Search

Background and Purpose: There is concern that subvisible aggregates in biotherapeutic drug products pose a risk to patient safety. We investigated the threshold of biotherapeutic aggregates needed to induce immunogenic responses., Methods and Results: Highly aggregated samples were tested in cell-based assays and induced cellular responses in a manner that depended on the number of particles. The threshold of immune activation varied by disease state (cancer, rheumatoid arthritis, allergy), concomitant therapies, and particle number. Compared to healthy donors, disease state patients showed an equal or lower response at the late phase (7 days), suggesting they may not have a higher risk of responding to aggregates. Xeno-het mice were used to assess the threshold of immune activation in vivo. Although highly aggregated samples (~ 1,600,000 particles/mL) induced a weak and transient immunogenic response in mice, a 100-fold dilution of this sample (~ 16,000 particles/mL) did not induce immunogenicity. To confirm this result, subvisible particles (up to ~ 18,000 particles/mL, containing aggregates and silicone oil droplets) produced under representative administration practices (created upon infusion of a drug product through an IV catheter) did not induce a response in cell-based assays or appear to increase the rate of adverse events or immunogenicity during phase 3 clinical trials., Conclusion: The ability of biotherapeutic aggregates to elicit an immune response in vitro, in vivo, and in the clinic depends on high numbers of particles. This suggests that there is a high threshold for aggregates to induce an immunogenic response which is well beyond that seen in standard biotherapeutic drug products., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Subcutaneous (SubQ) injection is a common administration route for biotherapeutics. However, limited tools are available for understanding the dynamic relationships between drug products and resident cells following injection. Advances in tissue engineering have enabled the production of in vitro skin models that recapitulate the morphological structure and functional activity of human skin. Here we explore the use of a commercially available skin model to investigate potential immune activation in response to subcutaneously injected biotherapeutics. Exposure to high levels of a mixture of process-related impurities (that are known potent immune system activators) induced a robust immune response from the skin model, as indicated by enhanced metabolic activity and increased secretion of 19 cytokines and chemokines. The skin model also responded to aggregated antibodies (generated by extreme mechanical stirring and pH-jump stress, which resulted in orders of magnitude higher particle numbers than that found in products), as shown by the secretion of several signature cytokines (GM-CSF, RANTES, and MCP-1). However, the magnitude of the responses to the aggregates were significantly lower than the response to the impurities. These results highlight the promising utility of in vitro skin models for investigating the potential immune response to process-related impurities and biotherapeutic attributes in a subcutaneous environment. The use of skin models for assessing drug safety may provide new insights to help guide drug product and process development, and potentially mitigate the risk of injection site reactions and systemic immunogenic responses that may compromise the safety and efficacy of subcutaneously administered drugs., Competing Interests: Declaration of Interest All authors are currently, or were previously, employees, shareholders, or both employees and shareholders, of Amgen where the work was conducted., (Copyright © 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

DNA is tightly wrapped around histone proteins in nucleosome core particles (NCPs) yet must become accessible for processing in the cell. This accessibility, a key component of transcription regulation, is influenced by the properties of both the histone proteins and the DNA itself. Small angle x-ray scattering with contrast variation is used to examine how sequence variations affect DNA unwrapping from NCPs at different salt concentrations. Salt destabilizes NCPs, populating multiple unwrapped states as many possible unwrapping pathways are explored by the complexes. We apply coarse-grained Monte Carlo methods to generate realistic sequence-dependent unwrapped structures for the nucleosomal DNA with thermal variations. An ensemble optimization method is employed to determine the composition of the overall ensemble as electrostatic interactions are weakened. Interesting DNA-sequence-dependent differences are revealed in the unwrapping paths and equilibrium constants. These differences are correlated with specific features within the nucleic acid sequences., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Chromatin remodelers are ATP-dependent motors that reorganize DNA packaging by disrupting canonical histone-DNA contacts within the nucleosome. Here, we show that the Chd1 chromatin remodeler stimulates DNA unwrapping from the edge of the nucleosome in a nucleotide-dependent and DNA sequence-sensitive fashion. Nucleosome binding, monitored by stopped flow, was complex and sensitive to nucleotide, with AMP-PNP promoting faster binding than ADP·BeF3-. Nucleosome unwrapping by Chd1, examined by bulk FRET, occurred in the presence and absence of nucleotide and did not require the Chd1 DNA-binding domain. In AMP-PNP conditions, Chd1 unwrapped one side of the Widom 601 DNA more easily than the other, consistent with previous observations of 601 asymmetry and indicating that Chd1 amplifies intrinsic sequence properties of nucleosomal DNA. Using small angle X-ray scattering (SAXS) with contrast variation, we found distinct DNA conformations depending on the nucleotide analog bound to Chd1: with AMP-PNP, DNA primarily unwrapped in-plane with the nucleosomal disk, whereas with ADP·BeF3-, a significant fraction showed distinctive out-of-plane unwrapping as well. Taken together, our findings show tight coupling between entry/exit DNA of the nucleosome and the Chd1 ATPase motor, suggesting that dynamic nucleosome unwrapping is coupled to nucleosome binding and remodeling by Chd1.

