| PD (parkinson's disease) is one of the most common neurodegenerative diseases, which affect 1%of the people over 65 years old. There are 170 million PD patients in our country, the figure will increase year by year with the aggravating of aging. So far, the pathogen of PD is unclear in most case. The LB, which was first described by Frederick Lewy in 1913, is present in essentially all cases of PD. The major protein constituent of LBs is alpha-synuclein (a-synuclein), a natively unfolded protein having high propensity for aggregation. Several studies have shown the presence of a-synuclein in the neuronal nuclei. However, no definite nuclear functions have been attributed to a-synuclein to date. It is not known if nuclear localization is the common property of a-synuclein or it is cause/consequence of PD pathology.In this study, we sought to address the following three aspects:the protein domains responsible for the nuclear import of a-synuclein; the way of a-synuclein import into the nuclear; the effect on cell function of a-synuclein in the nuclear.First of all, the bioinformatics were used to predict both the NLS in a-synuclein and the protein domains responsible for the nuclear localization of a-synuclein. Using deletion mutant forms of a-synuclein, we identified protein domains that are responsible for the observed nuclear import of a-synuclein in PC 12 cells.Secondly, the nuclear import inhibition,RNAi (RNA interference)-. Immunofluorescence,Immunoprecipitatio,Real Time PCR (real-time quantitative polymerase chain reaction) and WB (western blot), were used to explore the way of a-synuclein import into the nuclei.Finally, in order to examine the ability of a-synuclein in the nucleus, we created constructs of a-synuclein tagged with either a nuclear localization sequence (NLS) or a nuclear export sequence (NES). We used these constructs to transfect PC12 cells, and screened with G418, then the stable cell lines which express a-synuclein are located in the nucleus or cytoplasm were acquired. The growth curve morphological analysis,cell cycle sensitivity to neurotoxic agents (MPP+) of the stable cell lines were analyzed. The a-synuclein-GFP fusion protein was expressed and purified with yeast, then the circular dichroism (CD) was used to study the effect of a-synuclein on DNA.The results showed that, both the N terminal and the C terminal were responsible for the nuclear import of a-synuclein. a-synuclein could import into the nuclei through nuclear pore complex (NPC), mediated by importin a and provided energy by hydrolysis of GTP. There was no effect on cell morphological whenα-synuclein import into the nuclei, but both the growth curve and the cell cycle were delayed. And the PC 12 cells with a-synuclein localization in nuclei were sensitivity to neurotoxic agents (MPP+). Moreover, the results of the circular dichroism showed that twoα-synuclein molecules were prone to combine with one GC box-like single nucleotide sequence.In summary, mediated by both the N terminal and the C terminal,α-synuclein could combine with importin a and importinβ, then import into the nuclei through nuclear pore complex (NPC), provided energy by hydrolysis of GTP.α-synuclein could compete with the transcription factor SP1 for the same GC rich region in promoter, then the process of both the E2F family protein interaction with the GC rich region mediated by the transcription factor SP1 and the cyclins/cdks complex formation were inhibited, so the cell cycle was disturbed, it led to the PC 12 cells with a-synuclein localization in nuclei more sensitivity to neurotoxic agents (MPP+).
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