Binding Characteristics of IFN-<i>α</i>Subvariants to IFNAR2-EC and Influence of the 6-Histidine Tag

Schmeisser, Hana; Kontsek, Peter; Esposito, Dominic; Gillette, William; Schreiber, Gideon; Zoon, Kathryn C.

doi:10.1089/jir.2006.26.866

Cited by 20 publications

(24 citation statements)

References 39 publications

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“…Expression and purification of IFN proteins were previously described, 17,21 purity evaluated by SDS-PAGE, and concentrations determined using Bradford protein quantification assay (Pierce) and absorbance at 280 nm (Nanodrop spectrophotometer, ThermoScientific). Biologic activities of IFN were tested in a cell line assay as described.…”

Section: Ifn and Ifn Constructsmentioning

confidence: 99%

“…Nondenaturing immobilized metal affinity chromatography (IMAC) purification from culture supernatant, dialysis and sample concentration and SDS-PAGE for quality control was performed. 21 …”

Section: Ifnar2-ecmentioning

confidence: 99%

“…Samples were analyzed by SDS-PAGE followed by Western blot as described. 21 Antibodies for STAT1 and pSTAT1 (Y701) were obtained from BDTransduction Lab and Cell Signaling Technology. Antibody for STAT2 was obtained from Upstate Biotechnology.…”

Section: Western Blotsmentioning

confidence: 99%

“…Biologic activities of IFN were tested in a cell line assay as described. 21 IFN hybrids and mutant constructs containing amino-terminal from parental IFN␣21b and the carboxy-terminal from parental IFN␣2c ( Figure 1A . All IFN constructs were tested for endotoxin levels (EU/mL Ͻ 1 at 0.6mg/mL).…”

mentioning

confidence: 99%

“…21 Components of parental IFN␣21b and IFN␣2c ( Figure 1A), which both suppress R5 HIV with only a single treatment after the macrophages have been exposed to virus ( Figure 1B, day 10 p24 levels shown), similar to recombinant IFN␣2a, 8,9 were compared with engineered hybrid molecules generated by combining the most active components of these 2 IFN molecules ( Figure 1A-C). 21 One hybrid molecule (HY1), containing N-terminal of IFN␣21b combined with amino acids 75-165 that include the C-helix (amino acids 75-95) of IFN␣2c, was most active at inhibiting HIV infection of macrophages ( Figure 1C). By comparison, HY2 containing N-terminal and C-helix regions of IFN␣21b and COOH-terminus of IFN␣2c, while inhibitory at 0.1-1 ng/mL, was less active than parental IFN or the other hybrids at 0.01 ng/mL ( Figure 1C).…”

mentioning

confidence: 99%

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Structural variants of IFNα preferentially promote antiviral functions

Vázquez

Schmeisser

Dolan

et al. 2011

Blood

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IFN␣, a cytokine with multiple functions in innate and adaptive immunity and a potent inhibitor of HIV, exerts antiviral activity, in part, by enhancing apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family members. Although IFN␣ therapy is associated with reduced viral burden, this cytokine also mediates immune dysfunction and toxicities. Through detailed mapping of IFN␣ receptor binding sites, we generated IFN␣ hybrids and mutants and determined that structural changes in the C-helix alter the ability of IFN to limit retroviral activity. Selective IFN␣ constructs differentially block HIV replication and their directional magnitude of inhibition correlates with APOBEC3 levels. Importantly, certain mutants exhibited reduced toxicity as reflected by induced indoleamine 2,3-dioxygenase (IDO), suggesting discreet and shared intracellular signaling pathways. Defining IFN structure and function relative to APOBEC and other antiviral genes may enable design of novel IFN-related molecules preserving beneficial antiviral roles while minimizing negative effects. (Blood. 2011;118(9): 2567-2577) IntroductionHighly active antiretroviral therapy (HAART) may not eradicate all long-lived and productive reservoirs of HIV, including macrophages, 1-3 and additional approaches to viral suppression continue to center on host cell-derived molecules that antagonize the virus life cycle. Although hundreds of host cell factors are required by HIV for successful infection 4 and may serve as potential intervention targets, considerable effort has focused on those endogenous factors that contribute to cellular resistance to HIV. Among the innate intracellular HIV antagonists are the apolipoprotein-B mRNA-editing enzyme-catalytic polypeptide-like 3 (APOBEC3) family of cytidine deaminases, 5,6 and members of the tripartite motif family (TRIM), 7 but HIV has the potential to effectively eliminate or neutralize these resistance factors.In vitro studies demonstrated that IFN␣ enhances innate antiviral molecules including APOBEC family members as a counter-maneuver to HIV viral infectivity factor (VIF). 8,9 Importantly, treatment of HIV and HCV coinfected patients with pegylated-IFN␣-2a (peg-IFN␣) and Ribavirin or HIV monoinfected patients with peg-IFN␣ only 10 resulted in enhanced APOBEC expression in PBMCs along with other IFN stimulated genes (ISGs). 10 These studies illustrate the potential to alter the virus-host imbalance and suppression of de novo synthesis of HIV. Despite these promising findings, the use of IFN␣ therapeutically is fraught with potential side effects, including cytotoxicity, immune dysregulation, neuropathicity and apoptosis. [11][12][13][14] Although the use of peg-IFN␣ with its longer half-life has enabled reductions in levels of administered cytokine on a less frequent regimen, toxicity remains a deterrent.Based on the potential utility of IFN␣ in the arsenal of anti-HIV therapies, we have attempted to determine whether its antiviral capacities can be dissociated from its toxic functions....

