The formation of neurofibrillary tangles and amyloid plaques accompanies the progression of Alzheimer's disease. Tangles are made of fibrillar aggregates formed by the microtubule-associated protein ...tau, whereas plaques comprise fibrillar forms of amyloid-beta (Aβ). Both form toxic oligomers during aggregation and are thought to interact synergistically to each promote the accumulation of the other. Recent in vitro studies have suggested that the monomeric nonphosphorylated full-length tau protein hinders the aggregation of Aβ1–40 peptide, but whether the same is true for the more aggregation-prone Aβ1–42 was not determined. We used in vitro and in vivo techniques to explore this question. We have monitored the aggregation kinetics of Aβ1–42 by thioflavine T fluorescence in the presence or the absence of different concentrations of nonphosphorylated tau. We observed that elongation of Aβ1–42 fibrils was inhibited by tau in a dose-dependent manner. Interestingly, the fibrils were structurally different in the presence of tau but did not incorporate tau. Surface plasmon resonance indicated that tau monomers bound to Aβ1–42 oligomers (but not monomers) and hindered their interaction with the anti-Aβ antibody 4G8, suggesting that tau binds to the hydrophobic central core of Aβ recognized by 4G8. Tau monomers also antagonized the toxic effects of Aβ oligomers in Caenorhabditis elegans. This suggests that nonphosphorylated tau might have a neuroprotective effect by binding Aβ1–42 oligomers formed during the aggregation and shielding their hydrophobic patches.
A wide variety of human diseases are associated with the formation of highly organized protein aggregates termed amyloid fibrils, whose growth (elongation) is due to the assembly of the basic ...molecular units (monomers) in a sequential polymerization process. Surface plasmon resonance (SPR) technology has been proposed as a powerful approach to study in detail the fibril elongation of some amyloidogenic peptides. In particular, the injection of monomers over immobilized fibrils allows to follow in real time, and on a very short time-scale, the kinetics of fibril growth. In the present study we confirmed and extended this application of SPR to Aβ₁–₄₂, hampered till now by the very pronounced aggregation propensity of this peptide, involved in Alzheimer disease. We took advantage of a new synthetic strategy (“depsi-peptide” technique) which allows to obtain reliable seed-free solutions (monomers) as well as fibrils of Aβ₁–₄₂. SPR data were consistent with a “dock-and-lock” mechanism underlying Aβ₁–₄₂ elongation process. The setup of an assay monitoring the elongation kinetics is very useful for investigating potential anti-amyloidogenic compounds. Moreover, the possibility to reliably immobilize both Aβ₁–₄₂ monomers and fibrils allows to measure the binding affinities of putative ligands for these different species. The approach applied here to Aβ₁–₄₂ might well be also applied to the study of other fibrillogenic peptides/proteins or to the study of polymerization reactions in general.
The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds to interfere with the biological processes exploited by the virus. ...Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.
We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint ...inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC
in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.
Traditional quantitative structure - property / activity relationships (QSPRs/QSARs) are based on representation of molecular structure by molecular graph or simplified molecular input-line entry ...system (SMILES). It is an attractive idea to develop predictive models for large molecules in general and for peptides in particular. However, the representation of these molecules by molecular graph or SMILES is problematic owing to large size of these molecules. A possible alternative of SMILES is the representation of peptides via sequence of abbreviations of amino acids.
Models for hemolysis and cytotoxicity of peptides are suggested. These models are based on representation of the peptides by sequences of amino acids. Correlation weights, which are calculated for each amino acid using the Monte Carlo method are basis for quantitative sequence - activity relationships (QSAR) for antimicrobial peptides. The correlation weights are the basis for optimal descriptors, which are correlated with experimental data for hemolysis and cytotoxicity. The basic hypothesis is that if optimal descriptors are correlated with endpoints of peptides for the training set, they should also correlate with the endpoints for validation set.
Checking up of correlations between the above-mentioned descriptors and antimicrobial activity of peptides (cytotoxicity or hemolysis) has shown that these models have good predictive potential.
Suggested approach can be used as a tool to develop predictive models of biological activity of peptides as a mathematical function of sequences of amino acids.
Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In ...this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.
