阅读说明：本技术 一种改善线粒体功能的粘红酵母去乙酰化酶蛋白2的应用 (Application of rhodotorula glutinis deacetylase protein 2 for improving mitochondrial function ) 是由 孙晗笑 刘梦鸽 利时雨 于 2018-09-05 设计创作，主要内容包括：本发明公开了一种改善线粒体功能的sirtuin2蛋白的应用及阐述其作用机制,属于生物技术领域。本发明将具有重构线粒体网络和改善哺乳动物衰老细胞表型功能的粘红酵母sirtuin2基因在哺乳动物细胞中重组表达。实验结果表明其可以提高融合蛋白Mfn1,Mfn2和分裂蛋白Drp1的表达水平,降低哺乳动物细胞的SA-β-gal活性水平,增强CK19、β1整合素的表达水平,降低细胞内ROS水平水平,明显提升细胞内ATP水平,改善线粒体形态,从而改善了线粒体网络和提高了细胞抵抗衰老的能力,为使用sirtuin2蛋白治疗线粒体功能障碍和预防衰老提供了新的策略。(The invention discloses application of a sirtuin2 protein for improving mitochondrial function and elucidating an action mechanism of the sirtuin2 protein, belonging to the technical field of biology.A rhodotorula glutinis sirtuin2 gene with functions of reconstructing a mitochondrial network and improving the phenotype of a mammal aged cell is recombined and expressed in the mammal cell.an experimental result shows that the expression level of fusion proteins Mfn1, Mfn2 and mitosin Drp1 can be improved, the SA- β -gal activity level of the mammal cell is reduced, the expression level of CK19 and β 1 integrins is enhanced, the intracellular ROS level is reduced, the intracellular ATP level is obviously improved, and the mitochondrial morphology is improved, so that the mitochondrial network is improved, the anti-aging capacity of the cell is improved, and a new strategy is provided for treating mitochondrial dysfunction and preventing aging by using the sirtuin2 protein.)

1. A mechanism and use of sirtuin2 protein for reconstituting mitochondrial networks and improving the phenotype of senescent cells in mammals.

2. The sirtuin2 protein having the functions of remodeling mitochondrial networks and improving the phenotype of senescent cells in mammals according to claim 1, wherein the intracellular mechanism of the sirtuin2 protein for improving the phenotype of cellular senescence and the mitochondrial networks is to increase the expression levels of fusion kinetic proteins Mfn1, Mfn2 and mitosin Drp1, decrease the level of SA- β -gal activity in mammalian cells, increase the expression levels of CK19, β 1 integrins, decrease the level of intracellular ROS, significantly increase the intracellular ATP level, and improve the mitochondrial morphology.

3. The use of a sirtuin2 protein prepared according to claim 2 for reconstituting a mitochondrial network and improving the phenotypic function of senescent cells in a mammal for preventing and/or treating diseases associated with mitochondrial dysfunction.

4. A functional food and/or pharmaceutical product for preventing mitochondrial dysfunction, comprising an effective amount of the sirtuin2 protein component having the functions of reconstituting mitochondrial network and improving the phenotype of senescent cells in mammals, prepared according to claim 2, as an active ingredient.

5. A functional food and/or pharmaceutical product for preventing and improving a phenotype of aging cells, comprising an effective amount of the sirtuin2 protein component having a function of reconstituting a mitochondrial network and improving a phenotype of aging cells in mammals, prepared according to claim 2, as an active ingredient.

6. A functional food and/or pharmaceutical product for preventing metabolic syndrome, comprising an effective amount of the sirtuin2 protein component having the functions of reconstituting mitochondrial network and improving the phenotype of senescent cells in mammals, prepared according to claim 2, as an active ingredient.

7. A functional food and/or pharmaceutical product for increasing mitochondrial function and reconstituting mitochondrial network during stem cell induced differentiation and tumor cell differentiation, comprising an effective amount of the sirtuin2 protein component having the effects of reconstituting mitochondrial network and improving the phenotypic function of senescent cells in mammals, prepared according to claim 2, as an active ingredient.

Technical Field

The present invention belongs to the field of biotechnology of strain protein application mechanism. More particularly, the present invention relates to a mechanism of rhodotorula glutinis sirtuin2 protein expressed by mammalian cells and its use, which has the functions of reconstituting mitochondrial networks and improving the phenotype of senescent cells in mammals.