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Section: Ifn and Ifn Constructsmentioning

confidence: 99%

“…Nondenaturing immobilized metal affinity chromatography (IMAC) purification from culture supernatant, dialysis and sample concentration and SDS-PAGE for quality control was performed. 21 …”

Section: Ifnar2-ecmentioning

confidence: 99%

Section: Western Blotsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Structural variants of IFNα preferentially promote antiviral functions

Vázquez

Schmeisser

Dolan

et al. 2011

Blood

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Structure, interactions and function of the N-terminus of cardiac myosin binding protein C (MyBP-C): who does what, with what, and to whom?

Pfuhl

Gautel

2012

J Muscle Res Cell Motil

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The thick filament protein myosin-binding protein-C shows a highly modular architecture, with the C-terminal region responsible for tethering to the myosin and titin backbone of the thick filament. The N-terminal region shows the most significant differences between cardiac and skeletal muscle isogenes: an entire Ig-domain (C0) is added, together with highly regulated phosphorylation sites between Ig domains C1 and C2. These structural and functional differences at the N-terminus reflect important functions in cardiac muscle regulation in health and disease. Alternative interactions of this part of MyBP-C with the head-tail (S1-S2) junction of myosin or to actin filaments have been proposed, but with conflicting experimental evidence. The regulation of myosin or actin interaction by phosphorylation of the cardiac MyBP-C N-terminus may play an additional role in length-dependent contraction regulation. We discuss here the evidence for these proposed interactions, considering the required properties of MyBP-C, the way in which they may be regulated in muscle contraction and the way they might be related to heart disease. We also attempt to shed some light on experimental pitfalls and future strategies.

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Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus

et al. 2018

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Feline interferon beta is a cytokine that belongs to the type I IFN family, with antitumor, antiviral and immunomodulatory functions. In this work, recombinant feline interferon beta (rFeIFNβ) was expressed in insect larvae that constitute important agronomic plagues. rFeIFNβ accumulated in the hemolymph of larvae infected with recombinant baculovirus and was purified by Blue-Sepharose chromatography directly from larval homogenates on day 4 post-infection. rFeIFNβ was recovered after purification with a specific activity of 1 × 10 IU mg. By this method, we obtained 8.9 × 10 IU of purified rFeIFNβ per larva. The product was biologically active in vitro, with an antiviral activity of 9.5 × 10 IU mL, as well as a potent antitumor activity comparable to that of the commercial FeIFNω. The glycosylation of rFeIFNβ was confirmed by peptide--glycosidase F digestion. Our findings provide a cost-effective platform for large-scale rFeIFNβ production in laboratory research or veterinary medicine applications.

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Binding Characteristics of IFN-αSubvariants to IFNAR2-EC and Influence of the 6-Histidine Tag

Cited by 20 publications

References 39 publications

Structural variants of IFNα preferentially promote antiviral functions

Structural variants of IFNα preferentially promote antiviral functions

Structure, interactions and function of the N-terminus of cardiac myosin binding protein C (MyBP-C): who does what, with what, and to whom?

Production of biologically active feline interferon beta in insect larvae using a recombinant baculovirus

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