Hanna Harant

Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload

Summary The ERs capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3 , a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPK−/− mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.

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 Sec61β. 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.

1α,25-Dihydroxyvitamin D3 decreases DNA binding of nuclear factor-κB in human fibroblasts

1α,25‐Dihydroxyvitamin D3 (1,25‐(OH)2‐D3), the active metabolite of vitamin D, can inhibit NF‐κB activity in human MRC‐5 fibroblasts, targeting DNA binding of NF‐κB but not translocation of its subunits p50 and p65. The partial inhibition of NF‐κB DNA binding by 1,25‐(OH)2‐D3 is dependent on de novo protein synthesis, suggesting that 1,25‐(OH)2‐D3 may regulate expression of cellular factors which contribute to reduced DNA binding of NF‐κB. Although NF‐κB binding is decreased by 1,25‐(OH)2‐D3 in MRC‐5 cells, IL‐8 and IL‐6 mRNA levels are only moderately downregulated, demonstrating that inhibition of NF‐κB DNA binding alone is not sufficient for optimal downregulation of these genes.

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

The 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 Sec61β, 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.

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.

Up-regulation of interleukin-1β-stimulated interleukin-8 in human keratinocytes by nitric oxide

Nitric oxide (NO) is able to regulate the expression of a number of inflammatory mediators. In this study, the effect of NO on the expression of the chemokine interleukin-8 (IL-8) by primary human keratinocytes and the lines KB and HaCaT was examined. Incubation with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) for 24 hr increased IL-8 protein only in HaCaT cells, partly due to the presence of constitutive interleukin-1 (IL-1). However, in combination with IL-1beta, SNAP enhanced both IL-8 mRNA and protein in all three cell types. Transfection of cells with an IL-8 promoter reporter gene construct showed that the effect of NO was at least partly due to transcriptional activation. Despite small variations in the response to NO by the three cell types, these results demonstrate that NO can up-regulate IL-1beta-stimulated IL-8 expression in human keratinocytes. This study provides a regulatory mechanism which may be important in the context of skin inflammation, and supports the role of NO as an inflammatory mediator in the skin.

Transcription of the caspase-14 gene in human epidermal keratinocytes requires AP-1 and NFκB

Caspase-14, a protease involved in skin barrier formation, is specifically expressed in epidermal keratinocytes (KCs). Here, we mapped three start sites of transcription of the human caspase-14 gene and analyzed the upstream chromosomal region for promoter activity. Reporter gene assays identified a core promoter region proximal to the first exon and a distal regulatory region which differentially suppressed promoter activity in KC and other cells. Sequence elements in the proximal promoter were bound by the transcription factors AP-1 (JunB, c-Jun, JunD, Fra-1 and Fra-2) and NFkappaB (p50 and RelB). Our data reveal the basic organization of the human caspase-14 promoter and suggest an important role of AP-1 and NFkappaB in the transcriptional control of caspase-14.

3. – Interleukin-3

Publisher Summary This chapter focuses on interleukin-3 (IL-3), which was originally identified as a multilineage hemopoietic growth factor that stimulates colony formation of erythroid, megakaryocytic, granulocytic, and monocytic lineages. Human IL-3 is encoded by a single mRNA transcript of approximately 1 kb length. Human IL-3 gene is located on chromosome 5 at 5q23–31, together with the genes encoding granulocyte-macrophage colony stimulating factor (GM-CSF), IL-4, IL-5, IL-9, macrophage colony-stimulating factor (M-CSF), MCSF receptor, and various other genes encoding growth factors or their receptors. IL-3 supports proliferation of myeloid precursors and also plays an essential role in supporting the survival of these cells by preventing apoptosis. The production of IL-3 is, in contrast to other hemopoietic growth factors, restricted to a few cell types, such as activated T lymphocytes and mast cells, thereby underlying its role in inducible but not constitutive hemopoiesis. IL-3 acts on mature end-stage myeloid cells, such as basophils and eosinophils, by priming histamine release and generation of leukotrienes by IgE-dependent and IgE-independent mechanisms, thereby suggesting an essential role in allergic responses. IL-3 also acts on monocytes and endothelial cells and thus plays an important role in inflammatory processes. The biological effects of IL-3 on hemopoiesis have led to its use in clinical trials, to support and accelerate hemopoiesis in myeloid disorders, and after high-dose chemotherapy or autologous bone-marrow transplantation.