Background

Evidence that lower eukaryote Sirtuin genes are life-related provides a basis for mammalian Sirtuin genes to influence mammalian life. Various experiments have demonstrated that SIRT1 can be used to resist aging. Recent studies have shown that brain-specific SIRT1 overexpression can extend the lifespan of mice and delay senescence. The embryo development and postpartum life of the inbred mouse lacking the SIRT1 are seriously damaged. A recent study demonstrated that outcross mice deleted of SIRT1 could not extend lifespan by energy restriction. This indicates that endogenous SIRT1 is required for CR-triggered life-span effects. The effect of SIRT2 on longevity has not been clear, but SIRT2 may prevent certain age-related diseases. SIRT3 is the first protein in the Sirtuin family to be found linked to mammalian aging, and a series of studies have reported that SIRT3 plays an important role as a mitochondrial important deacetylase in maintaining mitochondrial function. SIRT achieves anti-aging under CR through protection against hearing impairment. In addition, SIRT3 was able to regulate ROS levels. To date, no studies have indicated a link between SIRT4 or SIRT5 genes and anti-aging, but the potential role of SIRT4 and SIRT5 in regulating mitochondrial metabolism, coupled with the potential impact of these Sirtuins in senescence-associated diseases.

Mitochondrial dynamics and mitochondrial morphological abnormalities are hallmarks of aging and are considered pathological causes of many age-related pathologies. Mitochondrial activity is related to mitochondrial quality control and is an asymmetric fission and fusion process. The reconstructed mitochondrial network promotes the interaction of mitochondria and other organelles, maintaining the metabolic activity of mitochondria. Sirtuins can regulate mitochondrial function through multiple pathways, it is not known whether Sir2 can directly realize the anti-aging of the body through the regulation of mitochondrial function, and how to control mitochondrial networks to realize the anti-aging is needed to be researched. Senescence is associated with loss of mitochondrial network homeostasis, but the cellular processes of biological senescence associated with these changes are still unclear. The dynamic remodeling of the mitochondrial network by fusion and fission promotes the maintenance of cellular homeostasis. Mitochondrial dynamics and mitochondrial morphological abnormalities are hallmarks of aging and are considered pathological causes of many age-related pathologies.

Mitochondria are constantly in cycles of fusion and fission, two diametrically opposed processes, essential to maintain the number and quality of mitochondria and exchange of materials between mitochondria, and in addition, normal mitochondrial membrane potential is necessary for fusion and fission. Mitochondrion increases the number of mitochondria, and mitochondrial fusion completes inter-mitochondrial protein complementation and information transfer. With age, excessive mitochondrial debris accumulates within the cell, inducing disease development. Mitochondrial fusion and division require the involvement and regulation of related proteins. The fusion of a normally functioning mitochondrion with another mitochondrion requires the involvement of the fusion proteins Mfnl, Mfn2 localized to the outer membrane and the atrophy of the optic nerve factor (OPA1) localized to the inner membrane. In particular, mitochondrial fission is a non-selective process requiring the involvement of the motor-related proteins 1 (Drpl), Fisl, MFF and MTP 18.

A set of 4698 feasible single gene-deleted strains was subjected to systematic analysis of yeast RLS and 238 single gene deletions were identified to prolong life, suggesting that single genes can also affect life. Notably, the deletion of the deacetylase gene of Saccharomyces cerevisiae, includesSIR2, 3, 4，HDA1,2,3，HOS1,2,3,4，HST1,2,3,4，RPd3, SAP1,30,155, 185, 190，4And the life cannot be prolonged, which indicates that the genes are necessary for maintaining the activity of the cells and can be related to the anti-aging of the cells. Rhodotorula glutinis is a known lipid producer, and the morphological change of cells is accompanied with proliferation and differentiation, a large amount of lipid droplets are formed when the cells grow well, the oil production is reduced when the cells age, and the cells are easier to observe than the growth phase of yeast cells, so the Rhodotorula glutinis a good research model. The Rhodotorula glutinis found to contain five Sir2 homologous genes by search at NCBI:hst3,hst4,sirtuin1, sirtuin2, sirtuin4. The yeast Sir2 family is highly homologous to human Sirtuins.

Therefore, whether the yeast Sir2 has the effect of reversing the cell senescence phenotype is discussed, and the influence of the changes of mitochondrion and fusion, mitophagy and mitochondrion dynamics on the cell activity and senescence in the process is researched; further validation in mammals. This study provides a new strategy for the prevention and treatment of diseases of mitochondrial dysfunction such as aging resistance or metabolic syndrome.

Disclosure of Invention

The invention aims to provide application of rhodotorula glutinis sirtuin2 protein and elucidation of the action mechanism of the rhodotorula glutinis sirtuin2 protein.

