Grace Ju

Conversion of the interleukin 1 receptor antagonist into an agonist by site-specific mutagenesis.

Interleukin 1 (IL-1) receptor antagonist (IL-1ra) is a naturally occurring protein that binds to the IL-1 receptor present on T cells, fibroblasts, and other cell types and acts to block IL-1-induced responses. IL-1ra is a pure antagonist and has no agonist activity in in vitro or in vivo systems. By site-specific mutagenesis, an analog of IL-1ra was created that contained a substitution of a single amino acid, Lys-145----Asp. This analog, IL-1ra K145D, exhibited partial agonist activity in the D10.G4.1 cell proliferation assay. The newly acquired agonist activity could not be neutralized by antisera to IL-1 alpha or IL-1 beta, but it could be blocked by a monoclonal antibody to the T-cell IL-1 receptor. The analog also showed agonist activity as assayed by increased prostaglandin E2 synthesis from CHO cells expressing recombinant mouse IL-1 receptor. These results with IL-1ra K145D demonstrate the importance of the region surrounding the corresponding Asp-145 residue in IL-1 beta for triggering the biological response to IL-1.

Functional analysis of the transcription control region located within the avian retroviral long terminal repeat.

We used several quantitative assays of in vivo transient gene expression to dissect the elements within the Rous sarcoma virus long terminal repeat (LTR) which constitute the retroviral transcription control region. Site-directed deletion mutagenesis was used to locate and define the enhancer and promoter elements within the LTR. In addition, we inserted exogenous DNA fragments into the LTR to examine the effects of position and sequence on the activity of these LTR transcriptional elements. The Rous sarcoma virus enhancer element, which we propose is located entirely within the LTR, was shown to activate both the beta-globin and retroviral LTR promoters when located in cis. We observed a striking correlation between the degree of activation and the distance between the retroviral promoter and enhancer elements. The LTR promoter element mediated the activation effect of the enhancer element, as LTR deletion mutants containing only the enhancer and TATA box region expressed little activity. The promoter region encoded a low but significant level of transcriptional activity even in the absence of an enhancer. Overall LTR transcriptional activity declined sharply with increasing distance between the LTR promoter and initiator elements. These results shed light on both the importance of the spatial arrangement of the sequence elements within this eucaryotic transcription control region and on the functional interrelationship between these elements.

NMR characterization of interleukin-2 in complexes with the IL-2Rα receptor component, and with low molecular weight compounds that inhibit the IL-2/IL-Rα interaction

Nuclear magnetic resonance (NMR) methods were employed to study the interaction of the cytokine Interleukin‐2 (IL‐2) with the α‐subunit of its receptor (IL‐2Rα), and to help understand the behavior of small molecule inhibitors of this interaction. Heteronuclear 1H‐15N HSQC experiments were used to identify the interaction surface of 15N‐enriched Interleukin‐2 (15N‐IL‐2) in complex with human IL‐2Rα. In these experiments, chemical shift and line width changes in the heteronuclear single‐quantum coherence (HSQC) spectra upon binding of 15N‐IL‐2 enabled classification of NH atoms as either near to, or far from, the IL‐2Rα interaction surface. These data were complemented by hydrogen/deuterium (H/D) exchange measurements, which illustrated enhanced protection of slowly‐exchanging IL‐2 NH protons near the site of interaction with IL‐2Rα. The interaction surface defined by NMR compared well with the IL‐2Rα binding site identified previously using mutagenesis of human and murine IL‐2. Two low molecular weight inhibitors of the IL‐2/IL‐2Rα interaction were studied: one (a cyclic peptide derivative) was found to mimic a part of the cytokine and bind to IL‐2Rα; the other (an acylphenylalanine derivative) was found to bind to IL‐2. For the interaction between IL‐2 and the acylphenylalanine, chemical shift perturbations of 15N and 15NH backbone resonances were tracked as a function of ligand concentration. The perturbation pattern observed for this complex revealed that the acylphenylalanine is a competitive inhibitor—it binds to the same site on IL‐2 that interacts with IL‐2Rα.

Insertion of a Structural Domain of Interleukin (IL)-1β Confers Agonist Activity to the IL-1 Receptor Antagonist IMPLICATIONS FOR IL-1 BIOACTIVITY

We showed previously that replacement of Lys-145 in the IL-1 receptor antagonist (IL-1ra) with Asp resulted in an analog (IL-1ra K145D) with partial agonist activity. To identify additional amino acids that affect IL-1 bioactivity, we created second site mutations in IL-1ra K145D. Substitutions of single amino acids surrounding position 145 were made; none of these substitutions increased the bioactivity of IL-1ra K145D. However, the insertion of the β-bulge (QGEESN) of IL-1β at the corresponding region of IL-1ra K145D resulted in a 3-4-fold augmentation of bioactivity. An additional increase in agonist activity was observed when the β-bulge was coexpressed with a second substitution (His-54 Pro) in IL-1ra K145D. We also show that the bioactivity of both IL-1ra K145D and the triple mutant IL-1ra K145D/H54P/QGEESN is dependent on interaction with the newly cloned IL-1 receptor accessory protein.

