Björn Rabe

Enzymatic Removal of Ribonucleotides from DNA Is Essential for Mammalian Genome Integrity and Development

Summary The presence of ribonucleotides in genomic DNA is undesirable given their increased susceptibility to hydrolysis. Ribonuclease (RNase) H enzymes that recognize and process such embedded ribonucleotides are present in all domains of life. However, in unicellular organisms such as budding yeast, they are not required for viability or even efficient cellular proliferation, while in humans, RNase H2 hypomorphic mutations cause the neuroinflammatory disorder Aicardi-Goutières syndrome. Here, we report that RNase H2 is an essential enzyme in mice, required for embryonic growth from gastrulation onward. RNase H2 null embryos accumulate large numbers of single (or di-) ribonucleotides embedded in their genomic DNA (>1,000,000 per cell), resulting in genome instability and a p53-dependent DNA-damage response. Our findings establish RNase H2 as a key mammalian genome surveillance enzyme required for ribonucleotide removal and demonstrate that ribonucleotides are the most commonly occurring endogenous nucleotide base lesion in replicating cells.

The soluble Interleukin 6 receptor: Generation and role in inflammation and cancer

Soluble cytokine receptors are frequently found in human serum, most of them possessing antagonistic properties. The Interleukin 6 receptor (IL-6R) is found as a transmembrane protein on hepatocytes and subsets of leukocytes, but soluble isoforms of the IL-6R (sIL-6R) are generated by alternative splicing or by limited proteolysis of the ADisintegrin And Metalloproteinases (ADAM) gene family members ADAM10 and ADAM17. Importantly, the sIL-6R in complex with its ligand Interleukin 6 (IL-6) has agonistic functions and requires cells expressing the signal transducing ß-receptor gp130 but not the membrane-bound IL-6R. We have called this process IL-6 trans-signaling. Naturally occurring isoforms of soluble gp130 (sgp130), which are generated by alternative splicing, are natural inhibitors of IL-6 trans-signaling, leaving IL-6 classic signaling via the membrane-bound IL-6R unaffected. We used recombinant sgp130Fc protein and recently generated transgenic mice expressing high levels of sgp130Fc to discriminate between classic and trans-signaling in vivo, and demonstrated that IL-6 trans-signaling is critically involved in generation and maintenance of several inflammatory and autoimmune diseases including chronic inflammatory bowel disease, rheumatoid arthritis, peritonitis and asthma, as well as inflammation-induced colon cancer.

IL-6 trans-signaling promotes pancreatitis-associated lung injury and lethality

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI.

Ribonuclease H2 mutations induce a cGAS/STING-dependent innate immune response.

Aicardi–Goutières syndrome (AGS) provides a monogenic model of nucleic acid‐mediated inflammation relevant to the pathogenesis of systemic autoimmunity. Mutations that impair ribonuclease (RNase) H2 enzyme function are the most frequent cause of this autoinflammatory disorder of childhood and are also associated with systemic lupus erythematosus. Reduced processing of either RNA:DNA hybrid or genome‐embedded ribonucleotide substrates is thought to lead to activation of a yet undefined nucleic acid‐sensing pathway. Here, we establish Rnaseh2bA174T/A174T knock‐in mice as a subclinical model of disease, identifying significant interferon‐stimulated gene (ISG) transcript upregulation that recapitulates the ISG signature seen in AGS patients. The inflammatory response is dependent on the nucleic acid sensor cyclic GMP‐AMP synthase (cGAS) and its adaptor STING and is associated with reduced cellular ribonucleotide excision repair activity and increased DNA damage. This suggests that cGAS/STING is a key nucleic acid‐sensing pathway relevant to AGS, providing additional insight into disease pathogenesis relevant to the development of therapeutics for this childhood‐onset interferonopathy and adult systemic autoimmune disorders.

Structure-guided Optimization of the Interleukin-6 Trans-signaling Antagonist sgp130

Binding of interleukin-6 (IL-6) to its specific receptor IL-6R is a prerequisite for the activation of the signal-transducing receptor glycoprotein 130 (gp130). A soluble form of the IL-6R (sIL-6R) in complex with IL-6 can activate cells lacking membrane-bound IL-6R (trans-signaling). IL-6-trans-signaling is counterbalanced by a naturally occurring, soluble form of gp130 (sgp130), whereby signaling via the membrane-bound IL-6R is not affected. Many inflammatory and neoplastic disorders are driven by IL-6 trans-signaling. By analysis of the three-dimensional structure of gp130 in complex with IL-6 and sIL-6R, we identified amino acid side chains in gp130 as candidates for the generation of sgp130 muteins with increased binding affinity to IL-6/sIL-6R. In addition, with information from modeling and NMR analysis of the membrane proximal domain of gp130, we generated a more stable variant of sgp130Fc. Proteins were tested for binding to the IL-6/sIL-6R-complex, for inhibition of IL-6/sIL-6R-induced cell proliferation and of acute phase gene expression. Several mutations showed an additive effect in improving the binding affinity of human sgp130 toward human IL-6/sIL-6R. Finally, we demonstrate the species specificity of these mutations in the optimal triple mutein (T102Y/Q113F/N114L) both in vitro and in a mouse model of acute inflammation.

