Hsp70l1 Biological Function Of A Preliminary Study

HSP70L1 (HSP70-like protein 1), a novel member of HSP70 subfamily, was identified by large-scale random sequencing of a human dendritic cells cDNA library in 2004. HSP70L1 is composed of 509 amino acids, and its calculated molecular weight is 54.8 kDa. HSP70L1 does not have either a transmembrane region or a signal peptide. The amino acid sequence of HSP70L1 has highest identity (90.5%) to mouse HSP70, and it is approximately 33.7% to 35.7% homologous to human HSP70 subfamily molecules. HSP70L1 can bind to common receptors (CD91, TLR2, TLR4) on DC, promoting DC maturation and stimulating secretion of the proinflammatory cytokines. HSP70L1 serves as a adjuvant with peptide immunizations, hybrided or fused with epitopes, promoting DC maturation and activating DC to produce cytokines, inducing antigen-specific Th1 responses and CD8+ CTL (cytotoxic T lymphocyte), results in significant inhibition of tumor growth or protecting the mice against virus challenge. HSP70L1 binds stably to MPP11, forming the RAC (ribosome-associated complex) of mammalian cells. RAC is involved in folding or maintaining nascent polypeptides in a folding-competent state, facilitating their transit through the ribosomal tunnel and preventing backward movement or clearing aggregated proteins from the ribosome. The other biology functions of HSP70L1 have remained elusive except as a member of RAC or an adjuvant.Here, recombinant fusion protein GST-HSP70L1 and His-HSP70L1 were expressed in E. coli. The rabbits were immunized with the purified GST-HSP70L1. The anti-serum was purified with purified His-HSP70L1. The anti-HSP70L1 polyclonal antibodies can recognize HSP70L1. HSP70L1 was detected by Western Blot analysis in various cell lines, appearing highest expression in K562 cell, indicating that Hsp70L1 may be homo-dimer. HSP70L1 forming homo-dimer was demonstrated by co-immunoprecipiation. K562 and Hela cells were transfected with the HSP70L1 RNAi vector (targeting its 766-784nt). The HSP70L1 knock down cell lines were screened by G418 selection. HSP70L1 RNAi cells grew more slowly compared to parental cells by MTT assay. In order to assess if the reduced cell growth observed in HSP70L1 RNAi cells was due to an altered progression through the different phases of the cell cycle, we used cell synchronization to study the transition from the G1 to S phases and from the G2 to M phases. Using mimosine coupled with FACS analyses, we noted HSP70L1 RNAi cells had slower G1 to S and G2 to M progression. Mitosis in cells is thought to be triggered by the cyclin-dependent kinase CDC2. Before mitosis, cyclinB-CDC2 complexes are held in an inactive state by phosphorylation of CDC2 at Thr14 and Tyr15, which is catalyzed by the protein kinases Wee1and Myt1. Dephosphorylation is carried out by the protein phosphatase CDC25C. By Western Blot, the expression of the CDC2 was similar in RNAi cells and parental cells, but a higher CDC2-pY15 was observed in RNAi cells. 293 cells co-expressing CDC2/CDC25C and HSP70L1, immunoprecipitates were found to co-precipitate HSP70L1 with CDC25C but not with CDC2. These results proposed that HSP70L1 regulated the activation of CDC2 by binding with CDC25C in the cell cycle progression. The activation of CDC2 also operates in the'DNA structure checkpoints'that arrest cells at the G2/M transition in response to DNA damage. HSP70L1 RNAi cells displayed a G2/M arrest compared to parental cells by treated with DNA damage agent CDDP, which indicated HSP70L1 links with the"DNA structure checkpoints". Invasion assays were done using Transwell with matrigel-coated filter containing 8μm pores. Hela/HSP70L1 RNAi cells had increased invasive capacity as compared with parental wild type cells. The result clearly showed that expression of HSP70L1 was sufficient to supress the in vitro invasion capacity. K562/HSP70L1 RNAi cells were less sensitive to docetaxel-induced apoptosis than parental cells, but Hela/HSP70L1 RNAi cells were more sensitive than parental cells to CDDP-induced and docetaxel-induced apoptosis. The different functions of HSP70L1 in apoptosis proposed HSP70L1 may be link with the activity and location of c-Abl nonreceptor protein-tyrosine phosphatase.c-Abl was identified as the cellular homolog of the v-Abl oncogene from the Abelson murine leukemia virus, and is ubiquitously expressed. c-Abl has three nuclear-localization signals (NLSs) a nuclear-export signal (NES), whick can mediated c-Abl shuttles between the nucleus and the cytoplasms. Introduction of the antisense sequence of c-Abl into cells results entering into S phase from G1 phase earlier in time. Overexpression of c-Abl inhibits growth by causing G1 phase arrest and apoptosis. C-Abl targets and phosphorylates WAVE2/3, and then regulates the Arp2/3-dependent actin remodeling. The tyrosine kinase c-Abl is activated by certain DNA-damaging agents. c-Abl binds to p73 in cells through its SH3 domain with the homo-oligomerization domain of p73. c-Abl phosphorylates p73 on a tyrosine residue at position 99 and stimulates p73-mediated transactivation and apoptosis. Therefore, c-Abl are important to cell proliferation, adhesion, apoptosis, stress responses and transformation.In yeast two-hybrid system, using full length c-Abl as bait protein to test partner, we got lots of the proteins can interact with c-Abl. HSP70L1 is a potential partner. The study of interaction between HSP70L1 and c-Abl can afford us the clue to study the biological functions of HSP70L1.In this study, the direct interaction between c-Abl and HSP70L1 was demonstrated by co-immunoprecipiation, in vitro binding assay and Far-Western blot. Then we found c-Abl bind HSP70L1 between the 429-469 by constructing truncation mutants and the interaction site with c-Abl in HSP70L1 (P433) by site mutation. Hela/HSP70L1 RNAi cells were transfected with the c-Abl/Arg RNAi vector (targeting 1346 to 1364nt ), screened by Hygromycin B selection. Knock down c-Abl leaded less sensitive to CDDP-induced apoptosis in Hela/HSP70L1 RNAi cells. The result indicated the the apoptosis sensitivity mediated by HSP70L1 knock down may be regulated by c-Abl. The interaction between HSP70L1 and PSMA7/FHL2 was demonstrated by co-immunoprecipiation, and both PSMA7 and FHL2 binds direct with and phosphorylated by c-Abl. These results afford us the clues to study the biological functions of HSP70L1.In the present study, we describe the role of the HSP70L1 (a new member of HSP70) in cell proliferation, cell cycle regulation and apoptosis by utilizing RNA interference technology. Moreover, we show that the activity of HSP70L1 is regulated by c-Abl nonreceptor protein-tyrosine phosphatase.