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•Three novel gelsolin amyloidogenic substitutions cluster at the G4:G5 interface.•Mutations impair stability through strand distortion, charge and steric repulsion.•Mutations do not ...increase sensitivity to furin, the first step of the canonical pathway.•In the unproteolysed form these variants tend to aggregate and are proteotoxic.•The variants aggregate via an alternative, likely proteolysis-independent, mechanism.
Gelsolin comprises six homologous domains, named G1 to G6. Single point substitutions in this protein are responsible for AGel amyloidosis, a hereditary disease causing progressive corneal lattice dystrophy, cutis laxa, and polyneuropathy. Although several different amyloidogenic variants of gelsolin have been identified, only the most common mutants present in the G2 domain have been thoroughly characterized, leading to clarification of the functional mechanism. The molecular events underlying the pathological aggregation of 3 recently identified mutations, namely A551P, E553K and M517R, all localized at the interface between G4 and G5, are here explored for the first time. Structural studies point to destabilization of the interface between G4 and G5 due to three structural determinants: β-strand breaking, steric hindrance and/or charge repulsion, all implying impairment of interdomain contacts. Such rearrangements decrease the temperature and pressure stability of gelsolin but do not alter its susceptibility to furin cleavage, the first event in the canonical aggregation pathway. These variants also have a greater tendency to aggregate in the unproteolysed forms and exhibit higher proteotoxicity in a C. elegans-based assay. Our data suggest that aggregation of G4G5 variants follows an alternative, likely proteolysis-independent, pathway.
Abstract Frontotemporal lobar degeneration (FTLD) can be sporadic or familial. The genes encoding the microtubule-associated protein tau ( MAPT ) and progranulin ( GRN ) are the most relevant genes ...so far known causing the hereditary forms. Following genetic screening of patients affected by FTLD, we identified 2 new MAPT mutations, P364S and G366R, the former in a sporadic case. In the study we report the clinical and genetic features of the patients carrying these mutations, and the functional effects of the mutations, analyzed in vitro in order to investigate their pathogenic character. Both mutations resulted in reduced ability of tau to promote microtubule polymerization; the P364S protein variant also showed a high propensity to aggregate into filaments. These results suggest a high probability that these mutations are pathogenic. Our findings highlight the importance of genetic analysis also in sporadic forms of FTLD, and the role of in vitro studies to evaluate the pathologic features of new mutations.
The monitoring of the blood levels of therapeutic antibodies and their immune responses is proposed to guide and optimize therapy with these expensive drugs. We describe a novel Surface Plasmon ...Resonance (SPR)-based assay suitable for the simultaneous determination of serum concentrations of infliximab and anti-infliximab antibodies. The real-time detection by SPR avoids the incubation/washing steps of commonly used methods, thus allowing faster and more reliable measurements, in particular for low-affinity anti-drug antibodies. This method proved to be highly reproducible and may be well applied to other biotherapeutics.
Background
The genetically engineered, humanized, bispecific monoclonal antibody emicizumab (Hemlibra) that mimics the cofactor activity of activated factor VIII (FVIII) has been approved for ...treatment of hemophilia A patients with and without inhibitor. In the pivotal premarketing clinical trials, emicizumab prophylaxis significantly reduced bleeding rates compared with previous treatments and was well tolerated. However, a consequence of this novel therapy may be the host immune response to a foreign protein.
Objective
Characterization of the neutralizing anti‐emicizumab antibody associated with the loss of treatment efficacy.
Patient
A pediatric hemophilia A patient with inhibitor enrolled in the HAVEN2 (Study of Emicizumab Administered Subcutaneously (SC) in Pediatric Participants With Hemophilia A and Factor VIII (FVIII) Inhibitors) clinical trial.
Methods
The anti‐emicizumab antibody has been characterized with Western blot and enzyme‐linked immunosorbent assay (ELISA). The antibody was affinity purified and sequenced. Binding affinity to full‐length and papain‐digested emicizumab was analyzed using surface plasmon resonance and byo‐layer interferometry.
Results
The neutralizing anti‐emicizumab antibody was highly polyclonal with high‐affinity binding mainly to the Fab portion of emicizumab with a small amount of binding to the Fc portion. Molecular interaction experiments between emicizumab and the purified antibody indicated the presence of at least two components with similar affinities.
Conclusions
Although the incidence of neutralizing anti‐emicizumab antibody is rare, this study highlights the importance of a close monitoring and the need of a simple laboratory assay to promptly detect these antibodies in patients with a history of poor drug efficacy.
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