Intron loss in interferon genes follows a distinct set of stages, and may confer an evolutionary advantage.

The promoter-intron-exon structure of genes evolve. While the structures of some IFN genes (e.g., piscine and amphibian Type I IFNs, most tetrapod IFN-λ genes) resemble those of other class II cytokines (e.g., interleukins-10, 19, 20, 22, 24, 26), the structures of other IFN genes differ significantly. Although all bony vertebrate IFN-γ genes lack the canonical third intron, and all amniote Type I IFN genes lack introns, only some IFN-λ genes lost their introns. Interestingly, these intronless IFN-λ genes are not preferentially related to one another nor are they clustered with canonical multi-intron IFN-λ genes. Hypothesizing that intronless IFN-λ genes repeatedly and independently evolved and transposed throughout the genome, we sought to understand the genetic processes involved in their intron loss and genomic migration. Utilizing the high conservation of the promoters, the UTRs and the ORFs of the IFN-λ genes, we collected data from two families of intronless IFN-λ genes, and developed a model supported by these data to explain how intronless IFN-λ genes evolved. (1) A cytoplasmic IFN-λ cDNA generated by reverse transcriptional activity enters the nucleus and attempts to recombine with its multi-exon progenitor. (2) Nuclear DNA synthesis at the 5' and 3' ends within recombination intermediates affixes the promoter onto the cDNA and preserves its 3' UTR. (3) Resolution of the recombination complex releases the promoter-associated cDNA. (4) The released intronless gene co-integrates with a highly duplicated sequence undergoing transposition. We propose that this process explains not only the evolution of the gene structure of IFN genes, but also the increased transposition of intronless genes in genomes, and may confer an evolutionary advantage.

Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes.

Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent's location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.

Analytical use of multi-protein Fluorescence Resonance Energy Transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes.

Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-γR1, IL-10R1 and IFN-λR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-λR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-γR1 and IFN-γR2 or between IFN-αR1 and IFN-αR2c increases, FRET among other pairs of proteins changes within each complex.

Improving the spectral analysis of Fluorescence Resonance Energy Transfer in live cells: application to interferon receptors and Janus kinases.

The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.

Ligand-independent interaction of the type I interferon receptor complex is necessary to observe its biological activity.

Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-αR1 and IFN-αR2c, or IFN-λR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-αR1 and IFN-αR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-αR1 and IFN-αR2c were sensitive to IFN-alpha only when IFN-αR1 and IFN-αR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-αR1 and IFN-αR2c. Both IFN-αR1 and IFN-αR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.

Historical developments in the research of interferon receptors.

Interferons (IFNs) were discovered 50 years ago independently by Isaacs and Lindemann and by Nagata and Kojima. When it was later realized that IFNs are active at very low concentrations, research began to determine how their powerful effects were generated from such a small initial signal. It has since been established that interferons, as well as all other cytokines, employ cell surface receptors to translate their presence in the serum to a potent cellular response to a viral infection. These receptor complexes are composed of multiple distinct glycosylated transmembrane polypeptides, a number of protein tyrosine kinases, and interact transiently with a large variety of other proteins including transcription factors, phosphatases, signaling repressors, and adaptor proteins coupling the receptor to alternative signaling pathways. Three major receptor complexes exist that are exclusive to each of three major classes of interferon. Even though the effects of each major class of interferon vary physiologically, each receptor complex interacts with its ligand in similar ways and activates similar signaling cascades. In this mini-review, we take a historical perspective at the major events in the characterization of interferon receptors, discussing interesting results that still need to be explained.

Protein arginine methyltransferases: evolution and assessment of their pharmacological and therapeutic potential.

Protein arginine N-methylation is a post-translational modification whose influence on cell function is becoming widely appreciated. Protein arginine methyltransferases (PRMT) catalyze the methylation of terminal nitrogen atoms of guanidinium side chains within arginine residues of proteins. Recently, several new members of the PRMT family have been cloned and their catalytic function determined. In this report, we present a review and phylogenetic analysis of the PRMT found so far in genomes. PRMT are found in nearly all groups of eukaryotes. Many human PRMT originated early in eukaryote evolution. Homologs of PRMT1 and PRMT5 are found in nearly every eukaryote studied. The gene structure of PRMT vary: most introns appear to be inserted randomly into the open reading frame. The change in catalytic specificity of some PRMT occurred with changes in the arginine binding pocket within the active site. Because of the high degree of conservation of sequence among the family throughout evolution, creation of specific PRMT inhibitors in pathogenic organisms may be difficult, but could be very effective if developed. Furthermore, because of the intricate involvement of several PRMT in cellular physiology, their inhibition may be fraught with unwanted side effects. Nevertheless, development of pharmaceutical agents to control PRMT functions could lead to significant new targets.

Evolution of the Class 2 cytokines and receptors, and discovery of new friends and relatives.

The sequencing of a wide variety of genomes and their transcripts has allowed researchers to determine how proteins or protein families evolved and how strongly during evolution a protein has been conserved. In this report, we analyze the evolution of the Class 2 ligands and their cognate receptors by analyzing Class 2 ligand and receptor chain gene sequences from a variety of DNA sequence databases. Both the Class 2 cytokines and receptor chains appear to have developed during the evolution of the chordate phyla: distant homologues of type I interferon (IFN) receptors are the only Class 2 cytokine receptors identified in the Ciona genomes, while a wide variety of Class 2 ligands and receptor chains are encoded in the currently available genomes of bony vertebrates (teleost fish, amphibians, reptiles, birds, mammals). Phylogenetic trees of ligands and ligand-binding receptor chains demonstrate that proteins involved in conferring antiviral activity diverged before those involved in adaptive immunity. Genes encoding IFNs and IFN receptors duplicated multiple times during chordate evolution, suggesting that duplication of genes encoding IFN activity conveyed an evolutionary advantage. Altogether, these data support a model whereby the original Class 2 cytokines and receptors evolved and duplicated during the evolution of the chordate innate immune response system; new receptor and ligand duplications evolved into signaling molecules to fulfill communication requirements of a highly specialized and differentiated vertebrate immune system. In addition, the genomic analysis led to the discovery of some new members of this family.

