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Information × Registration Number 0216U000896, 0115U004200 , R & D reports Title Modeling of molecular processes underlying Parkinson's disease in yeast cells and analyzing the impact of exogenous and endogenous factors on the processes of aggregation and degradation of human alpha-synuclein popup.stage_title Head Stasyk Oleh Volodymyrovych, Кандидат біологічних наук Registration Date 15-03-2016 Organization Institute of cell biology, National Academy of Sciences of Ukraine popup.description2 The object of study - the process of aggregation and degradation of the protein alpha-synuclein in model strains of methylotrophic yeast Hansenula polymorpha under the influence of exogenous and endogenous factors. The purpose of the first research phase - the construction of model strains for the study of molecular mechanisms of formation and degradation of alpha -sinuclein aggregates using cells of methylotrophic yeast H. polymorpha. As a result of this work the vector for expression of the chimeric open reading frame yEGFP3-SNCA and the strain of methylotrophic yeast H. polymorpha, which is produced of human alpha-synuclein labeled green fluorescent protein (EGFP), were constracted. The presence in the genome of the recombinant strains of SNCA gene ORF, encoding the human alpha-synuclein, was confirmed by Southern blot analysis and polymerase chain reaction in real time. The presence of heterologous protein alpha synuclein labeled EGFP in the cells strains of the model was confirmed by fluorescent microscopy and Western blot analysis. To simulate aging of neurons at Parkinson's disease, which is inherent in glucose hypometabolism, we have constructed strains producing human alpha synuclein, which are characterized by impaired glucose transport. To study the role of the intensity of glucose metabolism in the cytotoxic effects of a heterologous protein alpha-synuclein in cells of model strains we have also constructed strain with constitutive expression of the gene HXT1, encoding low-affinity glucose transporter and SNCA gene, encoding the human alpha-synuclein. In operation, we have found that the production and accumulation of the human alpha-synuclein in cells of methylotrophic yeast H. polymorpha causes damage of growth and adversely affects the cell viability. It is found that virtually all of our derived model strains did not form the visible alpha-synuclein amyloid . A mutant strain gcr1-2-SNCA, which has damaged the catabolite repression and glucose transport, has been characterized by enhanced aggregation of fluorescently labeled alpha-synuclein. Such a difference in the ability to aggregate was not due to damage the metabolism of glucose. Aggregates were also observed in the case of growing the cells in a medium with glycerol. The molecular mechanism of this interesting effect should be investigated. Keywords: alpha-synuclein man methylotrophic yeast, Parkinson's disease Product Description popup.authors Бобак Ярослав Петрович Денега Ірина Олегівна Переверзєва Галина Георгіївна Стасик Олег Володимирович Стасик Олена Георгіївна popup.nrat_date 2020-04-02 Close