The Discovery And Neuroprotective Mechanism Study Of HDAC6Specific Inhibitor

Neurodegenerative disease is an age-related disease, which could be caused by many pathogenic factors, including mitochondrial dysfunction, Oxidative stress, Misfolding protein aggregation, Inflammation, immune dysfunction genetic mutations and so on. In recent years studies have shown that the regulation of protein acetylation modification plays an important role in the process of neuroprotection and promoting differentiation of endogenous neural stem cells. The inhibitors of Histone deacetylase6(HDAC6), a member of HDAC Ⅱb, can promote neuroprotection and axonal regeneration, which inferred that HDAC6is a potential drug target against neurodegenerative disease. But there are little available HDAC6selective inhibitor and the roles of specific HDAC6inhibitor in regulating of oxidative stress are still not very clear. So here, we focus not noly on the discovery of novel selective HDAC6inhibitor, but also the mechanism of oxidative stress regulated by HDAC6inhibitor.,First, GST-HDAC6fusion protein was expressed with insect expression system and purified with affinity method. The (Boc-Lys(-acetyl)-AMC) synthetic substrate was used to evaluate the deacetylase activity of purified protein. SAHA was used as the positive control. And the assay was optimized that the Z’-factor is about0.60, indicating that the molecular-based high-throughput screening model for HDAC6inhibitors was successfully established.Next, compounds from Professor Fajun Nan and Jingkang Shen on the model of HDAC1, HDAC3and HDAC6were screened. And compounds numbered LGP09295, LGP09286, LGP07073and LGP07074were identified as HDAC6-specific inhibitors in both molecular and cellular level.Finally, the neuroprotective activity of these novel HDAC6-specific inhibitors was evaluated. Besides the positive comound tubastatin A, the other two compounds LGP07073and LGP07074can significantly release the H2O2-induced cell death in PC12cells and primary cortical neuron cells..Peroxiredoxin (of Prx) Ⅰ and of Prx Ⅱ directly acetylated by HDAC6has been reported to be involved in the process of HDAC6inhibitors agaisnt oxidative stress. Here, Tubastatin A was used for the mechanism study. And our results showed that XBP1s also play important role in the process.Firstly, Tubastatin A could protect PC12cells and primair neurons cells of rat from H2O2treatment. High levels of reactive oxygen damage cells are belived to be associated with neurodegenerative disorders. To minimize the damage, hosts evolve non-enzymatic and enzymatic antioxidant defenses, the latter include catalase, superoxide dismutases (SOD), peroxidases (Prx) and so on. So, anti-oxidative stress related genes were detected with Q-PCR in cells treated with Tubastatin A and H2O2. The resuts showed that tubastatin A can improve the expression of catalase, PRDX5and Pdia5genes after H2O2treated, which has been reported be regulated by transcription factor X-box binding protein-1(XBP1).Then the other known XBP1s regulated ERAD related gene includingEedm1was also upregulated in both Tubastatin A and H2O2treatment, which indicated the XBP1s transcription factor activity was upregulated. Knockdown XBP1expression with siRNA method could down regulate the expression of catalase, PRDX5, Pdia5and ERAD genes, and reduce the cellular protective effect of Tubastatin A. Over-expression of XBP1s with adenovirus could upregulate the expression of Catalase、PRDX5、Pdia5and directly protect PC12cells from H2O2treatment. It is the first time that XBPls play an important role in the tubastatin A against oxidative damage.Furthermore, the mechanism of Tubastatin A enhancing the transcriptional activity of XBPls was studied. Tubastatin A can increase the exogenous XBP1s expression, compared with DMSO control, but has no obvious effect on the transcription and splicing of XBP1. Overexpressing wild type HDAC6could reduce the effect of Tubastatin A on the exogenous XBP1s expression. And only overexpressing wild type can decrease the exogenous XBP1s expression, but HDAC6mutation without deacetylase activity has no effect. These results confirmed that the upregulation of XBPls of Tubastatin A was dependent on the HDC6deacetylase activityCo-IP experiments confirmed that XBP1s can direct interact with HDAC6, when both of them co-overexpressed in HEK293T cells. And deletion mutant Co-IP results confirmed that the HUB region of HDAC6is necessary for their interaction. And the acetylation level of XBPls is increased obviously after tubastatin A treatment. These results showed that XBPls is a novel substrate of HDAC6. And the XBPls acetylation sites of K241, K257, K276and K297were identified with bioinformatics prediction and protein mass spectrometry method.Finally, the relationship between the upregulation of XBPls’s protein levels and its protein degradation process was evalated. It has been suggested that degradation of XBPls is through the proteasome pathway. CHX was used to prevented protein synthesis. In the present of Tubastatin A, the degradation rate of XBPls was slowed down, compared with DMSO control. MG132was used to inhibited proteasome ativity. In the present of MG132, there are no significant difference in the XBPls protein expression between the Tubastatin A and DMSO control. These results showed that tubastatin A increased the level of XBPls by slowing down the degradation of XBPls. Our next work is to mutate four candidated acetylated sites and studying the impact of these sites on the protein degradation and its regulation of protein transcriptional activity.In summary, we established a high-throughput screening model of HDAC6-specific small molecule inhibitors, on which we screened out a number of small molecule inhibitors with new structure and neuroprotective effects. This provide metarial basis for drug development of neuroprotective effect. We use the positive inhibitor of HDAC6to research the neuroprotective function and mechanism, and found that tubastatin A can reverse H2O2-induced oxidative damage of nerve cells, where XBPls play an important role by directly interacted and deacetylated with HDAC6and acetylation modificaiton regulated protein stability. This work provided new clues for the mechanism of the neuroprotective function and potent therapy idea for neurodegenerative diseases.