Human Genes Rab24, Rab8b, And Pdlim5 Of Functional Studies

Rab proteins comprise a large family of Ras superfamily (small GTP-binding proteins), which are involved in vesicle trafficking process and very important in protein sorting, trafficking and docking. Despite much progress made in determining the role of several Rab proteins in discrete vesicular transport events, the function of most members of the Rab family has not yet been elucidated. Among these, Rab24 is an atypical Rab protein whose function is poorly defined. It is known that several attributes distinguish this protein from other members of the Rab family. To date, there is still no much progress in studying the function of Rab24 from mouse, and it remains unknown that what kind of signaling pathway Rab24 may be involved in. The cDNA sequence of human RAB24 was released from NCBI database in 2002, but very little functional study about this human gene has been reported these years.The RAB24 cDNA was successfully isolated from the human fetal brain cDNA library constructed by our lab, and some fundamental function study on this gene was accomplished by our endeavor. The main study results are as follows:(1) It is found that in total, there are four splice variants for human RAB24 based on bioinformatics analysis. Human EST searching and RT-PCR results showed that these four splice variants are all ubiquitously expressed in human tissues.(2) Interestingly, RAB24 is localized predominantly in nucleus of COS7 cells, with small quantities in perinuclear region; but in AD293 cells, RAB24 is distributed in cytoplasm, mainly around the nuclear, which is consistent with previous reports. This difference of subcellular distribution of RAB24 in these two cell lines imply that Rab24 should play some different role in human and monkey cells.(3) In COS7 cells, Mutant type of RAB24, RAB24 (D123I), shifted to form intracellular inclusions, mainly localized on perinuclear region, which is also remarkably different from previously study reports.(4) In AD293 cells, the distribution pattern of mutant type of RAB24, RAB24(G66D), RAB24 (T120N) and RAB24 (HH202II) seems to be different form that of the wild type since they are distributed in the whole cells, mainly in the nuclear, which imply that RAB24 may operate through a novel subcellular localization mechanism that is quite distinct from that of other Rab proteins. But the mutant sites, Y17A and S67Q, have no effect on the subcellular distribution of RAB24.(5) Based on yeast two-hybrid system, interaction between human RAB24 and two proteins, named CYCLOPHILIN A and GABARAP, were detected and identified by yeast mating and fluorescent co-localization. These interactions suggested that RAB24 should share some common signaling pathways with the two significant proteins.(6) GTPase activity assay indicate that RAB24 has a very low GTPase activity, which is is consistent to previous related reports based on nonhuman Rab24. Two mutant types of RAB24, RAB24 (Y17A) and RAB24 (S67Q), result in the dramatic increase of GTPase activity of RAB24, which suggested that this two amino acids sites could contribute to the low GTPase activity of RAB24.(7) Apoptosis assay for RAB24 indicate that RAB24 is not involved in this cell event.Rab8b is also a member of Rab family, which is found when investigating the intracellular molecular machinery of secretory granule fusion in mast cells and basophils from rat, and little study has been ongoing from that time. Although the cDNA of human RAB8B was released from NCBI database in 2000, no progress has been made on its function research to date. RAB8B cDNA was successfully isolated from the human fetal brain cDNA library constructed by our lab, and some fundamental study was performed. RAB8B is localized on 15q22.2, in total containing eight exons and spaning approximately 76kb on the genome. RAB8B encodes a protein containing 207 amino acids, which including almost all the domains (except RAN domain) of Ras superfamily. RT-PCR experiment showed that RAB8B is ubiquitously expressed in human tissues, especially showing higher expression level in heart, brain, placenta, lung, liver and testis. The highest abundance seems to be in testis implys that RAB8B may play some special role in this tissue. An...