Ligand-induced Assembling of the Type I Interferon Receptor on Supported Lipid Bilayers

Lamken, Peter; Lata, Suman; Gavutis, Martynas; Piehler, Jacob

doi:10.1016/j.jmb.2004.05.059

Cited by 115 publications

(173 citation statements)

References 47 publications

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“…The peak corresponding to YNS was occluded by either receptor separately (orange and red lines), as well as by both receptors together (dotted black line), while forming higher M r heterodimers and ternary complexes, respectively. It should be noted that the retention times of IFNAR1-EC and its complexes are shorter than expected from their calculated molecular mass (R1, 47 kDa; R2, 24.5 kDa; IFN, 18.5 kDa; R2 ϩ IFN, 43 kDa), due to glycosylation of the protein during production (22). The indicated receptor binding and ternary complex formation for YNS is possible due to its higher affinity to IFNAR1-EC, whereas occlusion of IFN␣2 WT by IFNAR1 was not detectable in this type of assay.…”

Section: Screening a Phage-displayed Ifn␣2 Library For Tight Ifnar1mentioning

confidence: 86%

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An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Kalie¹,

Jaitin²,

Abramovich

et al. 2007

Journal of Biological Chemistry

122

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All ␣-interferons (IFN␣) bind the IFNAR1 receptor subunit with low affinity. Increasing the binding affinity was shown to specifically increase the antiproliferative potency of IFN␣2. Here, we constructed a phage display library by randomizing three positions on IFN␣2 previously shown to confer weak binding to IFNAR1. The tightest binding variant selected, comprised of mutations H57Y, E58N, and Q61S (YNS), was shown to bind IFNAR1 60-fold tighter compared with wild-type IFN␣2, and 3-fold tighter compared with IFN␤. Binding of YNS to IFNAR2 was comparable with wild-type IFN␣2. The YNS mutant conferred a 150-fold higher antiproliferative potency in WISH cells compared with wild-type IFN␣2, whereas its antiviral activity was increased by only 3.5-fold. The high antiproliferative activity was related to an induction of apoptosis, as demonstrated by annexin V binding assays, and to specific gene induction, particularly TRAIL. To determine the potency of the YNS mutant in a xenograft cancer model, we injected it twice a week to nude mice carrying transplanted MDA231 human breast cancer cells. After 5 weeks, no tumors remained in mice treated with YNS, whereas most mice treated with wild-type IFN␣2 showed visible tumors. Histological analysis of these tumors showed a significant antiangiogenic effect of YNS, compared with wild-type IFN␣2. This work demonstrates the application of detailed biophysical understanding in the process of protein engineering, yielding an interferon variant with highly increased biological potency. Type I interferons (IFNs)3 are multifunctional cytokines that orchestrate the antiviral innate immunity in vertebrates, acting in virtually every nucleated cell. They belong to the helical cytokine superfamily (1), and consist of at least 17 different members in humans: 13 different IFN␣ subtypes, IFN␤, IFN, and IFN, all of which share significant amino acid homology (2).All type I interferons induce their biological effects through a common receptor, composed of two distinct transmembrane proteins, namely IFNAR1 and IFNAR2 (3-5). Upon formation of the ternary complex, the interferon signal is transduced through receptor-associated Janus family kinases (JAK), which activate the signal transducers and activators of transcription (STAT) proteins. These, in turn, form homo-and heterodimers that translocate to the nucleus, where they directly regulate the transcription of specific interferon responsive genes (6).In addition to their important roles at the first line of defense against viral infections (7) and in modulation of the adaptive immune system (8), type I interferons are capable of inducing a significant antiproliferative activity (9). Consequently, interferons are widely used in the clinic for the treatment of multiple sclerosis and chronic hepatitis (10, 11), as well as for a number of malignancies (12). However, the efficiency of interferon treatment in the majority of cancers is modest, limited by side effects and varying resistance of malignant cells to this treatment (13,14).Despite their...

