IL-33 is a nuclear cytokine from the IL-1 family that plays important roles in health and disease. Extracellular IL-33 activates a growing number of target cells, including group 2 innate lymphoid cells, mast cells and regulatory T cells, but it remains unclear whether intracellular nuclear IL-33 has additional functions in the nucleus. Here, we used a global proteomic approach based on high-resolution mass spectrometry to compare the extracellular and intracellular roles of IL-33 in primary human endothelial cells, a major source of IL-33 protein in human tissues. We found that exogenous extracellular IL-33 cytokine induced expression of a distinct set of proteins associated with inflammatory responses in endothelial cells. In contrast, knockdown of endogenous nuclear IL-33 expression using two independent RNA silencing strategies had no reproducible effect on the endothelial cell proteome. These results suggest that IL-33 acts as a cytokine but not as a nuclear factor regulating gene expression in endothelial cells.

Edited by Velia FowlerMicrotubules are nucleated from multiprotein complexes containing ␥-tubulin and associated ␥-tubulin complex proteins (GCPs). Small complexes (␥TuSCs) comprise two molecules of ␥-tubulin bound to the C-terminal domains of GCP2 and GCP3. ␥TuSCs associate laterally into helical structures, providing a structural template for microtubule nucleation. In most eukaryotes ␥TuSCs associate with additional GCPs (4, 5, and 6) to form the core of the so-called ␥-tubulin ring complex (␥TuRC). GCPs 2-6 constitute a family of homologous proteins. Previous structural analysis and modeling of GCPs suggest that all family members can potentially integrate into the helical structure. Here we provide experimental evidence for this model. Using chimeric proteins in which the N-and C-terminal domains of different GCPs are swapped, we show that the N-terminal domains define the functional identity of GCPs, whereas the C-terminal domains are exchangeable. FLIM-FRET experiments indicate that GCP4 and GCP5 associate laterally within the complex, and their interaction is mediated by their N-terminal domains as previously shown for ␥TuSCs. Our results suggest that all GCPs are incorporated into the helix via lateral interactions between their N-terminal domains, whereas the C-terminal domains mediate longitudinal interactions with ␥-tubulin. Moreover, we show that binding to ␥-tubulin is not essential for integrating into the helical complex.In all eukaryotes, microtubules are nucleated from specialized multiprotein complexes containing ␥-tubulin and associated proteins (1-3). These complexes resemble small rings by electron microscopy and are thus called ␥-tubulin ring complexes (␥TuRCs) 4 (4 -7). Closer inspection revealed that these ␥TuRCs are helices of one turn, with the two ends overlapping. They are ubiquitous and essential for viability: the growth of new microtubules is crucial to drive mitotic spindle formation and cell division. ␥TuRCs are mainly composed of ␥-tubulin and of proteins of the GCP (␥-tubulin complex protein) family. GCPs are characterized by sequence homology in two specific regions, also referred to as the grip1 and grip2 motifs (8). Five members of this family are known: GCPs 2, 3, 4, 5, and 6. GCPs 2 and 3 associate with ␥-tubulin to form a V-shaped subcomplex, called ␥-tubulin small complex (␥TuSC). GCPs 2 and 3 constitute the arms of the V, interacting laterally via their N-terminal domains (Fig. 1, A and B). The C-terminal domains are located at the two tips of the V, each binding one molecule of ␥-tubulin (9). In the budding yeast Saccharomyces cerevisiae, ␥TuSCs are directly recruited to the spindle pole body (yeast centrosome equivalent) by the protein Spc110. Oligomers of Spc110 interact with the basis of the V-shaped ␥TuSCs and stabilize their lateral association (10 -13). Likely, seven ␥TuSCs assemble stepwise into a helix of one turn plus a small overlap (14). In this helical array, the ␥-tubulin molecules are exposed to form a platform from which ␣/␤-tubulin dimers assemble into...