Purification of SUMO-1 modified IκBα and complex formation with NF-κB.

Covalent modification of proteins with SUMO (Small Ubiquitin-like MOdifier) affects many cellular processes, including transcriptional regulation, DNA repair and signal transduction. Although hundreds of SUMO targets have been identified, many biological outcomes of protein sumoylation remain poorly understood. In particular, biochemical and structural analysis can only be easily conducted if highly pure sumoylated substrates are available. Purification of sumoylated substrates in vitro or in bacteria have been previously reported but separating the sumoylated protein from the undesired unmodified fraction is often technically challenging, inefficient and time consuming. Here we develop a new vector system for in vivo sumoylation in Escherichia coli which improves purification of sumoylated proteins. We describe the purification of IκBα, its sumoylation, the subsequent separation and purification of the modified and the unmodified forms and the purification of the complex IκBα-SUMO-1/NF-κB. After a first GST affinity chromatography and GST-tag removal, a unique metal-ion affinity chromatography using a 6xHis-SUMO-1 tag results in mgs of highly pure SUMO-1 modified IκBα. Our pure SUMO-1 modified IκB/NF-κB complex could be a useful tool to identify new interaction partner specific of the SUMO-1 modified IκBα form. This approach may be extended to other SUMO substrates not isolable by classical chromatography techniques.

Transfer of flexibility between ankyrin repeats in IkappaB* upon formation of the NF-kappaB complex.

The mechanism of inhibition of the transcriptional activator nuclear factor kappaB (NF-kappaB) by the inhibitor IkappaB* is central to the understanding of the control of transcriptional activity via this widely employed pathway. Previous studies suggested that IkappaB* , a modular protein with an NF-kappaB binding domain consisting of six ankyrin repeat domains (ANKs), shows differential flexibility, with ANK 1-4 apparently more rigid in solution in the absence of NF-kappaB than ANK 5 and 6. Here we report NMR studies that confirm the enhanced flexibility of ANK 5 and 6 in free IkappaB* . Upon binding of NF-kappaB, ANK 5 and 6 become well structured and rigid, but, somewhat surprisingly, other domains of the IkappaB* , which were relatively rigid in the free protein, become significantly more flexible. Due to the high molecular masses of the component proteins and the complexes, we employ a hierarchical experimental plan to maximize the available information on local flexibility in the ankyrin repeat domains. Backbone resonances of the 221-residue IkappaB* protein were assigned firstly in a smaller construct consisting of ankyrin repeats 1-4. These assignments could be readily transferred to the spectra of the construct containing six repeats, both free and complexed with various combinations of the NF-kappaB p50 and p65 domains. Transverse relaxation optimized spectroscopy-type NMR experiments on differentially labeled proteins enabled information on backbone structure and dynamics to be obtained, even in complexes with molecular masses approaching 100 kDa. Changes in the flexibility and stability of the various ankyrin repeat domains of IkappaB* complex formation take a variety of forms depending on the position of the domain in the complex, providing a variety of examples of the structural and functional utility of intrinsically unstructured or partly folded protein domains.

A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis.

BACKGROUND: Airway inflammation and infection are early events in cystic fibrosis (CF) pathogenesis. The existence of an imbalance in the immune cell population of the CF fetal airway before infection remains completely unknown. OBJECTIVE: The aim of this study was to determine whether early signs of inflammation are observed in CF airways during human fetal development. METHODS: Tracheas and lungs were collected from 21 CF and 16 non-CF fetuses. In tissue sections, the numbers of neutrophils, mast cells, macrophages, and B and T lymphocytes were quantitatively analyzed by means of image cytometry. The presence of IL-4, IL-6, IL-8, IL-10, RANTES, IFN-gamma, TNF-alpha, and NF kappa B and its inhibitor I kappa B-alpha was qualitatively evaluated by immunofluorescent staining. RESULTS: During fetal airway development, epithelial and glandular differentiation, as well as the distribution of inflammatory markers, was similar in CF and non-CF tissues. Significant differences between CF and non-CF fetal airways were observed only in the numbers of mast cells and macrophages. In the CF trachea, the mast cell number increased slowly but continuously, whereas in the non-CF trachea this number rapidly reached a plateau. In the CF lung, the macrophage number increased with time, whereas in the non-CF lung it decreased. CONCLUSION: Although no intrinsic inflammation was demonstrated, we observed a distinct appearance of mast cells and macrophages in CF airways in comparison with non-CF airways during fetal development. These 2 cell populations were greater in CF airways at a late stage of fetal development, suggesting their possible involvement in the early onset of inflammation in CF infants.