.1 A New Mammalian Proteasome Subunit Hrpn13/adrm1/gp110 Function Of 2-ii Protein Ywk / Aplp2 In Spermatogenic Cells, Functional Studies And Its Expression In Tumor Cells,

Protein degradation in eukaryotic cells is involved in regulation of metabolism, progression through the cell division cycle, antigen presentation to lymphocytes and other important functions. The ubiquitin-proteasome system (UPS) is responsible for much of the regulated proteolysis in the cell. The 26S proteasome is a huge protein complex of approximately 2.5 MDa composed of one proteolytically active 20S proteasome and two 19S regulatory particles (RP), each attached to one end of the 20S proteasome. In addition to the genuine proteasome subunits, several molecules that associate with proteasomes and play auxiliary roles have been identified. Most of the proteasome studies have been carried out using yeast cells, especially budding yeasts. To date, most mammalian proteasomes and the counterparts of yeast proteasome subunits have been identified. To better define its composition and associated regulatory proteins, Qiu et al developed affinity methods to rapidly purify 26S proteasomes from mammalian cells. By this approach, they discovered a novel 45 kDa (407 residues) subunit of its 19S regulatory complex, hRpn13 (previously termed ADRM1 or GP110), which was previously reported as a heavily glycosylated membrane protein and regulates cell adhesion. It can be incorporated into the 26S proteasome by its N-terminal half and bind directly to the proteasome-associated deubiquitinating enzyme (DUBs), UCH37, by the C-terminal half. Yeast proteasome subunit Daq1/Rpn13, an ortholog of hRpn13, is highly homologous to the N-terminal Xoom, a 404 amino-acid protein required for the embryonic development of frogs.Here we report hRpn13 is homologous to yeast Daq1/Rpn13 and conserved in eukaryotes, which may have many binding partners and be involved in many biological functions. ADRM1/hRpn13 is expressed in various mouse tissues examined by Western blot, especially higher in testis, brain, pancreas and spleen. Interestingly, higher molecular weight bands were detected in several tissues, including brain, pancreas, liver, heart and skeletal muscle, probably because of the post-translational modifications of ADRM1/hRpn13. By GST pull down assay, Qiu et al found that the C-terminal half of hRpn13 binds directly to the proteasome-associated deubiquitinating enzyme (DUBs), UCH37. To confirm the interaction between hRpn13 and UCH37, we constructed and expressed His-hRpn13 and its C-terminal half, the full-length GST-fusion UCH37 and two truncated proteins: GST-UCH37-N-terminal (A227-329) and GST-UCH37-C-terminal (â–³1-226) in E. coli. When purified, His-hRpn13 was incubated with either truncated or the full-length GST-UCH37, both the full-length and the C-terminal half, but not the N-terminal half of UCH37, could pull down hRpn13. That is, UCH37, through its C-terminal half, binds to the C-terminal half of hRpn13. When purified GST-UCH37 was incubated with purified His-tagged S5a, S5a could be pulled down by GST-UCH37, but not GST, suggesting that UCH37 binds to S5a. Thus, there are two, probably complementary, mechanisms by which UCH37 can be recruited to the proteasome.It is reported that UCH37 acts as a deubiquitylating enzyme and we then investigate the effect of hRpn13 in activity of the deubiquitylating enzyme, UCH37. As a positive control, His-tagged hRpn13 (purified in insect cells by Qiu et al) could significantly promote the activity of UCH37. Then the purified full-length hRpn13 from E. coli was incubated with purified UCH37, the hydrolysis of Ub-amc by UCH37 increased by 69%. In contrast, under similar conditions, the C-terminal half of hRpn13 had no effect on the activity of UCH37.Qiu et al showed that the C-terminal half of hRpn13 appears to induce cell death. In order to clarify the mechanism of this effect, we asked whether the C-terminal half of hRpn13 (which itself does not bind to the proteasome) might interact with full-length hRpn13. GST pull-down assays demonstrated that unlike UCH37, hRpn13 did not bind to its C-terminal half in vitro. Thus, the C-terminal half of hRpn13 induces cell