Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara 252-5258, Japan.
Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 5-10-1 Fuchinobe, Chuo-ku, Sagamihara 252-5258, Japan.
Biochim Biophys Acta Gen Subj. 2022 Feb;1866(2):130049. doi: 10.1016/j.bbagen.2021.130049. Epub 2021 Oct 30.
Living organisms are subject to various mechanical stressors, such as high hydrostatic pressure. Empirical evidence shows that under high pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. Here, we demonstrate that superoxide dismutase 1 (Sod1) plays a role in resisting high pressure for cell growth. Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. When these mutant cells were cultured under 25 MPa, their intracellular O levels increased while sod1∆ mutant genome stability was unaffected. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O generator paraquat. The sod1∆ mutant is known to exhibit methionine and lysine auxotrophy. However, excess methionine addition or overexpression of the lysine permease gene LYP1 did not counteract high-pressure sensitivity in the sod1 mutants, suggesting that their amino acid availability might be intact under 25 MPa. Interestingly, an exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria appeared to partially restore the high-pressure growth ability in the sod1 mutants. Taken these results together, we suggest that high pressure enhances O production and Sod1 within the IMS plays a role in scavenging O allowing the cells to grow under high pressure.
Empirical evidence shows that under high hydrostatic pressure, the oxidative stress response is activated in Saccharomyces cerevisiae. However, the mechanisms involved in its antioxidant systems are unclear. In the current study, we aimed to explore the role of superoxide dismutase 1 (Sod1) in yeast able to grow under high pressure.
Wild type and sod1 mutant cells were cultured in high-pressure chambers under 25 MPa (~250 kg/cm). The SOD activity in whole cell extracts and 6His-tagged Sod1 recombinant proteins was analyzed using an SOD assay kit. The O generation in cells was estimated by fluorescence staining.
Mutants lacking Sod1 or Ccs1, the copper chaperone for Sod1, displayed growth defects under 25 MPa. Of the various SOD1 mutations associated with familial amyotrophic lateral sclerosis, H46Q and S134N substitutions diminished SOD activity to levels comparable to those of catalytically deficient H63A and null mutants. The high-pressure sensitive sod1 mutants were also susceptible to sublethal levels of the O generator paraquat. Exclusive localization of Sco2-Sod1 to the intermembrane space (IMS) of mitochondria partially restored the high-pressure growth ability in the sod1 mutants.
High pressure enhances O production and Sod1 within the IMS plays a role in scavenging O allowing the cells to grow under high pressure.
Unlike external free radical-generating compounds, high-pressure treatment appeared to increase endogenous O levels in yeast cells. Our experimental system offers a unique approach to investigating the physiological responses to mechanical and oxidative stresses in human body.
生物体受到各种机械应激源的影响，如高静水压力。经验证据表明，在高压下，酿酒酵母中的氧化应激反应被激活。然而，其抗氧化系统中涉及的机制尚不清楚。在这里，我们证明超氧化物歧化酶 1（Sod1）在细胞生长中抵抗高压中起作用。缺乏 Sod1 或 Sod1 的铜伴侣 Ccs1 的突变体在 25 MPa 下显示出生长缺陷。与家族性肌萎缩侧索硬化症相关的各种 SOD1 突变中，H46Q 和 S134N 取代将 SOD 活性降低到与催化缺陷的 H63A 和缺失突变体相当的水平。当这些突变细胞在 25 MPa 下培养时，它们的细胞内 O 水平增加，而 sod1Δ突变体基因组稳定性不受影响。高压敏感的 sod1 突变体也容易受到亚致死水平的 O 生成剂百草枯的影响。已知 sod1Δ 突变体表现出蛋氨酸和赖氨酸营养缺陷。然而，过量添加蛋氨酸或过表达赖氨酸通透酶基因 LYP1 并不能抵消 sod1 突变体的高压敏感性，这表明它们的氨基酸可用性在 25 MPa 下可能完整。有趣的是，Sco2-Sod1 到线粒体的内膜间隙（IMS）的特有定位似乎部分恢复了 sod1 突变体在高压下的生长能力。综上所述，我们认为高压增强了 O 的产生，而 IMS 内的 Sod1 发挥作用清除 O，使细胞能够在高压下生长。
经验证据表明，在高静水压力下，酿酒酵母中的氧化应激反应被激活。然而，其抗氧化系统中涉及的机制尚不清楚。在目前的研究中，我们旨在探索超氧化物歧化酶 1（Sod1）在能够在高压下生长的酵母中的作用。
野生型和 sod1 突变细胞在 25 MPa（~250 kg/cm）的高压室中培养。使用 SOD 测定试剂盒分析全细胞提取物和 6His 标记的 Sod1 重组蛋白中的 SOD 活性。通过荧光染色估计细胞中的 O 生成。
缺乏 Sod1 或 Sod1 的铜伴侣 Ccs1 的突变体在 25 MPa 下显示出生长缺陷。与家族性肌萎缩侧索硬化症相关的各种 SOD1 突变中，H46Q 和 S134N 取代将 SOD 活性降低到与催化缺陷的 H63A 和缺失突变体相当的水平。高压敏感的 sod1 突变体也容易受到亚致死水平的 O 生成剂百草枯的影响。Sco2-Sod1 到线粒体的内膜间隙（IMS）的特有定位部分恢复了 sod1 突变体在高压下的生长能力。
高压增强了 O 的产生，而 IMS 内的 Sod1 发挥作用清除 O，使细胞能够在高压下生长。
与外部自由基生成化合物不同，高压处理似乎增加了酵母细胞内的内源性 O 水平。我们的实验系统提供了一种独特的方法来研究人体对机械和氧化应激的生理反应。
