Seiichi Mizushima

CD40 signalings play crucial roles in B-cell function. To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-beta receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246-269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230-245 were required. Overexpression of TRAF6 activates transcription factor NFkappaB, and its TRAF-C domain suppresses NFkappaB activation triggered by CD40 lacking residues 246-277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230-245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Human inter-alpha-trypsin-inhibitor (ITI) is a serine proteinase inhibitor with a molecular weight of 220 kDa which consists of 3 different polypeptides. The constitutive components are 2 heavy chains (H1 and H2 chains) and 1 light chain (L chain), and its inhibitory activity is considered to be derived from this L chain. It has also been reported that this L chain is almost identical to the trypsin inhibitor (UTI) occurring in human urine. We examined the gene expression of the ITI constitutive peptides in human tissues using the reverse transcription (RT) -PCR technique. As a result, the genes of the H1 chain were found to be expressed in various tissues, particularly strongly in the liver. On the other hand, the genes of the H2 chain were found to be strongly expressed in the adrenal glands, brain, kidneys, and lungs, as well as the liver. Further, the PCR amplification product of the L chain was strongly detected not only in the liver but also in the pancreas, kidneys, lungs, stomach and testes. These results suggest the possibility that the major tissue which produces ITI is the liver, and the H chains and L chain (UTI) are produced as a component of ITI- related proteins in other tissues as well as in the liver.