CIISB Research Highlights Archive

Model for ameloblast-specific autoantibody production in patients with coeliac disease.

Jakub Abramson Research Group

Significance

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation—amelogenesis. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta. Defects in enamel formation are also found in patients with autoimmune polyglandular syndroze type-1 (APS-1), caused by AIRE deficiency, and in patients diagnosed with coeliac disease. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Gruper, Y., Wolff, A.S.B., Glanz, L. et al. Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Nature 624, 653–662 (2023). https://doi.org/10.1038/s41586-023-06776-0

Close-up view of FMA-binding pocket with residues creating the active site in stick representation. Key hydrogen bonds are shown as dashed yellow lines.

Martin Marek Research Group

Significance

NanoLuc, a superior β-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques are combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O₂ via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

Nemergut, M., Pluskal, D., Horackova, J. et al. Illuminating the mechanism and allosteric behavior of NanoLuc luciferase.

Nat Commun 14, 7864 (2023). https://doi.org/10.1038/s41467-023-43403-y

Ultrahigh binding affinity of bambusuril receptors toward halides is achieved and continuously increases with increasing electron-withdrawing power of groups installed on its benzyl substituents, as measured by ¹H and ¹⁹F NMR spectroscopy.

Vladimír Šindelář Research Group

Significance

Inspired by nature, artificial hydrogen bond-based anion receptors have been developed to achieve high anion selectivity; however, their binding affinity is usually low. The potency of these receptors is usually increased by the introduction of aryl substituents, which withdraw electrons from their binding site through the resonance effect. Here, we show that the polarization of the C(sp³)-H binding site of bambusuril receptors, and thus their potency to bind anions, can be modulated by the inductive effect. The presence of electron-withdrawing groups on benzyl substituents of bambusurils significantly increases their binding affinities to halides, resulting in the strongest iodide receptor reported to date with an association constant greater than 10¹³ M⁻¹ in acetonitrile. A Hammett plot showed that while the bambusuril affinity toward halides linearly increases with the electron-withdrawing power of their substituents, their binding selectivity remains essentially unchanged.

Tuning CH Hydrogen Bond-Based Receptors toward Picomolar Anion Affinity via the Inductive Effect of Distant Substituents, M. Chvojka, D. Madea, H. Valkenier, V. Šindelář

Angew. Chem. Int. Ed. 2023, e202318261. https://doi.org/10.1002/anie.202318261

Double-strand breaks (DSBs) are the most severe type of DNA damage. Long et al. show that hSSB1 is modified and forms a trimeric SOSS1 complex that comes to DSBs in an R-loop-dependent manner. At DSBs, SOSS1 and RNA polymerase II form liquid-like repair compartments. Depletion of the SOSS1 impairs DNA repair.

Monika Gullerova Research Group

Significance

Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid- like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.

Long, QL; Sebesta, M; Sedova, K; Haluza, V; Alagia, A; Liu, ZC; Stefl, R; Gullerova, M: The phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks.

Cell Reports 42, 113439, https://doi.org/10.1016/j.celrep.2023.113489

c) Three-dimensional (3D) reconstruction showing an electron density map with γ-conglutin model in top and side views (PDB: 4pph) fitted as a ring-like hexamer assemble.

Jaroslaw Czubinsky Research Group

Significance

Despite extensive research carried out on lupin seed γ-conglutin neither its mechanism of action as a hypoglycaemic nutraceutical compound nor its physiological role for the plant has been unveil. This article revealed a pH-dependent reversible association/dissociation equilibrium involving monomer, dimer and hexamer of Lupinus angustifolius γ-conglutin. The interaction between different oligomeric forms of this protein is reversible, and spectroscopic studies showed that the intact structure of γ-conglutin was preserved under the tested environmental conditions tested (pH 4.5–7.5). The obtained results prove that the hexameric form was preferred under basic conditions and was stabilised by a number of bonds formed upon association of individual protomers. The simultaneous occurrence of several oligomeric forms at a given pH value was shown, and their share was strongly driven by protein concentration. The main changes in oligomerisation of γ-conglutin take place in a pH range of 4.5–6.0, correlating with the pKa_R values of the amino acid residues of His (6.0), Glu (4.1), and Asp (3.9). Moreover, a structural model of the protein in hexamer assembly was obtained based on small-angle X-ray scattering (SAXS) and negative staining cryo-electron microscopy (cryo-EM) analyses. The presented study provides essential knowledge about the colloidal dynamics and stability of γ-conglutin in solution, improving our understanding of fundamental environmental factors that could affect the health-promoting activity of this lupin seed protein.

Czubinksi, J., Kubíčková, M., et al. pH-Dependent oligomerisation of γ-conglutin: A key element to understand its molecular mechanism of action.

Food Hydrocol. 147, Part A, 109386 (2024) https://doi.org/10.1016/j.foodhyd.2023.109386

a, b Mapping the binary interaction between RSM and RPA32C by NMR titrations. ¹⁵N labeled RSM titrated with zero to fourfold molar addition of RPA32C (a) and the reverse (b).

Kostas Tripsianes and Lumír Krejčí Research Groups

Significance

Biomolecular polyelectrolyte complexes can be formed between oppositely charged intrinsically disordered regions (IDRs) of proteins or between IDRs and nucleic acids. Highly charged IDRs are abundant in the nucleus, yet few have been functionally characterized. Here, we show that a positively charged IDR within the human ATP-dependent DNA helicase Q4 (RECQ4) forms coacervates with G-quadruplexes (G4s). We describe a three-step model of charge-driven coacervation by integrating equilibrium and kinetic binding data in a global numerical model. The oppositely charged IDR and G4 molecules form a complex in the solution that follows a rapid nucleation-growth mechanism leading to a dynamic equilibrium between dilute and condensed phases. We also discover a physical interaction with Replication Protein A (RPA) and demonstrate that the IDR can switch between the two extremes of the structural continuum of complexes. The structural, kinetic, and thermodynamic profile of its interactions revealed a dynamic disordered complex with nucleic acids and a static ordered complex with RPA protein. The two mutually exclusive binding modes suggest a regulatory role for the IDR in RECQ4 function by enabling molecular handoffs. Our study extends the functional repertoire of IDRs and demonstrates a role of polyelectrolyte complexes involved in G4 binding.

Papageorgiou, A.C., Pospisilova, M., Cibulka, J. et al. Recognition and coacervation of G-quadruplexes by a multifunctional disordered region in RECQ4 helicase.

Nat Commun 14, 6751 (2023). https://doi.org/10.1038/s41467-023-42503-z