death, probably by preventing UCH37, but not hRpn13, from binding to the proteasome.Thus in human 26S proteasomes, hRpn13 appears to be important for the recruiting of UCH37, the proteasome-associated deubiquitinating enzyme, to the 19S complex (its N-terminal half can be incorporated into the 26S proteasome and the C-terminal half of hRpn13 binds directly to UCH37), as a bridge between UCH37 and the proteasome, and enhances its isopeptidase activity. YWK-â…¡protein is a typeâ… sperm membrane protein which was initially identified as the target antigen to a monoclonal antibody (mAb) raised against proteins extracted from human sperm and was found to localize on the equatorial sector of human sperm head. It is highly homologous to the Amyloid Protein Precursor (APP) which is related to Alzheimer's desease, containing a segment with high homology (70.6%) to the transmembrane-cytoplasmic region of APP. The protein is subsequently referred to as the human placenta amyloid precursor protein homologue (APPH) and more similar to rat amyloid precursor like- protein 2 (APLP2), which are apparently species-specific forms of the same component. In vitro study demonstrates that the recombinant polypeptide of the cytoplasmic region of YWK-â…¡protein/APLP2 can be phosphorylated by PKC and cdc2 kinase and interacts with the GTP-binding protein G_o. The coupling of the polypeptide and G_o protein suggests that the YWK-â…¡protein/APLP2 is more like a G_o-coupled receptor and facilitates the G_o-mediated signal transduction pathway in germ cells undergoing differentiation and gamete interaction. It was found that YWK-â…¡protein/APLP2 can interact with Mullerian Inhibiting Substance (MIS) in yeast two-hybide system. The interaction between YWK-â…¡protein/APLP2 and MIS was verified by GST Pull-down assay and Surface Plasmon Resonance (SPR) in vitro. It was found that there are binding sites of MIS on mature sperm; however, there is no evidence shown that MIS typeâ…¡receptor (MISRâ…¡) is expressed on sperm. Mullerian inhibiting substance (MIS) has recently been implicated in multiple cellular functions including promotion of cell survival, but the receptor(s) and signaling pathways involved remain elusive. We suppose that MIS may initially interact with the YWK-â…¡protein/APLP2 and affect the motility and viability of human sperm by stimulating the G_o-mediated signal transduction pathway subsequently.In a previous study, we have investigated the possibility of YWK-â…¡protein/APLP2, previously shown to interact physically with MIS and G_o protein, being a receptor mediating the cell survival effect of MIS. However, the mechanism of action of MIS on sperm remains unclear since an MIS typeâ…¡receptor has not been shown to be present in germ cells. We thus hypothesized that YWK-â…¡protein/APLP2 may be a Go-coupled receptor involved in mediating the observed cell survival enhancing effect of MIS in sperm.In the present study, base on previous study, we gained some further findings as follows: 1) MIS typeâ…¡receptor (MISRII) mRNA is abundant in human and mouse testis but not in COS-7 cells, CHO cells and human sperm. These data implicated that COS-7 and CHO cells can be used as the cell model which provided the possibility of YWK-â…¡protein/APLP2 as a receptor mediating the effect of MIS in cell survival control. 2) In YWK-â…¡-overexpressing COS-7 cells, just like CHO cells, MIS activates the G_o-coupled ERK1/2 signaling pathway. These results provide further support for our previous conclusion, so, we presume, the YWK-â…¡-overexpressed CHO cells are appropriate for the purpose of this study. 3) In YWK-â…¡-overexpressing CHO cells, MIS promotes cell survival with altered levels of p53 and caspase-3. That is to say, MIS-enhanced cell viability was due to anti-apoptotic activity. On the other hand, we found YWK-â…¡protein/APLP2 is high expressed in pancreatic cancer by tissue chip. Further studies can be carried out to investigate the immunogenicity and possible application of YWK-â…¡protein/APLP2 in tumor immunotherapy.Taken together, the present study has demonstrated a new G_o-coupled receptor for MIS in mediating ERK1/2 activation leading to anti-apoptotic activity or cell survival.