Identification of KLHDC2 as an efficient proximity-induced degrader of K-RAS, STK33, β-catenin, and FoxP3

Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, β-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells

Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction

Antibody-PROTAC conjugate enables selective degradation of receptor-interacting serine/threonine-protein kinase 2 (RIPK2) in HER+ cell lines

Proteolysis targeting chimeras (PROTACs) are a family of heterobifunctional molecules that are now realising their promise as a therapeutic strategy for targeted protein degradation. However, one limitation of existing designs is the lack of cell-selective targeting of the protein degrading payload. This manuscript reports a cell-targeted approach to degrade receptor-interacting serine/threonine-protein kinase 2 (RIPK2) in HER2+ cell lines. An antibody-PROTAC conjugate is prepared containing a protease cleavable linkage between the antibody and the corresponding degrader. Potent RIPK2 degradation is observed in HER2+ cell lines, whereas an equivalent anti-IL4 antibody-PROTAC conjugate shows no degradation at therapeutically relevant concentrations. No RIPK2 degradation was observed in HER2- cell lines for both bioconjugates. This work demonstrates the potential for cell-selective delivery of PROTAC scaffolds by engaging with signature extracellular proteins expressed on the surface of particular cell types

Histones induce epithelial and endothelial cell death.

<p>(A) A549 cells were treated for 16 h with different concentrations of histone type II-A, and the cell morphology was evaluated. (B) A549 cell numbers were counted after treatment with various concentrations of histones for 16 h. (C) HUVEC or A549 cells were treated with 200 µg/ml histones for 16 h or left untreated (control). (D) HUVEC were treated for 16 h with different concentrations of histones, and the extent of cytotoxity was measured. B and D are representative data of three independent experiments, and in A and C pictures are representative pictures from three independent experiments at 20× magnification.</p

Histone antibodies and polysialic acid decrease NET-mediated cytotoxicity.

<p>(A) NET were pre-incubated with different antibodies against histones (DNA/H1, H2A, H2B, H3, citrullinated H3 [cit H3], H4) or with isotype-matched control antibodies. Antibody-treated NET or NET alone (−) were incubated with A549 cells for 16 h to analyze the cytotoxicity. Shown are representative data of three independent experiments (mean SD), ***<i>p</i><0.001 and ns = non-significant. (B) Histones or (C) NET were pre-incubated with antibody against histone H4 or polysialic acid (PSA), followed by incubation with A459 cells for 16 h to analyze the cytotoxicity. Note that polysialic acid considerably decreased both histone- and NET-mediated cytotoxicity.</p

APC decreases epithelial cytotoxicity induced by histones but not by NET.

<p>(A) Histones (200 or 100 µg/ml), pre-incubated for 1 h at 37°C in the absence or presence of 100 nM human APC, were incubated with A549 cells for 16 h, followed by analysis of cytotoxicity. (B) NET were incubated with APC (mass ratio APC: NET proteins, 1∶5, 1∶2 and 1∶1) or without APC for 1 h at 37°C, followed by incubation with A549 cells for 16 h and measurement of cytotoxicty. APC alone or active-site blocked APC (APC+PPACK) were incubated with A549 cells for control. (C) DNase-digested and (D) undigested forms of NET were pre-incubated with 100 nM APC for 20 to 80 min before incubation with A549 cells for 16 h, followed by determination of cytotoxicty. Shown are representative data of four independent experiments (mean SD), ***<i>p</i><0.001 and ns = non-significant.</p

NET induce cytotoxicity in epithelial and endothelial cells independent of digestion.

<p>(A) The extent of cytotoxicity was measured after treatment of A549 cells for 16 h with undigested NET (−), completely (DNase), partially digested (MNase) or boiled forms of NET. The same concentration of DNA alone as DNA-NET (3.4 µg/ml) as well as DNase or MNase alone were used as controls. Shown are representative data of five independent experiments (mean SD), ***<i>p</i><0.001, and ns = non-significant. (B) The degree of cytotoxicity was measured after treatment of HUVEC, HPAEC, AT-II or MLE-12 cells for 16 h with undigested NET (−) or completely (DNase) forms of NET as well as DNA alone. Shown are representative data of three (except for AT-II, n = 2) independent experiments (mean SD), ns = non-significant.</p