The technical scheme adopted by the invention for solving the technical problem is as follows:

1. construction of plasmids transfection and cell treatment

1.1 construction of plasmids

1.2 H₂O₂HaCaT cells before and after treatment of transfection

1.3 plasmid transfection

1.4 identification of the protein of interest

2. Cell proliferation assay (MTT method)

2.1 SA- β -gal Activity assay

2.2 detection of cell CK19, β 1 integrin, CK10 protein expression level

2.3 Western Blot technique for detecting protein expression level

2.4 RT-PCR technology for mRNA level detection

3. Mitochondrial mass analysis

3.1 Transmission Electron microscopy analysis of mitochondrial ultrastructure

3.2 Mito-Tracker analysis of mitochondrial morphology

3.3 mitochondrial kinetic protein and autophagy protein expression levels

3.4 Western Blot technique for detecting the expression level of mitochondrial fusion split proteins Mfn1, Mfn2, Drp1, autophagy-related proteins

3.5 RT-PCR technology for detecting autophagy-related protein mRNA expression level

3.6 detection of intracellular ROS content by DCFH-DA Probe

3.7 ATP and enzyme levels in HaCaT cells after treatment

3.8 measurement of ATP Change Using ATP kit

3.9 determination of SOD, CAT, ATP synthetase levels with the kit

The invention discloses a Rhodotorula glutinis sirtuin2 gene with functions of reconstructing mitochondrial network and improving phenotype of mammal senescent cell and its action mechanism, belonging to biotechnology field, the invention recombinates and expresses Rhodotorula glutinis sirtuin2 gene with functions of reconstructing mitochondrial network and improving phenotype of mammal senescent cell in HaCat cell of human.

Drawings

FIG. 1 is a diagram showing a plasmid construction map and electrophoresis results in the examples, wherein M: DNA Marker; 1: pFLAg-CMV-2-Rt-sirtuin2(6283bp)；2：pFlag-CMV-2-Rt-sirtuin4(6106bp)；3：pFlag-CMV-2(4679bp)。

FIG. 2 shows the relationship between hydrogen peroxide concentration and cell survival rate in the examplesp<0.05, **p<0.01 vs.Normal group.

FIG. 3 shows the results of the expression and identification of recombinant plasmid proteins in examples.

FIG. 4 shows HaCaT cell viability in the examples.

FIG. 5 shows the galactosidase activity (bar.50 μm) of HaCaT cells in the examplesp<0.05, **p<0.01 vs.Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group.

FIG. 6 shows the expression levels of CK19, CK10 and β 1 integrin proteins in HaCaT cells of examples, wherein 1: Normal and 2: H₂O_2;3: Empty; 4: co-sirtuin

FIG. 7 shows the expression levels of CK19, CK10, β 1 integrin mRNA of HaCaT cells in examplesp<0.05, **p<0.01vs. Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group.

FIG. 8 shows the intracellular ROS levels (bar.20 μm) measured by DCFH-DA in the examples. *p<0.05, **p<0.01 vs.Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group.

FIG. 9 shows the ATP levels of HaCaT cells in examplesp<0.05, **p<0.01 vs. Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group.

FIG. 10 is mitochondrial mass assessment (200X) of HaCaT cells in examples.

FIG. 11 shows the expression levels of Mfn1, Mfn2 and Drp1 proteins in HaCaT cells in examples, in which 1: Normal and 2: H₂O_2;3: Empty; 4: co-sirtuin; 5:Rt-sirtuin4; 6:Rt-sirtuin2。

FIG. 12 shows the expression levels of proteins LC3-II, LC3-I and PINK1 in HaCaT cells of examples, wherein 1: Normal and 2: H₂O_2;3: Empty; 4: co-sirtuin; 5:Rt-sirtuin2;6:Rt-sirtuin4;7: co-sirtuin+3MA。

FIG. 13 shows the expression levels of LC3 and PINK1 mRNA of HaCaT cells in examplep<0.05, **p<0.01 vs.Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group,^△ p<0.05,^△△ p<0.01 vs. H₂O₂+co-sirtuin group.

FIG. 14 is the mitochondrial and cytoplasmic Parkin protein expression in the examples, where 1: Normal and 2: H₂O_2;3: Empty; 4: co-sirtuin; 5:Rt-sirtuin2; 6:Rt-sirtuin4;7: co-sirtuin+3MA

FIG. 15 measurement of SOD, CAT and ATP synthetase activities of HaCaT cells in examplep<0.05, **p<0.01 vs.Normal group,^# p<0.05,^## p<0.01 vs. H₂O₂group,^△ p<0.05,^△△ p<0.01 vs. H₂O₂+co-sirtuin group.

Detailed Description

The present invention will be described in further detail with reference to examples.