KU812 cells provide a novel in vitro model of the human IL-33/ST2L axis: Functional responses and identification of signaling pathways

Activation of interleukin-1 family receptor ST2L by its ligand interleukin-33 (IL-33) is an important component in inflammatory responses. Peripheral blood basophils, recognized as major effector cells in allergic inflammation that play a role in both innate and adaptive immunity, are activated by IL-33 through ST2L. However, studies are challenging due to the paucity of this cell population, representing less than 1% of peripheral blood leukocytes. We identified a basophil-like chronic myelogenous leukemia cell line, KU812, that constitutively expresses ST2L and demonstrates functional responses to IL-33 stimulation. IL-33 induced production of multiple inflammatory mediators in KU812 cells that were blocked by anti-ST2L and anti-IL-33 antibodies. The interaction of IL-33 and ST2L activated NF-kappaB, JNK, and p38 MAPK, but not ERK1/2 signaling pathways. Studies using pharmacological inhibitors to IKK-2 and MAP kinases revealed that one of the functional responses, IL-33-induced IL-13 production, was regulated through NF-kappaB, but not JNK or p38 MAPK signaling. The requirement of NF-kappaB was confirmed by IKK-2 knockdown using shRNA. KU812 represents the first human cell line-based in vitro model of the IL-33/ST2L axis and provides a valuable tool to aid in understanding the mechanism and significance of IL-33 and ST2L interaction and function.

Induction of lymphokine-activated killing with reduced secretion of interleukin-1β, tumor necrosis factor-α, and interferon-γ by interleukin-2 analogs

AbstractBackground: Interleukin-2 (IL-2)-based immunotherapy has been shown to effect clinical responses in 15–35% of patients with metastatic renal cell carcinoma or melanoma. Despite its clinical efficacy, many clinicians refrain from using IL-2 because of the associated toxicity. This toxicity is believed to be mediated by such secondary cytokines as IL-1, tumor necrosis factor (TNF), and interferon (IFN)-γ, which are produced by the patients IL-2-stimulated peripheral blood mononuclear cells (PBMCs). Methods: Human PBMCs were stimulated with 1 nM wild-type recombinant IL-2 (rIL-2) or IL-2 analogs (R38A or F42K) that preferentially bind to the intermediate affinity IL-2 receptor (IL-2R). PBMCs were activated for lymphokine-activated killer (LAK) activity in 4-h51Cr-release assays, using Daudi target cells. Cytokine content in the culture supernatants was determined by enzyme-linked immunosorbent assay. Results: Both R38A and F42K were capable of generating substantial LAK activity. Maximal specific lysis was 54% for PBMCs activated by R38A and 52% for F42K-stimulated cells, in contrast to 64% for rIL-2. In addition, analog-stimulated PBMCs secreted 59% of the IL-1β, 25% of the TNF-α, and only 8% of the IFN-γ produced in response to rIL-2 (allp<0.01 compared with rIL-2-stimulated secretion; one-way ANOVA). Conclusions: IL-2 analogs that preferentially bind the intermediate-affinity IL-2R retain the capacity to induce substantial LAK activity despite a greatly reduced secondary cytokine production. Therefore, such IL-2 analogs may provide an effective, yet less toxic means of cancer immunotherapy.

The Role of Avian Retroviral Ltrs in the Regulation of Gene Expression and Viral Replication

Publisher Summary This chapter discusses the structure and role of the long terminal repeats (LTRs) of the avian sarcoma-leukosis viruses (ASV-ALV). Many of the characteristics of the ASV-ALVs are typical of all retroviruses. Retroviruses comprise a diverse group of viruses which nevertheless share an unusual set of genetic and biological features. These include a unique strategy for replication, the ability to induce neoplasia by subverting cellular genes, and an intimate association and dependence on the host cell. The latter two properties reflect the ability of these viruses to direct the stable integration into the host chromatin of a DNA copy of their RNA genome (the provirus). Molecular analyses of retroviral DNA intermediates have established the presence of a DNA element repeated at both ends of the provirus. This important structure is designated as the long terminal repeat or LTR. The functional attributes of the LTRs are discussed in the context of the expression of both viral and cellular genes. Finally, the implications for pathology and the possible applications for directed gene expression in heterologous systems are also discussed in the chapter.

Pyrazolobenzodiazepines: Part I. Synthesis and SAR of a potent class of kinase inhibitors

A novel series of pyrazolobenzodiazepines 3 has been identified as potent inhibitors of cyclin-dependent kinase 2 (CDK2). Their synthesis and structure-activity relationships (SAR) are described. Representative compounds from this class reversibly inhibit CDK2 activity in vitro, and block cell cycle progression in human tumor cell lines. Further exploration has revealed that this class of compounds inhibits several kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. Together, these properties suggest a compelling basis for their use as antitumor agents.

Discovery of a Highly Potent, Orally Active Mitosis/Angiogenesis Inhibitor R1530 for the Treatment of Solid Tumors

A new series of 7,8-disubstituted pyrazolobenzodiazepines based on the lead compound 1 have been synthesized and evaluated for their effects on mitosis and angiogenesis. Described herein is the design, synthesis, SAR, and antitumor activity of these compounds leading to the identification of R1530, which was selected for clinical evaluation.

Expression of transfected DNA in avian cells can be enhanced in trans by retroviral infection.

Using a quantitative S1 nuclease protection assay, we demonstrated that acute or chronic infection of avian cells enhances expression of an exogenously introduced rat preproinsulin II gene by approximately equal to 50-fold. The degree of enhancement is shown to vary with the transfection technique used but is independent of the transcription control region of the transfected gene. We conclude that retroviral infection of avian cells enhances expression of transfected DNA in trans by facilitating the uptake of DNA rather than by activating the transfected promoter.