Unraveling Viral Interleukin-6 Binding to gp130 and Activation of STAT-Signaling Pathways Independently of the Interleukin-6 Receptor

ABSTRACT Human herpesvirus 8 encodes a viral version of interleukin-6 (vIL-6) which shows 25% sequence homology with human IL-6. In contrast to human IL-6, which first binds to the IL-6 receptor (IL-6R) and only subsequently associates with the signal transducing receptor subunit gp130, vIL-6 has been shown to directly bind to gp130 without the need of IL-6R. As a functional consequence, vIL-6 can activate far more target cells in the body since all cells express gp130, but only cells such as hepatocytes and some leukocytes express IL-6R. We sought to understand which amino acid sequences within the vIL-6 protein were responsible for its ability to bind and activate gp130 independent of IL-6R. As a first approach, we constructed chimeric IL-6 proteins in which all known gp130 interacting sites (sites II and III) were sequentially transferred from vIL-6 into the human IL-6 protein. To our surprise, human IL-6 carrying all gp130 interacting sites from vIL-6 did not show IL-6R-independent gp130 activation. Even more surprisingly, the loop between helix B and C of vIL-6, clearly shown in the crystal structure not to be in contact with gp130, is indispensable for direct binding to and activation of gp130. This points to an IL-6R induced change of site III conformation in human IL-6, which is already preformed in vIL-6. These data indicate a novel activation mechanism of human IL-6 by the IL-6R that will be important for the construction of novel hyperactive cytokine variants.

Abrogation of Viral Interleukin-6 (vIL-6)-Induced Signaling by Intracellular Retention and Neutralization of vIL-6 with an Anti-vIL-6 Single-Chain Antibody Selected by Phage Display

ABSTRACT Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposis sarcoma as well as primary effusion lymphoma and multicentric Castlemans disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.

Shedding of Endogenous Interleukin-6 Receptor (IL-6R) Is Governed by A Disintegrin and Metalloproteinase (ADAM) Proteases while a Full-length IL-6R Isoform Localizes to Circulating Microvesicles.

Background: A soluble form of IL-6 receptor mediates pathogenic IL-6 trans-signaling. Results: ADAM10 and ADAM17 release IL-6 receptor from both human and murine monocytes/macrophages, whereas in the blood IL-6 receptor is also present on microvesicles. Conclusion: Shedding of endogenous IL-6 receptor is similar in humans and mice. Significance: Microvesicle release represents a novel mode of soluble IL-6 receptor generation with potential clinical implications. Generation of the soluble interleukin-6 receptor (sIL-6R) is a prerequisite for pathogenic IL-6 trans-signaling, which constitutes a distinct signaling pathway of the pleiotropic cytokine interleukin-6 (IL-6). Although in vitro experiments using ectopically overexpressed IL-6R and candidate proteases revealed major roles for the metalloproteinases ADAM10 and ADAM17 in IL-6R shedding, the identity of the protease(s) cleaving IL-6R in more physiological settings, or even in vivo, remains unknown. By taking advantage of specific pharmacological inhibitors and primary cells from ADAM-deficient mice we established that endogenous IL-6R of both human and murine origin is shed by ADAM17 in an induced manner, whereas constitutive release of endogenous IL-6R is largely mediated by ADAM10. Although circulating IL-6R levels are altered in various diseases, the origin of blood-borne IL-6R is still poorly understood. It has been shown previously that ADAM17 hypomorphic mice exhibit unaltered levels of serum sIL-6R. Here, by quantification of serum sIL-6R in protease-deficient mice as well as human patients we also excluded ADAM10, ADAM8, neutrophil elastase, cathepsin G, and proteinase 3 from contributing to circulating sIL-6R. Furthermore, we ruled out alternative splicing of the IL-6R mRNA as a potential source of circulating sIL-6R in the mouse. Instead, we found full-length IL-6R on circulating microvesicles, establishing microvesicle release as a novel mechanism for sIL-6R generation.

Polo-like Kinase 2, a Novel ADAM17 Signaling Component, Regulates Tumor Necrosis Factor α Ectodomain Shedding

Background: The metalloprotease ADAM17 emerged as the main sheddase of several cytokines and cytokine receptors. Results: The acidophilic kinase PLK2 interacts with and phosphorylates ADAM17 in mammalian cells. Conclusion: PLK2 represents a novel cellular interaction partner of ADAM17 modulating its activity. Significance: Regulation of ADAM17 activity is essential for inflammatory responses. ADAM17 (a disintegrin and metalloprotease 17) controls pro- and anti-inflammatory signaling events by promoting ectodomain shedding of cytokine precursors and cytokine receptors. Despite the well documented substrate repertoire of ADAM17, little is known about regulatory mechanisms, leading to substrate recognition and catalytic activation. Here we report a direct interaction of the acidophilic kinase Polo-like kinase 2 (PLK2, also known as SNK) with the cytoplasmic portion of ADAM17 through the C-terminal noncatalytic region of PLK2 containing the Polo box domains. PLK2 activity leads to ADAM17 phosphorylation at serine 794, which represents a novel phosphorylation site. Activation of ADAM17 by PLK2 results in the release of pro-TNFα and TNF receptors from the cell surface, and pharmacological inhibition of PLK2 leads to down-regulation of LPS-induced ADAM17-mediated shedding on primary macrophages and dendritic cells. Importantly, PLK2 expression is up-regulated during inflammatory conditions increasing ADAM17-mediated proteolytic events. Our findings suggest a new role for PLK2 in the regulation of inflammatory diseases by modulating ADAM17 activity.

Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17.

Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.