Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB.

The effect of orphan nuclear receptor Nur77 overexpression on activation of an interleukin-2 (IL-2) promoter-luciferase construct was analyzed in the human leukemic cell line Jurkat. Cotransfection of the IL-2 promoter construct together with the Nur77 expression plasmid resulted in a significant repression of IL-2 promoter activation compared to control cells. The repression by Nur77 requires the N-terminal activation function-1 domain. The repressive effect of Nur77 on IL-2 promoter activation is mediated through inhibition of the transcription factor complex nuclear factor-kappaB (NF-kappaB), since blocking or alteration of the IL-2 NF-kappaB binding sites resulted in abrogation of the repressive effect of Nur77. Moreover, further examination of a reporter gene construct containing multiple copies of the IL-2 CD28 response element (CD28RE) showed that Nur77 can inhibit transactivation mediated by the NF-kappaB components p65 and c-Rel. However, no effect of Nur77 was seen on p65-mediated transactivation of a construct containing multiple NF-kappaB binding sites of the HIV LTR. Our data suggest that Nur77 is able to block activation through NF-kappaB when bound to low-affinity NF-kappaB binding sites, such as those located in the IL-2 promoter

Human macrophage inflammatory protein-3α/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-α via a non-standard NF-κB site

AbstractThe 5′-flanking sequences of the human macrophage inflammatory protein-3α/CCL20 gene were cloned and transfected into G-361 human melanoma cells in a luciferase reporter construct. Tumor necrosis factor-α (TNF-α) treatment stimulated luciferase expression, and promoter truncations demonstrated that TNF-α inducibility is conferred by a region between nt −111 and −77, which contains a non-standard nuclear factor-κB (NF-κB) binding site. The requirement for NF-κB was demonstrated as follows: (i) mutations in this NF-κB site abrogated TNF-α responsiveness; (ii) TNF-α activated a construct containing two copies of the CCL20 NF-κB binding site; (iii) overexpression of NF-κB p65 activated the CCL20 promoter; (iv) NF-κB from nuclear extracts of TNF-α-stimulated cells bound specifically to this NF-κB site

The translocation inhibitor CAM741 interferes with vascular cell adhesion molecule 1 signal peptide insertion at the translocon.

The cyclopeptolide CAM741 selectively inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), a process that is dependent on its signal peptide. In this study we identified the C-terminal (C-) region upstream of the cleavage site of the VCAM1 signal peptide as most critical for inhibition of translocation by CAM741, but full sensitivity to the compound also requires residues of the hydrophobic (h-) region and the first amino acid of the VCAM1 mature domain. The murine VCAM1 signal peptide, which is less susceptible to translocation inhibition by CAM741, can be converted into a fully sensitive signal peptide by two amino acid substitutions identified as critical for compound sensitivity of the human VCAM1 signal peptide. Using cysteine substitutions of non-critical residues in the human VCAM1 signal peptide and chemical cross-linking of targeted short nascent chains we show that, in the presence of CAM741, the N- and C-terminal segments of the VCAM1 signal peptide could be cross-linked to the cytoplasmic tail of Sec61beta, indicating altered positioning of the VCAM1 signal peptide relative to this translocon component. Moreover, translocation of a tag fused N-terminal to the VCAM1 signal peptide is selectively inhibited by CAM741. Our data indicate that the compound inhibits translocation of VCAM1 by interfering with correct insertion of its signal peptide into the translocon

Crystallization and preliminary X-ray crystallographic study of interleukin-8

AbstractInterleukin-8 (neutrophil-activating factor; NAP-1) has been crystallized by the vapour diffusion technique to give single crystals suitable for three-dimensional structural study at a resolution higher than 2.4 Å. The crystals belong to the space group P3121 or P3221 and have unit cell dimensions a = b = 40.9 Å, c = 90.3 Å

Inhibition of vascular endothelial growth factor cotranslational translocation by the cyclopeptolide CAM741.

The cyclopeptolide CAM741 inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), which is dependent on its signal peptide. We now describe the identification of the signal peptide of vascular endothelial growth factor (VEGF) as the second target of CAM741. The mechanism by which the compound inhibits translocation of VEGF is very similar or identical to that of VCAM1, although the signal peptides share no obvious sequence similarities. By mutagenesis of the VEGF signal peptide, two important regions, located in the N-terminal and hydrophobic segments, were identified as critical for compound sensitivity. CAM741 alters positioning of the VEGF signal peptide at the translocon, and increasing hydrophobicity in the h-region reduces compound sensitivity and causes a different, possibly more efficient, interaction with the translocon. Although CAM741 is effective against translocation of both VEGF and VCAM1, the derivative NFI028 is able to inhibit only VCAM1, suggesting that chemical derivatization can alter not only potency, but also the specificity of the compounds

Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1.

Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61beta. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery