Study On The Function Of Adapter Protein NRBP And A Novel Isoform Of Caspase-10

In this thesis, we focus on the research about the situation and function of two proteins: adaptor protein NRBP and caspase-10g, which are associated with cancer and some immune diseases. The functional study as well as detailed investigation into their truly mechanism will be helpful to develop the drug target of relative diseases.NRBP is a novel adaptor protein which consists of 535 amino acids, it contains a potential Src homology 2 (SH2) domain binding region, a kinase-like domain, a nuclear receptor binding motif, a nuclear localization and nuclear export sequences. Although the study on its function is limited, the identified NRBP putative domains suggests that NRBP may potentially mediate the convergence of disparate signaling pathways. Here, we firstly employed a yeast-two hybrid screening and identified that a key gene activation and growth regulator Jab1 interacts with NRBP, the CO-IP assay in HEK293 T cells further confirms this physical interaction.Jun activation domain-binding protein 1 (Jab1), initially identified as a coactivator of AP-1, is the fifth component of the COP9 signalosome complex. It interacts with the activation domain of c-Jun,induces the phosphorylation of c-Jun and activates AP-1 transcriptional activity. We identified that NRBP inhibited Jab1-induced phosphorylation of c-Jun and AP-1 activation. We futher found that NRBP potently inhibited PMA- or TCR-induced AP-1 activation. AP-1 is an integral component of NFAT, they always cooperates with each other to specifically control gene transcription. Overexpression of NRBP potently blocked NFAT activity induced by TCR or PMA/Ionomycin. However, it did not significantly affect CD69 induction in response to TCR or PMA in T cells. So we propose that the adaptor NRBP might act as a downstream regulator of TCR stimulation, likely through Jab1.Taken together, our data suggest that NRBP may be an important negative regulator of Jab1-mediated functions such as gene transcription and tumor progression. Our discovery of the physical and functional interaction between NRBP and Jab1 provides insights into the mechanism of NRBP in AP-1 pathway.Caspase-10 is an initiator caspase in the death receptor (DR)-dependent apoptotic pathway. So far six splice variants (caspase-10 a～f) have been identified. The second part of the thesis is about a novel isoform of caspase-10 named caspase-10g. Caspase-10g consists of 247 amino acids, it has the DED-containing prodomain and does not contain the large or small subunit. A caspase-10g-specific exon is present between exon 5 and exon 6, which results in a protein product truncated shortly after the prodomain. We also found that caspase-10g was widely expressed in normal human tissues and various tumor cell lines using the RT-PCR assay. Furthermore, the protein expression of endogenous caspase-10g in both HeLa and Jurkat cells was strongly confirmed. After the confirmation of the novel isoform, we next design a series of experimental assays to find the function of caspase-10g. Overexpression of caspase-10g led to significant activation of NF-κB reporter in a dose- and time-dependent manner. Further assay showed that the ability of caspase-10g, as a prodomain-only isoform of caspase-10, to activate NF-κB was relatively greater than the protease-dead mutant of caspase-10a,which is consistent with previous studies. Moreover, caspase-10g, unlike the protease-active caspase-10a, only promotes apoptosis slightly and it has no effect on Fas-mediated apoptosis. Taken together, these results suggest that caspase-10g, as a prodomain-only isoform of caspase-10, may have complicated regulatory roles in the apoptosis signaling pathway. The further research on caspase-10 may help us take it as a novel target of tumor or immune diseases.