Insights into the stability of a therapeutic antibody Fab fragment by molecular dynamics and its stabilization by computational design

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions, while retaining their specific mode of action. Gaining a deeper understanding of the drivers of instability across different stress conditions, will potentially enable the engineering of protein scaffolds that are inherently manufacturable and stable. Here, we compared the structural robustness of a humanized antibody fragment (Fab) A33 using atomistic molecular dynamics simulations under two different stresses of low pH and high temperature. RMSD calculations, structural alignments and contact analysis revealed that low pH unfolding was initiated through loss of contacts at the constant domain interface (CL-CH1), prior to CL domain unfolding. By contrast, thermal unfolding began with loss of contacts in both the CL-CH1 and variable domain interface (VL-VH), followed by domain unfolding of CL and also of VH, thus revealing divergent unfolding pathways. FoldX and Rosetta both agreed that mutations at the CL-CH1 interface have the greatest potential for increasing the stability of Fab A33. Additionally, packing density calculations found these residues to be under-packed relative to other inter-domain residues. Two salt bridges were identified that possibly drive the conformational change at low pH, while at high temperature, salt bridges were lost and reformed quickly, and not always with the same partner, thus contributing to an overall destabilization. Sequence entropy analysis of existing Fab sequences revealed considerable scope for further engineering, where certain natural mutations agreed with FoldX and Rosetta predictions. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR), which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, which provide interesting targets for future protein engineering work aimed at stabilizing to both thermal and pH-stresses simultaneously

Agrotechnologies towards Ecotechnologies the three pillars for developing Eco-design

International audienceTo boost agrotechnologies towards ecotechnologies ("environmental technologies" according to ETAP programme of EU, or "more ecologically productive technologies" in the context of agriculture), we need to strengthen a "triple bottom" system: -To take into account, in "Life Cycle Analysis" methodologies, the natural variability in time and space of these applications in land use. - To develop an overall approach for realistic machinery qualification, in order to feed the environmental burdens accurately through relevant data bases collected on agrotechnologies in real action. - To work on Eco-innovation processes, by deepening specific innovation tools and methods, for implementation of innovative solutions chosen according to LCA results. This paper presents the concept, develops the methods and illustrates them by examples of results on organic spreading technologies

Computational Design To Reduce Conformational Flexibility and Aggregation Rates of an Antibody Fab Fragment

Computationally-guided semi-rational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilise proteins to aggregation under some conditions, previous studies suggest that such an approach is limited because thermal transition temperatures (Tm) and the fraction of protein unfolded (fT) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the Tm. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, that reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilising mutations to regions of the A33 Fab surface structure, that were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations, and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation, and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔGND for each potential point mutation. Additional destabilising variants were prepared as controls to evaluate the prediction accuracy, and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of eighteen variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased Tm values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by Tm. However, six of the twelve stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate

The mechanisms of action of interleukin-1 on rabbit intestinal epithelial cells.

Interleukin-1 (IL-1) is an inflammatory mediator that increases Cl- secretion in intestinal epithelial cells. To identify the signal transduction pathway(s) involved in IL-1's action, cells were treated with IL-1 and the levels of cyclooxygenase (COX) enzymes, prostaglandin E2 (PGE2) and phospholipase A2-activating protein (PLAP), and the activity of phospholipase A2 (PLA2) were measured. IL-1 caused concentration- and time-dependent increases in the levels of PLA2 activity, and/or in the levels of PLAP, COX-2 and PGE2. The IL-induced increase in PGE2 levels was biphasic, with the first peak due to the increase in PLAP levels, and the second peak due to the increase in COX-2 levels. This increase in PGE2 levels may provide a mechanism for acute and chronic inflammation in the intestine

Comparison of the pH- and thermally-induced fluctuations of a therapeutic antibody Fab fragment by molecular dynamics simulation

