Christopher D. Krause

Interferons, interferon-like cytokines, and their receptors

Summary:  Recombinant interferon‐α (IFN‐α) was approved by regulatory agencies in many countries in 1986. As the first biotherapeutic approved, IFN‐α paved the way for the development of many other cytokines and growth factors. Nevertheless, understanding the functions of the multitude of human IFNs and IFN‐like cytokines has just touched the surface. This review summarizes the history of the purification of human IFNs and the key aspects of our current state of knowledge of human IFN genes, proteins, and receptors. All the known IFNs and IFN‐like cytokines are described [IFN‐α, IFN‐β, IFN‐ε, IFN‐κ, IFN‐ω, IFN‐δ, IFN‐τ, IFN‐γ, limitin, interleukin‐28A (IL‐28A), IL‐28B, and IL‐29] as well as their receptors and signal transduction pathways. The biological activities and clinical applications of the proteins are discussed. An extensive section on the evolution of these molecules provides some new insights into the development of these proteins as major elements of innate immunity. The overall structure of the IFNs is put into perspective in relation to their receptors and functions.

Identification and functional characterization of a second chain of the interleukin-10 receptor complex.

Interleukin‐10 (IL‐10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL‐10Rα or IL‐10R1), was identified previously. We report here that, although human IL‐10 binds to the human IL‐10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co‐expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL‐10R1 chain renders hamster cells sensitive to IL‐10. The IL‐10:CRFB4 complex was detected by cross‐linking to labeled IL‐10. In addition, the IL‐10R1 chain was able to be co‐immunoprecipitated with anti‐CRF antibody when peripheral blood mononuclear cells were treated with IL‐10. These results demonstrate that the CRFB4 chain is part of the IL‐10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL‐10R2 or IL‐10Rβ chain, serves as an accessory chain essential for the active IL‐10 receptor complex and to initiate IL‐10‐induced signal transduction events.

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 Å resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.

Partial Interferon-γ Receptor Signaling Chain Deficiency in a Patient with Bacille Calmette-Guérin and Mycobacterium abscessus Infection

Complete deficiency of either of the two human interferon (IFN)-gamma receptor components, the ligand-binding IFN-gammaR1 chain and the signaling IFN-gammaR2 chain, is invariably associated with early-onset infection caused by bacille Calmette-Guérin vaccines and/or environmental nontuberculous mycobacteria, poor granuloma formation, and a fatal outcome in childhood. Partial IFN-gammaR1 deficiency is associated with a milder histopathologic and clinical phenotype. Cells from a 20-year-old healthy person with a history of curable infections due to bacille Calmette-Guérin and Mycobacterium abscessus and mature granulomas in childhood were investigated. There was a homozygous nucleotide substitution in IFNGR2, causing an amino acid substitution in the extracellular region of the encoded receptor. Cell surface IFN-gammaR2 were detected by flow cytometry. Cellular responses to IFN-gamma were impaired but not abolished. Transfection with the wild-type IFNGR2 gene restored full responsiveness to IFN-gamma. This is the first demonstration of partial IFN-gammaR2 deficiency in humans.

Interferon-γ receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death

Theheterodimeric interferon (IFN)‐γ receptor (IFN‐γR) is formed of two chains. Here we show that the binding chain (IFN‐γR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN‐γR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN‐γR2 determined the number of receptor complexes that transduced the IFN‐γ signal and resulted in a different response to IFN‐γ. After IFN‐γ stimulation, high IFN‐γR2 membrane expression induced rapid activation of signal transducer and activator of transcription‐1 (STAT‐1) and high levels of interferon regulatory factor‐1 (IRF‐1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT‐1, lower levels of IRF‐1, and induction of proliferation. Because the forced expression of IFN‐γR2 on T cells switched their response to IFN‐γ from proliferative to apoptotic, we concluded that the surface expression of IFN‐γR2 determines whether a cell stimulated by IFN‐γ undergoes proliferation or apoptosis.

Seeing the light: Preassembly and ligand-induced changes of the interferon Gamma receptor complex in cells

Our experiments were designed to test the hypothesis that the cell surface interferon γ receptor chains are preassembled rather than associated by ligand and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer, a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation, and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand interferon γ. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

Chaperone Hsp27, a Novel Subunit of AUF1 Protein Complexes, Functions in AU-Rich Element-Mediated mRNA Decay

ABSTRACT Controlled, transient cytokine production by monocytes depends heavily upon rapid mRNA degradation, conferred by 3′ untranslated region-localized AU-rich elements (AREs) that associate with RNA-binding proteins. The ARE-binding protein AUF1 forms a complex with cap-dependent translation initiation factors and heat shock proteins to attract the mRNA degradation machinery. We refer to this protein assembly as the AUF1- and signal transduction-regulated complex, ASTRC. Rapid degradation of ARE-bearing mRNAs (ARE-mRNAs) requires ubiquitination of AUF1 and its destruction by proteasomes. Activation of monocytes by adhesion to capillary endothelium at sites of tissue damage and subsequent proinflammatory cytokine induction are prominent features of inflammation, and ARE-mRNA stabilization plays a critical role in the induction process. Here, we demonstrate activation-induced subunit rearrangements within ASTRC and identify chaperone Hsp27 as a novel subunit that is itself an ARE-binding protein essential for rapid ARE-mRNA degradation. As Hsp27 has well-characterized roles in protein ubiquitination as well as in adhesion-induced cytoskeletal remodeling and cell motility, its association with ASTRC may provide a sensing mechanism to couple proinflammatory cytokine induction with monocyte adhesion and motility.

Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation.

ABSTRACT AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.

Preassembly and ligand-induced restructuring of the chains of the IFN-gamma receptor complex: the roles of Jak kinases, Stat1 and the receptor chains.

We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-γ) receptor complex is preassembled 1. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-γR1 results in a conformational change localized to IFN-γR1. Jak1 but not Jak2 is required for the two chains of the IFN-γ receptor complex (IFN-γR1 and IFN-γR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Signaling by Covalent Heterodimers of Interferon-γ EVIDENCE FOR ONE-SIDED SIGNALING IN THE ACTIVE TETRAMERIC RECEPTOR COMPLEX

Interferon-γ (IFN-γ) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-γ that bind only one IFN-γR1 chain per dimer molecule (called a monovalent IFN-γ) to see if the interaction of IFN-γ with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-γ yielded two monovalent IFN-γs, γm-γ and γ-γm, which cross-link to only a single soluble IFN-γR1 molecule in solution and on the cell surface. Monovalent IFN-γ competes fully with wild type IFN-γ for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-γ. Monovalent IFN-γ had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1α produced by monovalent IFN-γs after 15 min were never more than half of those produced by either wild type or covalent IFN-γs in human cell lines. These data indicate that while monovalent IFN-γ activates only one-half of a four-chain receptor complex, this is sufficient for Stat1α activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-γ and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.