Modulation of the activation of Stat1 by the interferon-gamma receptor complex.

The activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex is responsible for the transcription of a significant portion of IFN-gamma induced genes. Many of these genes are responsible for the induction of an apoptotic state in response to IFN-gamma. In the absence of Stat1 activation, IFN-gamma instead induces a proliferative response. Modifying Stat1 activation by IFN-gamma may have pharmacological benefits. We report that the rate of activation of Stat1 can be altered in HeLa cells by overexpressing either the IFN-gammaR1 chain or the IFN-gammaR2 chain. These alterations occur in hematopoietic cell lines: Raji cells and monocytic cell lines, which have average and above-average IFN-gammaR2 surface expression, activate Stat1 similarly to HeLa cells and HeLa cells overexpressing IFNgammaR2, respectively. The rapid Stat1 activation seen in HeLa cells can be inhibited by overexpressing a chimeric IFN-gammaR2 chain that does not bind Jak2 or (when high concentrations of IFN-gamma are used) by overexpressing IFN-gammaR1. These data are consistent with a model in which the recruitment of additional Jak2 activity to a signaling complex accelerates the rate of Stat1 activation. We conclude that the rate of activation of Stat1 in cells by IFN-gamma can be modified by regulating either receptor chain and speculate that pharmacological agents which modify receptor chain expression may alter IFN-gamma receptor signal transduction.

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-gamma) 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-gammaR1 results in a conformational change localized to IFN-gammaR1. Jak1 but not Jak2 is required for the two chains of the IFN-gamma receptor complex (IFN-gammaR1 and IFN-gammaR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-gammaR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-gammaR2 with IFN-gammaR1. These results agree with a detailed model of the IFN-gamma receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.

Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid.

INTRODUCTION: Local synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably. METHODS: To follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5. RESULTS: Murine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth. CONCLUSIONS: Our modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.

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

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--Ser(15), Ser(78), and Ser(82)-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.

Chaperone Hsp27, a novel subunit of AUF1 protein complexes, functions in AU-rich element-mediated mRNA decay.

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.

FBXO11/PRMT9, a new protein arginine methyltransferase, symmetrically dimethylates arginine residues.

We have identified a protein, FLJ12673 or FBXO11, that contains domains characteristically present in protein arginine methyltransferases (PRMTs). Immuno-purified protein expressed from one of the four splice variants in HeLa cells and in Escherichia coli exhibited methyltransferase activity. Monomethylarginine, symmetric, and asymmetric dimethylarginine (SDMA, ADMA) were formed on arginine residues. Accordingly, we have designated the protein PRMT9. PRMT9 is the third member of the PRMT family that forms SDMA modifications in proteins. Structurally, this protein is distinct from all other known PRMTs implying that convergent evolution allowed this protein to develop the ability to methylate arginine residues and evolved elements conserved in PRMTs to accomplish this.

Interactions among the components of the interleukin-10 receptor complex.

We used fluorescence resonance energy transfer previously to show that the interferon-gamma (IFN-gamma) receptor complex is a preformed entity mediated by constitutive interactions between the IFN-gammaR2 and IFN-gammaR1 chains, and that this preassembled entity changes its structure after the treatment of cells with IFN-gamma. We applied this technique to determine the structure of the interleukin-10 (IL-10) receptor complex and whether it undergoes a similar conformational change after treatment of cells with IL-10. We report that, like the IFN-gamma receptor complex, the IL-10 receptor complex is preassembled: constitutive but weaker interactions occur between the IL-10R1 and IL-10R2 chains, and between two IL-10R2 chains. The IL-10 receptor complex undergoes no major conformational changes when cells are treated with cellular or Epstein-Barr viral IL-10. Receptor complex preassembly may be an inherent feature of Class 2 cytokine receptor complexes.

Interferons, interferon-like cytokines, and their receptors.

Recombinant interferon-alpha (IFN-alpha) was approved by regulatory agencies in many countries in 1986. As the first biotherapeutic approved, IFN-alpha 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-alpha, IFN-beta, IFN-epsilon, IFN-kappa, IFN-omega, IFN-delta, IFN-tau, IFN-gamma, 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.

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 gamma 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 gamma. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

Interleukin-10 and related cytokines and receptors.

The Class 2 alpha-helical cytokines consist of interleukin-10 (IL-10), IL-19, IL-20, IL-22, IL-24 (Mda-7), and IL-26, interferons (IFN-alpha, -beta, -epsilon, -kappa, -omega, -delta, -tau, and -gamma) and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29). The interaction of these cytokines with their specific receptor molecules initiates a broad and varied array of signals that induce cellular antiviral states, modulate inflammatory responses, inhibit or stimulate cell growth, produce or inhibit apoptosis, and affect many immune mechanisms. The information derived from crystal structures and molecular evolution has led to progress in the analysis of the molecular mechanisms initiating their biological activities. These cytokines have significant roles in a variety of pathophysiological processes as well as in regulation of the immune system. Further investigation of these critical intercellular signaling molecules will provide important information to enable these proteins to be used more extensively in therapy for a variety of diseases.