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Abstract: The human immunoglobulin G (IgG) class is the most prevalent antibody in serum, with the IgG1 subclass being the most abundant. IgG1 is comprised of two Fab regions connected to a Fc region through a 15-residue hinge peptide. Two glycan chains are conserved in the Fc region in IgG, however their importance for the structure of intact IgG1 has remained unclear. Here, we subjected glycosylated and deglycosylated monoclonal human IgG1 (designated as A33) to a comparative multidisciplinary structural study of both forms. Following deglycosylation using PNGase F, analytical ultracentrifugation showed that IgG1 remained monomeric and the sedimentation coefficients s020,w of IgG1 decreased from 6.45 S by 0.16-0.27 S. This change was attributed to the reduction in mass following glycan removal. X-ray and neutron scattering revealed changes in the Guinier structural parameters after deglycosylation. While the radius of gyration RG was unchanged, the cross-sectional radius of gyration, RXS-1, increased by 0.1 nm and the commonly occurring distance peak M2 of the distance distribution curve P(r) increased by 0.4 nm. These changes revealed that the Fab-Fc separation in IgG1 was perturbed following deglycosylation. To explain these changes, atomistic scattering modelling based on Monte Carlo simulations resulted in 123,284 and 119,191 trial structures for glycosylated and deglycosylated IgG1 respectively. From these, 100 X-ray and neutron best-fit models were determined. For these, principal component analyses identified five groups of structural conformations that were different for glycosylated and deglycosylated IgG1. The Fc region in glycosylated IgG1 showed a restricted range of conformations relative to the Fab regions, while the Fc region in deglycosylated IgG1 showed a broader conformational spectrum. These more variable Fc conformations account for the loss of binding to the FcγR receptor in deglycosylated IgG1.

Open Access

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Abstract: Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.

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Abstract: Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T cell-mediated anti-tumor response. Systemic administration of anti-4-1BB-agonistic IgGs, althougheffective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, anti-tumor efficacy, and toxicity in vivo. Results: In the presence of a T cell receptor signal, the trimerbody provided potent T cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant anti-tumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small-cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1-blocker led to increased IFNg secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. Conclusions: These results demonstrate the non-toxic broad anti-tumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most cancer patients while avoiding Fc-mediated adverse reactions.

Open Access

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Abstract: The SCFSKP2 ubiquitin ligase relieves G1 checkpoint control of CDK-cyclin complexes by promoting p27KIP1 degradation. We describe reconstitution of stable complexes containing SKP1-SKP2 and CDK1-cyclin B or CDK2-cyclin A/E, mediated by the CDK regulatory subunit CKS1. We further show that a direct interaction between a SKP2 N-terminal motif and cyclin A can stabilize SKP1-SKP2-CDK2-cyclin A complexes in the absence of CKS1. We identify the SKP2 binding site on cyclin A and demonstrate the site is not present in cyclin B or cyclin E. This site is distinct from but overlapping with features that mediate binding of p27KIP1 and other G1 cyclin regulators to cyclin A. We propose that the capacity of SKP2 to engage with CDK2-cyclin A by more than one structural mechanism provides a way to fine tune the degradation of p27KIP1 and distinguishes cyclin A from other G1 cyclins to ensure orderly cell cycle progression.

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Abstract: Dupuytren's Disease (DD) is a common fibroproliferative disease of the palmar fascia. We previously identified a causal association with a non-synonymous variant (rs1042704, p.D273N) in MMP14 (encoding MT1-MMP). In this study, we investigated the functional consequences of this variant, and demonstrated that the variant MT1-MMP (MT1-N273) exhibits only 17% of cell surface collagenolytic activity compared to the ancestral enzyme (MT1-D273). Cells expressing both MT1-D273 and MT1-N273 in a 1:1 ratio, mimicking the heterozygous state, possess 38% of the collagenolytic activity compared to the cells expressing MT1-D273, suggesting that MT1-N273 acts in a dominant negative manner. Consistent with the above observation, patient-derived DD myofibroblasts with the alternate allele demonstrated around 30% of full collagenolytic activity detected in ancestral G/G genotype cells, regardless of the heterozygous (G/A) or homozygous (A/A) state. Small angle X-ray scattering analysis of purified soluble Fc-fusion enzymes allowed us to construct a 3D-molecular envelope of MT1-D273 and MT1-N273, and demonstrate altered flexibility and conformation of the ectodomains due to D273 to N substitution. Taking together, rs1042704 significantly reduces collagen catabolism in tissue, which tips the balance of homeostasis of collagen in tissue, contributing to the fibrotic phenotype of DD. Since around 30% of the worldwide population have at least one copy of the low collagenolytic alternate allele, further investigation of rs1042704 across multiple pathologies is needed.