Hsd-34 (trim69) Protein Function

Spermatogenesis is a complex process,in which undifferentiated spermatogonia divide and differentiate into mature spermatozoa.During the process,many genes are transcribed and translated under precise temporal and spatial regulation.A large number of cell-specific and developmental stage-specific proteins were accurately degraded.Therefore,the ubiquitin-proteasome pathway plays a key role in spermatogenesis.Research had found that the ubiquitin-proteasome system had some new functions,such as the regulation of protein activity and their subcellular localization.Recent studies have found that depend on its non-degradation function,the ubiquitin system was also involved in the regulation of cell growth,apoptosis,and aging.The method of laser capture microdissection(LCM) combined with suppressive subtractive hybridization(SSH) was used to isolate the differentially expressed genes during germ cell differentiation.A new gene designated as HSD34 was obtained and characterized.HSD-34 cDNA is 1544bp.Its open reading frame consists of 1506bp,which is encoding a 500 amino acids protein.The GenBank Accession No.of HSD-34 is AY305385 and localizes in human chromosome 15(Location:15q21.1).HSD34,containing a RING finger domain,a B-box,and a coiled-coil motif in the N-terminal region,belongs to the TRIM (Tripartite motif) or RBCC(RING,B-Box,coiled-coil) family.The identity of HSD-34 and its mouse homolog is about 91%.The mouse homolog of HSD-34 is observed with higher expression in mouse testis,heart,liver and brain tissue.RING finger domain plays a key role in transferring the ubiquitin to specific substrate and RING protein itself.Therefore,the RING domain is a character of ubiquitin ligase E3. Ubiquitin ligase E3 can ubiquitinate itself.We construct a series of point mutants within the RING finger domain and some truncates.With the in vivo ubiquitination assay,we found that HSD-34 could ubiquitinate itself,which showed that HSD-34 was a new ubiquitin ligase E3. Meanwhile,RING finger domain is the active domain of HSD-34;C61 and C64 sites are the key sites of this domain.Using a yeast two-hybrid assay,we had screened the interaction proteins of HSD-34.We use a truncate,which had been removed of the N-terminal RING finger domain(246bp),as the bait gene.Four positive clones were found,namely,the cell cycle checkpoint protein Hus1 (GenBank Accession No.NM-004507);RhoE(GenBank Accession No.NM-00516 8); Bcl-2A1(GenBank Accession No.NM-00404 9);and IL-2RB(GenBank Accession No. NM-0008 78).We had chosen the checkpoint protein Hus1 for further study because checkpoint protein Hus1 interacts with two other checkpoint protein Rad9 and Rad1,which could form a G2/M cell cycle checkpoint complex,Rad9-Hus1-Rad1(9-1-1) complex.Rad9-Hus1 -Rad1 complex is confirmed to be involved in DNA repair and apoptosis.Using the co-immunoprecipitation method,we had proved HSD-34 could interact with Hus1.Immunofluorescence and confocal laser scanning showed that HSD-34 co-localized with Hus1 in HeLa cells,which also confirmed the interaction between these two proteins.HSD-34 protein was distributed in cytoplasm and nucleus.In cytoplasm,HSD-34 was mainly surrounding the nucleus as a ring;while in nucleus,it was in a speckled pattern.The deletions of different domain could result in different localization of this protein,for example, RING deletions mutant was surrounded the membrane;RING-B deletions mutant was gathered the membrane more apparently,and stretched out small feet from the membrane; B30.2 deletions was also surrounded the membrane;RBCC deletions was aggregated within the cytoplasm just as HSD-34,but the border was not clear;C61A/C64A mutant was diffused throughout the cell,completely lost the distribution pattern of HSD-34 protein.The degradation experiment showed that HSD-34 did not participate in the degradation of Hus1 through the proteasome pathway.The transfection of HSD-34 plasmid into HeLa cells could cause the increased expression of Hus1 protein,which the level of phosphorylated Hus1 protein was also significantly increased.At the same time,HSD-34 could also active caspase-3 pathway,which could lead to apoptosis.These results suggested that HSD-34 can regulate the function of Hus1 protein in apoptosis.Morphology analysis found that HSD-34 can promote cell apoptosis.Fluorescence microscopy found that GFP-HSD-34 fusion protein was first distributed as a ring in the cytoplasm;then was transferred to the nucleus,where it could cause the condensation and fragmentation of the nucleus.Then we had speculated that,HSD-34 protein was synthesized in the cytoplasm,and shuttled into the nucleus,from where it exerted its important role in apoptosis.After the transfection of HSD-34 into HeLa cells,we detected different protein levels at different time points.We found that the expression of active caspase-9,active caspase-3,Hus1 and phosphorylation Hus1 were elevated upon the induction of HSD-34 within those test cells. However,the expression of p53 did not change between the test group and the control group.The pathDetect(?) cis-reporting system was used to detect if HSD-34 could affect the cellular signal transduction pathways,such as NF-κB,NFAT,CRE,AP1,STAT1 pathway.The results showed that HSD-34 can activate CRE cellular signal transduction pathway.These results suggested that HSD-34 may induce apoptosis through caspase-9,caspase-3 pathway;at the same time,HSD-34 could activate the CRE signaling pathway within the nucleus.After that,the expression of Hus1 could be up-regulated and phosphorylated.The phosphorylated Hus1 could activate the apoptosis pathway and lead the cells to apoptosis. This process is a p53-independent pathway.