Characterization and Quantitation of NF-κB Nuclear Translocation Induced by Interleukin-1 and Tumor Necrosis Factor-α

113

The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 M, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipidbound peptide. In solution, the peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Cell-penetrating Peptide Derived from Human Lactoferrin with Conformation-dependent Uptake Efficiency

Duchardt

Ruttekolk

Verdurmen

et al. 2009

113

“…These assays measure activation of individual signaling elements by measuring their localization in the nuclear versus cytoplasmic compartment in resting or stimulated cells (22). For our assays, STAT1-and STAT3-specific detection reagents were used.…”

Section: Expression Of Receptor Subunits-recentmentioning

confidence: 99%

Interleukins 19, 20, and 24 Signal through Two Distinct Receptor Complexes

Parrish-Novak¹,

Xu²,

Brender

et al. 2002

242

Reporter, proliferation, and direct STAT activation assays using cell lines expressing transfected receptors revealed differences between the receptor complexes. IL-19 and IL-24 also exhibited growth inhibition on a cell line endogenously expressing all three receptor subunits, an effect that was seen at cytokine levels two orders of magnitude above those required for STAT activation or proliferation. These results demonstrate that, although this subclass exhibits receptor complex redundancy, there are differences in ligand/receptor interactions and in signal transduction that may lead to specificity and a distinct biology for each cytokine.

“…studies have demonstrated that in IL-1␤-stimulated fibroblasts, the kinetics of translocation of p65-RELA from the cytoplasm to the nucleus correlate with induction of nuclear NF-B DNA binding activity (45). 2 To verify that EGFPRELA accurately reflects the nuclear translocation of endogenous RELA, transfected cells were treated with varying doses of IL-1␤ for 30 min, and the distribution of the fusion protein was analyzed (Fig.…”

Section: Real Time Analysis Of Nuclear Translocation Of Egfp-rela-prementioning

confidence: 99%

Activation of Nuclear Factor κB in Single Living Cells

Carlotti

Chapman

Dower

et al. 1999

We have studied the dynamics of nuclear translocation during nuclear factor B activation by using a p65(RELA)-enhanced green fluorescent protein (EGFP) fusion construct. Quantitation of expression levels indicates that EGFPRELA can be detected at physiological concentrations of about 60,000 molecules per cell. Stimulation of transfected fibroblasts with interleukin (IL)-1␤ caused nuclear translocation of EGFPRELA, typically resulting in a 30-fold increase in nuclear protein at maximum induction and a concomitant 20% decrease in cytoplasmic levels. The response of individual cells to IL-1␤ was graded, and the kinetics of nuclear translocation were dependent on the dose of IL-1␤ and the level of EGFPRELA expression. The rate of nuclear uptake was saturable, and the time lag for uptake increased at higher EGFPRELA expression levels. Furthermore, nuclear translocation was reduced at less than saturating doses of IL-1␤ suggesting that the pathway is limited by incoming signals. The response to IL-1␤ was biphasic, demonstrating a decline in nuclear import rate at expression levels above three to four times endogenous. This correlated with the anti-apoptotic function of EGFP-RELA which was more prominent at low expression levels and demonstrated successively less protection at higher levels. In comparison, transfection of p50 had no effect on the level of apoptosis and demonstrated some toxicity in combination with EGFPRELA.Inflammatory responses involve the rapid and coordinated activation of a diverse set of genes. This activation is initiated by a variety of agonists including bacterial lipopolysaccharide, phorbol esters, IL-1, 1 and tumor necrosis factor-␣ (1-6) and in addition by stress (7,8) and cell adhesion (9, 10). These agents dynamically regulate cytoplasmic signal transduction networks dependent on developmental stage-and cell type-specific expression of extracellular receptors and intracellular adaptor proteins. A major downstream target of these networks is the NF-B family of transcription factors (11-15) that has been shown to possess both pro-and anti-apoptotic functions dependent on cell type (16, 17).NF-B transcription factors are hetero-or homodimers of a family of related proteins characterized by the Rel homology domain. They form two subfamilies, those containing activation domains (p65-RELA, RELB, and c-REL) and those lacking activation domains (p50, p52) (18). The prototypical NF-B is a heterodimer of p65 (RELA) and p50 (NF-B1). Additionally, a family of at least seven inhibitory subunits, IB, characterized by multiple ankyrin-like repeats has been described (19,20). The paradigm for NF-B activation is nuclear translocation of p50 (NF-B1)/p65 (RELA) heterodimers following IB␣ degradation. Specifically, IB␣ binds to these dimers, thereby masking their nuclear localization signals and preventing nuclear import. Activation of cytoplasmic signal transduction pathways results in the phosphorylation and then ubiquitination and subsequent degradation of IB␣, allowing the dimers to move to the nucleus w...