Successful development of protein therapeutics depends critically on achieving stability under a range of conditions. A deeper understanding of the drivers of instability across different stress conditions, will enable the engineering of more robust protein scaffolds. We compared the impacts of low pH and high temperature stresses on the structure of a humanized antibody fragment (Fab) A33, using atomistic molecular dynamics simulations, using a recent 2.5 Å crystal structure. This revealed that low-pH induced the loss of native contacts in the domain CL. By contrast, thermal stress led to 5–7% loss of native contacts in all four domains, and simultaneous loss of >30% of native contacts in the VL-VH and CL-CH interfaces. This revealed divergent destabilising pathways under the two different stresses. The underlying cause of instability was probed using FoldX and Rosetta mutation analysis, and packing density calculations. These agreed that mutations in the CL domain, and CL-CH1 interface have the greatest potential for stabilisation of Fab A33. Several key salt bridge losses underpinned the conformational change in CL at low pH, whereas at high temperature, salt bridges became more dynamic, thus contributing to an overall destabilization. Lastly, the unfolding events at the two stress conditions exposed different predicted aggregation-prone regions (APR) to solvent, which would potentially lead to different aggregation mechanisms. Overall, our results identified the early stages of unfolding and stability-limiting regions of Fab A33, and the VH and CL domains as interesting future targets for engineering stability to both pH- and thermal-stresses simultaneously

GPI-anchor signal sequence influences PrPC sorting, shedding and signalling, and impacts on different pathomechanistic aspects of prion disease in mice

The cellular prion protein (PrPC) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrPC-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrPC in vivo is limited We exchanged the PrPC GPI-anchor signal sequence of for that of Thy-1 (PrPCGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrPCGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrPC. Interestingly, after prion infection, mice expressing PrPCGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrPC, influencing the development of prion disease

Comprehensive miRNome-wide profiling in a neuronal cell model of synucleinopathy implies involvement of cell cycle genes

Growing evidence suggests that epigenetic mechanisms like microRNA-mediated transcriptional regulation contribute to the pathogenesis of parkinsonism. In order to study the influence of microRNAs (miRNAs), we analyzed the miRNome 2 days prior to major cell death in α-synuclein-overexpressing Lund human mesencephalic neurons, a well-established cell model of Parkinson\u27s disease (PD), by next-generation sequencing. The expression levels of 23 miRNAs were significantly altered in α-synuclein-overexpressing cells, 11 were down- and 12 upregulated

An Expanded Conformation of an Antibody Fab Region by X-Ray Scattering, Molecular Dynamics and smFRET Identifies an Aggregation Mechanism

Protein aggregation is the underlying cause of many diseases, and also limits the usefulness of many natural and engineered proteins in biotechnology. Better mechanistic understanding and characterization of aggregation-prone states, is needed to guide protein engineering, formulation, and drug-targeting strategies that prevent aggregation. While several final aggregated states - notably amyloids - have been characterized structurally, very little is known about the native structural conformers that initiate aggregation. We used a novel combination of small-angle X-ray scattering (SAXS), atomistic molecular dynamics (MD) simulations, single-molecule FRET (smFRET), and aggregation-prone region (APR) predictions, to characterize structural changes in a native humanized Fab A33 antibody fragment, that correlated with the experimental aggregation kinetics. SAXS revealed increases in the native state radius of gyration, Rg, of 2.2% to 4.1%, at pH 5.5 and below, concomitant with accelerated aggregation. In a cutting-edge approach, we fitted the SAXS data to full molecular dynamics simulations from the same conditions, and located the conformational changes in the native state to the constant domain of the light chain (CL). This CL displacement was independently confirmed using smFRET measurements with two dual-labeled Fabs. These conformational changes were also found to increase the solvent exposure of a predicted aggregation-prone region (APR), suggesting a likely mechanism through which they promote aggregation. Our findings provide a means by which aggregation-prone conformational states can be readily determined experimentally, and thus potentially used to guide protein engineering, or ligand binding strategies, with the aim of stabilizing the protein against aggregation

A study of patent thickets

Report analysing whether entry of UK enterprises into patenting in a technology area is affected by patent thickets in the technology area