Themis (Thymocyte expressed molecule involved in selection), a member of a family of proteins with unknown functions, is highly conserved among vertebrates. Here we found that Themis is expressed in high amounts in thymocytes between the pre-T cell receptor (TCR) and positive selection checkpoints, and in low amounts in mature T cells. Themis-deficient thymocytes exhibit defective positive selection, which results in reduced numbers of mature thymocytes. Negative selection is also impaired in Themis-deficient mice. A higher percentage of Themis-deficient T cells exhibit CD4+CD25+Foxp3+ regulatory and CD62LloCD44hi memory phenotypes than in wild-type mice. Supporting a role for Themis in TCR signaling, this protein is phosphorylated quickly after TCR stimulation, and is needed for optimal TCR-driven Ca2+ mobilization and Erk activation.

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.

IB␤, one of the major IB proteins, is only partially degraded in response to most extracellular signals. However, the molecular mechanism of this event is unknown. We show here that IB␤ exists in at least two different forms: one that is bound to the NF-B dimer and the other bound to both NF-B and B-Ras, a Ras-like small G protein. Removal of cellular B-Ras enhances whereas excess B-Ras blocks induced IB␤ degradation. Remarkably, B-Ras functions in both GDP-and GTP-bound states, and mutations of the conserved guanine-binding residues of B-Ras abrogate its ability to block degradation of IB␤. B-Ras also directly blocks the in vitro phosphorylation of IB␤ by IKK␤. These observations suggest that IB␤ in the ternary complex is resistant to degradation by most signals. We suggest that specific signals, in addition to those that activate only IKK, are essential for the complete degradation of IB␤.The dimeric NF-B transcription factors are inhibited in quiescent cells through stable association with inhibitor IBs. A large number of extracellular stimuli transmit signals to relieve this inhibition (2,17,38). Almost all of these signals lead to the activation of a specific kinase known as IB kinase (IKK), which phosphorylates IB. Phosphorylated IB proteins are degraded by the sequential actions of ubiquitin ligase and the 26S proteasome releasing free NF-B (23).The major IB proteins, IB␣ and IB␤, resemble each other in both primary sequence and tertiary structure, with the exception of a 40-residue-long insert present within the ankyrin repeat 3 in IB␤. However, these two proteins exhibit one major functional difference (17,35,36). While signal-induced degradation of IB␣ is responsible for rapid NF-B activation, prolonged activation of NF-B, which is essential for certain biological functions such as T-cell activation, requires IB␤ degradation (1). Several pathological conditions, such as asthma, cystic fibrosis, and viral and bacterial infection, also require prolonged 4,10,19,22,29,33,35). We do not know why prolonged NF-B activation requires IB␤ degradation. Two other functional properties of IB␤ distinguish it from IB␣. Unlike IB␣, IB␤ does not fully degrade in response to most stimuli, and IB␤/NF-B complexes are exclusively cytoplasmic in resting cells (16,17,25,34). How these two properties contribute to persistent NF-B activation through IB␤ degradation is not known. A recent report shows that different MEKK kinases recruit IKK to IB␣/NF-B and IB␤/NF-B complexes in tumor necrosis factor alpha (TNF-␣)-activated cells (31). This suggests that the compositions of IB␣ and IB␤ complexes are different, which may lead to their differential functional properties.Yeast two-hybrid screening has identified two Ras-like small GTPases, B-Ras1 and -2, as inhibitors of NF-B transcriptional activity (14). B-Ras proteins belong to the Ras family due to their high sequence similarity (5, 6, 39). However, there are some critical differences in the sequences of B-Ras. In addition to the fact that these two proteins lack lipid modific...

Alport syndrome (AS) is a genetic disease characterized by haematuric glomerulopathy variably associated with hearing loss and anterior lenticonus. It is caused by mutations in the COL4A3, COL4A4 or COL4A5 genes encoding the α3α4α5(IV) collagen heterotrimer. AS is rare, but it accounts for >1% of patients receiving renal replacement therapy. Angiotensin-converting enzyme inhibition slows, but does not stop, the progression to renal failure; therefore, there is an urgent requirement to expand and intensify research towards discovering new therapeutic targets and new therapies. The 2015 International Workshop on Alport Syndrome targeted unmet needs in basic science, genetics and diagnosis, clinical research and current clinical care. In three intensive days, more than 100 international experts including physicians, geneticists, researchers from academia and industry, and patient representatives from all over the world participated in panel discussions and breakout groups. This report summarizes the most important priority areas including (i) understanding the crucial role of podocyte protection and regeneration, (ii) targeting mutations by new molecular techniques for new animal models and potential gene therapy, (iii) creating optimal interaction between nephrologists and geneticists for early diagnosis, (iv) establishing standards for mutation screening and databases, (v) improving widespread accessibility to current standards of clinical care, (vi) improving collaboration with the pharmaceutical/biotech industry to investigate new therapies, (vii) research in hearing loss as a huge unmet need in Alport patients and (viii) the need to evaluate the risk and benefit of novel (including ‘repurposing’) therapies on an international basis.

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