Unlike tailed bacteriophages, which use a preformed tail for transporting their genomes into a host bacterium, the ssDNA bacteriophage ΦX174 is tailless. Using cryo-electron microscopy and time-resolved small-angle X-ray scattering, we show that lipopolysaccharides (LPS) form bilayers that interact with ΦX174 at an icosahedral fivefold vertex and induce single-stranded (ss) DNA genome ejection. The structures of ΦX174 complexed with LPS have been determined for the pre- and post-ssDNA ejection states. The ejection is initiated by the loss of the G protein spike that encounters the LPS, followed by conformational changes of two polypeptide loops on the major capsid F proteins. One of these loops mediates viral attachment, and the other participates in making the fivefold channel at the vertex contacting the LPS., Competing Interests: The authors declare no conflict of interest.

The nucleosome core particle (NCP) is the basic structural unit for genome packaging in eukaryotic cells and consists of DNA wound around a core of eight histone proteins. DNA access is modulated through dynamic processes of NCP disassembly. Partly disassembled structures, such as the hexasome (containing six histones) and the tetrasome (four histones), are important for transcription regulation in vivo. However, the pathways for their formation have been difficult to characterize. We combine time-resolved (TR) small-angle X-ray scattering and TR-FRET to correlate changes in the DNA conformations with composition of the histone core during salt-induced disassembly of canonical NCPs. We find that H2A-H2B histone dimers are released sequentially, with the first dimer being released after the DNA has formed an asymmetrically unwrapped, teardrop-shape DNA structure. This finding suggests that the octasome-to-hexasome transition is guided by the asymmetric unwrapping of the DNA. The link between DNA structure and histone composition suggests a potential mechanism for the action of proteins that alter nucleosome configurations such as histone chaperones and chromatin remodeling complexes., Competing Interests: The authors declare no conflict of interest.

Human pyruvate kinase isoform M2 (PKM2) is a glycolytic enzyme isoform implicated in cancer. Malignant cancer cells have higher levels of dimeric PKM2, which is regarded as an inactive form of tetrameric pyruvate kinase. This perceived inactivity has fueled controversy about how the dimeric form of pyruvate kinase might contribute to cancer. Here we investigate enzymatic properties of PKM2(G415R), a variant derived from a cancer patient, which we show by size-exclusion chromatography and small-angle X-ray scattering to be a dimer that cannot form a tetramer in solution. Although PKM2(G415R) binds to fructose 1,6-bisphosphate (FBP), unlike the wild type this PKM2 variant shows no activation by FBP. In contrast, PKM2(G415R) is activated by succinyl-5-aminoimidazole-4-carboxamide-1-ribose 5'-phosphate (SAICAR), an endogenous metabolite that we previously showed correlates with an increased level of cell proliferation and promotes protein kinase activity of PKM2. Our results demonstrate an important and unexpected enzymatic activity of the PKM2 dimer that likely has a key role in cancer progression.

Understanding how DNA carries out its biological roles requires knowledge of its interactions with biological partners. Since DNA is a polyanionic polymer, electrostatic interactions contribute significantly. These interactions are mediated by positively charged protein residues or charge compensating cations. Direct detection of these partners and/or their effect on DNA conformation poses challenges, especially for monitoring conformational dynamics in real time. Small-angle x-ray scattering (SAXS) is uniquely sensitive to both the conformation and local environment (i.e. protein partner and associated ions) of the DNA. The primary challenge of studying multi-component systems with SAXS lies in resolving how each component contributes to the measured scattering. Here, we review two contrast variation (CV) strategies that enable targeted studies of the structures of DNA or its associated partners. First, solution contrast variation enables measurement of DNA conformation within a protein-DNA complex by masking out the protein contribution to the scattering profile. We review a specific example, in which the real-time unwrapping of DNA from a nucleosome core particle is measured during salt-induced disassembly. The second method, heavy atom isomorphous replacement, reports the spatial distribution of the cation cloud around duplex DNA by exploiting changes in the scattering strength of cations with varying atomic numbers. We demonstrate the application of this approach to provide the spatial distribution of monovalent cations (Na + , K + , Rb + , Cs + ) around a standard 25-base pair DNA. The CV strategies presented here are valuable tools for understanding DNA interactions with its biological partners.

The modulation of DNA accessibility by nucleosomes is a fundamental mechanism of gene regulation in eukaryotes. The nucleosome core particle (NCP) consists of 147 bp of DNA wrapped around a symmetric octamer of histone proteins. The dynamics of DNA packaging and unpackaging from the NCP affect all DNA-based chemistries, but depend on many factors, including DNA positioning sequence, histone variants and modifications. Although the structure of the intact NCP has been studied by crystallography at atomic resolution, little is known about the structures of the partially unwrapped, transient intermediates relevant to nucleosome dynamics in processes such as transcription, DNA replication and repair. We apply a new experimental approach combining contrast variation with time-resolved small angle X-ray scattering (TR-SAXS) to determine transient structures of protein and DNA constituents of NCPs during salt-induced disassembly. We measure the structures of unwrapping DNA and monitor protein dissociation from Xenopus laevis histones reconstituted with two model NCP positioning constructs: the Widom 601 sequence and the sea urchin 5S ribosomal gene. Both constructs reveal asymmetric release of DNA from disrupted histone cores, but display different patterns of protein dissociation. These kinetic intermediates may be biologically important substrates for gene regulation., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.