Research On The Functions Of HSP70 And Its Interacting Protein HIP In Stress Adaptation Establish

Adaptation is characterized as stressful elicit a complex array of behavioral and physiological changes believed to contribute to optimal coping of the organism with the situation. As a result of stress, cellular and molecular response could result in either adaptation or damage in cells, which depended on the stress load. This implied that biomolecule that can inhibit and repair stress damage were just the important regulating molecule and proteins. HSPs(heat shock/stress protein, HSP)was a group of proteins which were rapidly expressed with organism coping with stress, including HSP90,HSP70,HSP40 and small HSP. HSP70 was a very famous member among HSP family, it played important roles in nascent polypeptide chain folding and transport and protein assembly and degradation, and HSP70 could also regulate biological function of many proteins and vital process, especially normal cell signaling and cell life cycle regulation. It must be paid much more attention that HSP70 could inhibit important cellular protein degeneration and degradation, repair damaged protein, stabilize cytoskeleton, improve cellular anti-oxidant ability, and protect enzyme activity, so HSP70 was a very important restoration protein. Previous study of our lab had demonstrated that high expressing level of HSP70 with heat shock can inhibit cell apoptosis to improve adaptation establish. So we presumed that HSP70 was an important protein which composed a system of protein damage and restoration with its regulation proteins to improve adaptation establish. So to explore the HSP70 expression pattern during adaptation, confirm its adaptation promotion, detect HSP70 interacting protein during adaptation, and interpret its biological function could not only deeply reveal biological mechanism of adaptation and its protein basis, but also have important significance in controlling stress damage and promoting adaptation.1. Expression pattern of HSP70 during adaptation and protecting role of HSP70 during stress damage. Intermittence chronic restraint stress was used to induce the construction of adaptation, and neuroendocrine hormone and myocardium damage degree were used as indexes to evaluate adaptation establish. The results showed that HPA response tend to be mild when adaptation rat were given a more intense stress. And plasma level of E,NE,GC and plasma LDH\CK-MB activity of stress group after adaptation were lower than more intense stress group. pathological observation showed that adaptation could significantly attenuate the damage of more intense stress. So those results showed that an adaptation model was established. At the same time, low GC concentration pretreatment can attenuate the damage of high GC concentration treatment, so an adaptation cell model was established.HSP70 level was detected by western blotting. The results showed that HSP70 level continuing increased within two weeks of construction of adaptation, adaptation rat with high HSP70 level could attenuate the damage of more intense stress. HSP70 expression pattern was similar in H9C2 cell with glucocorticoids treatment. These results implied that HSP70 have an important role in adaptation. We also found that HSP70 could protect H9C2 cell from GC inducing cell damage with over expression and inhibition of HSP70. These results confirmed that HSP70 had an important role on adaptation attenuating the damage of more intense stress.2. Screening for HSP70 interacting proteins during adaptation43 proteins were acquired with bioinformatics technology including: co-chaperones: HSF1,HSPBP1,BAG1,BAG2,BAG3,BAG4,HIP,HSP40,CHIP,HOP,DNAJA3,DNAJB11,TTC1, proteins in apoptosis pathway: PDCD8,FANCC,IKBKG,TP53,APAF1,BCL2,HCFC1,RHOA, cell cycle regulating proteins: APEX1,MAP3K5,PPP1R15A,CDK9,EIF2AK1,NR3C1,EIF2AK2,APOB,PKC, cell differentiation correlated proteins:MSR1,PTMA,TIF1,KRT7, embryo development correlated proteins: SOX9,PEX5, protein translation and transport correlated proteins: BAT3,TCERG1,TID1,TOM7, energy metabolism correlated proteins: NQO1,SLC5A1.pGBKT7-Hsp70 was used as a bait to screen cDNA library with yeast two hybrid technology. HIP was acquired after yeast two hybrid screen, repeat identification and sequencing. Co-immunonoprecipitation of complexes captured using antibody against HSP70 showed that HIP bound to HSP70 in rat adaptation animal model and H9C2 cell with GC treatment. So these results showed that HIP also had important role in adaptation with its interaction with HSP70.3. Mechanism of protection role of HSP70 and its interacting protein during stress damageWestern blotting results showed that HIP level continuing increased within two weeks of construction of adaptation. HIP expression pattern was similar in H9C2 cell with glucocorticoids treatment. HIP could also protect H9C2 cell from GC inducing damage with over expressing and inhibiting HIP expression. Mutant analysis and cotransfection research showed that HIP and HSP70 could protect H9C2 cell from GC inducing damage through their interaction. Luciferase report gene system showed that HIP and HSP70 could decrease the GR transcription activity, this may be a mechanism of HSP70 and HIP protecting H9C2 cell from GC inducing damage.In conclusion, HSP70 had important roles in adaptation establish, and HIP was an important HSP70 interacting protein in adaptation, which can reduce the transcription activity of GR, protect H9C2 cells from GC induced damage, and promote adaptation establish.