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Section: Screening a Phage-displayed Ifn␣2 Library For Tight Ifnar1mentioning

confidence: 86%

“…IFNAR1-EC was expressed in Sf9 insect cells and purified as described before (22), and was a generous gift from Jacob Piehler.…”

mentioning

confidence: 99%

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Kalie¹,

Jaitin²,

Abramovich

et al. 2007

Journal of Biological Chemistry

122

View full text Add to dashboard Cite

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“…No expression of mRNA encoding a cell-associated IFN-lR1 variant with a truncated (23) 100 (46) 0.6 -5 (45)(46)(47)(48)(49) > 0.4 (45,46,48,49) + + + + + + 10 µg/ml À1 IFN-l1 and 500 U ml À1 IFN-b1, which had been preincubated for 2 h with 0, 1 and 10 mg ml À1 sIFNlR1-Fc. Major histocompatibility complex class I (MHC I) cellsurface expression was measured by flow cytometry.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, the K D value of 73 nM indicated an approximately 100 times lower binding than that for the interaction of IL-22 with its potently inhibiting soluble receptor IL-22BP, as previously determined by our laboratory. 43 Moreover, the sIFN-lR1-Fc-IFN-l1 binding had a similar (IFN-a1) or lower (IFN-a2, IFN-b) affinity compared with the binding of the extracellular domain of the R1-type IFN receptor subunit IFN-aRB to the respective type I IFN [44][45][46][47][48] ( Figure 7a). Our yet preliminary data also indicated that the affinity of sIFN-lR1-Fc to IFN-l1 was similar to the affinity to IFN-l2.…”

Section: Target Cells Of Type III Interferons K Witte Et Almentioning

confidence: 97%

Despite IFN-λ receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type III interferons: implications for therapeutic applications of these cytokines

et al. 2009

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Interferon (IFN)-l1, -2 and -3 (also designated as interleukin (IL)-29, IL-28a and IL-28b) represent a new subfamily within the class II cytokine family. They show type I IFN-like antiviral and cytostatic activities in affected cells forming the basis for IFN-l1 therapy currently under development for hepatitis C infection. However, many aspects of IFN-ls are still unknown. This study aimed at identifying the target cells of IFN-ls within the immune system and the skin. Among skin cell populations, keratinocytes and melanocytes, but not fibroblasts, endothelial cells or subcutaneous adipocytes turned out to be targets. In contrast to these target cells, blood immune cell populations did not clearly respond to even high concentrations of these cytokines, despite an IFN-l receptor expression. Interestingly, immune cells expressed high levels of a short IFN-l receptor splice variant (sIFN-lR1/sIL-28R1). Its characterization revealed a secreted, glycosylated protein that binds IFN-l1 with a moderate affinity (K D 73 nM) and was able to inhibit IFN-l1 effects. Our study suggests that IFN-l therapy should be suited for patients with verrucae, melanomas and non-melanoma skin cancers, apart from patients with viral hepatitis, and would not be accompanied by immune-mediated complications known from type I IFN application.

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“…Our method is similar to the previously-described simultaneous display of receptor subunits on sensor chips coated with lipid bilayers. Using this latter method, interactions have been evaluated kinetically for interferon and receptor subunits simultaneously anchored through fused His tags to exposed lipid head groups containing immobilized metal chelate [36] [37].…”

Section: Development Of Biosensor Surface To Investigate Receptor Assmentioning

confidence: 99%

Slow-dissociation effect of common signaling subunit β on IL5 and GM-CSF receptor assembly

et al. 2008

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Receptor activation by IL5 and GM-CSF is a sequential process that depends on their interaction with a cytokine-specific subunit α and recruitment of a common signaling subunit β (βc). In order to elucidate the assembly dynamics of these receptor subunits, we performed kinetic interaction analysis of the cytokine-receptor complex formation by a surface plasmon resonance biosensor. Using the extracellular domains of receptor fused with C-terminal V5-tag, we developed an assay method to co-anchor α and βc subunits on the biosensor surface. We demonstrated that dissociation of the cytokine-receptor complexes was slower when both subunits were co-anchored on the biosensor surface than when α subunit alone was anchored. The slow-dissociation effect of βc had a greater impact on GM-CSF receptor stabilization than that of IL5. The effects were abolished by alanine replacement of either Tyr18 or Tyr344 residue in βc, which together constitute key parts of a cytokine binding epitope. The data argue that βc plays an important role in preventing the ligandreceptor complexes from rapidly dissociating. This slow-dissociation effect of βc explains how, when multiple βc cytokine receptor α subunits are present on the same cell surface, selective βc usage can be controlled by sequestration in stabilized cytokine-α-βc complexes.

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Ligand-induced Assembling of the Type I Interferon Receptor on Supported Lipid Bilayers

Cited by 115 publications

References 47 publications

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

An Interferon α2 Mutant Optimized by Phage Display for IFNAR1 Binding Confers Specifically Enhanced Antitumor Activities

Despite IFN-λ receptor expression, blood immune cells, but not keratinocytes or melanocytes, have an impaired response to type III interferons: implications for therapeutic applications of these cytokines

Slow-dissociation effect of common signaling subunit β on IL5 and GM-CSF receptor assembly

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