AIT is also associated with a significant adverse events profile, including the potential to cause severe, systemic allergic reactions. One alternative to traditional whole-antigen AIT is peptide immunotherapy, which uses small synthetic peptide immunoregulatory epitopes (SPIRE) representing T cell epitopes from the allergen of interest. Peptide immunotherapy is being developed for the treatment of allergic and autoimmune diseases where pathogenesis is T cell-dependent. Short, soluble, monomeric peptide fragments avoid the problem of IgE-mediated adverse events (since the peptides will not cross-link allergen-specific IgE on the surface of effector cells such as mast cells and basophils). However, such peptides retain the capability to induce T cell tolerance and immunoregulation. In early clinical trials, efficacy has been demonstrated months to years after the cessation of a short course of treatment, supporting the conclusion that this approach is disease-modifying, changing the natural history of the disease. The improved safety profile of short peptides allows for larger molar-equivalent doses to be administered in shorter time frames than AIT; treatment can be completed in as few as four intradermal injections, while efficacy persists for two years or more.

Dimerization Is Not a Determining Factor for Functional High Affinity Human Plasminogen Binding by the Group A Streptococcal Virulence Factor PAM and Is Mediated by Specific Residues within the PAM a1a2 Domain

A emm53 subclass of Group A Streptococcus pyogenes (GAS) interacts tightly with human plasma plasminogen (hPg) and plasmin (hPm) via the kringle 2 (K2hPg) domain of hPg/hPm and the N-terminal a1a2 regions of a GAS coiled-coil M-like protein (PAM). Previous studies have shown that a monomeric PAM fragment, VEK30 (residues 97-125 + Tyr), interacted specifically with isolated K2hPg. However, the binding strength of VEK30 (KD = 56 nm) was ∼60-fold weaker than that of full-length dimeric PAM (KD = 1 nm). To assess whether this attenuated binding was due to the inability of VEK30 to dimerize, we defined the minimal length of PAM required to dimerize using a series of peptides with additional PAM residues placed at the NH2 and COOH termini of VEK30. VEK64 (PAM residues 83-145 + Tyr) was found to be the smallest peptide that adopted an α-helical dimer, and was bound to K2hPg with nearly the same affinity as PAM (KD = 1-2 nm). However, addition of two PAM residues (Arg(126)-His(127)) to the COOH terminus of VEK30 (VEK32) maintained a monomeric peptidic structure, but exhibited similar K2hPg binding affinity as full-length dimeric PAM. We identified five residues in a1a2 (Arg(113), His(114), Glu(116), Arg(126), His(127)), mutation of which reduced PAM binding affinity for K2hPg by ∼ 1000-fold. Replacement of these critical residues by Ala in the GAS genome resulted in reduced virulence, similar to the effects of inactivating the PAM gene entirely. We conclude that rather than dimerization of PAM, the five key residues in the binding domain of PAM are essential to mediate the high affinity interaction with hPg, leading to increased GAS virulence.

Secreted amyloid β-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid β-protein (Aβ) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of Aβ-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of Aβ on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aβ could be therapeutically advantageous, it is important to understand the full range of soluble Aβ derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aβ species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aβ40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aβ-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aβ. These N-terminally extended Aβ-containing monomeric fragments are distinct from soluble Aβ oligomers formed from Aβ1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing β-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aβ-containing species can arise from APP processing events N-terminal to the classical β-secretase cleavage site.