人Polo样激酶1活性缺失突变体和结构域在293细胞中的瞬时表达

目的:构建人Polo样激酶1(Plk1)活性缺失突变体及结构域突变体的真核表达载体,并在293细胞中表达。方法:用二次PCR方法扩增Plk1基因并点突变,将82位赖氨酸突变为精氨酸,定向克隆到pcDNA3-Flag载体中;用普通PCR方法扩增Plk1激酶区域及Polo盒区域(PBD)基因,定向克隆到pcDNA3-Flag载体中;将上述质粒转染293细胞进行瞬时表达,Western印迹检测Plk1蛋白的表达。结果:构建了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD真核表达质粒,在293细胞中均可有效表达,蛋白相对分子质量分别为68×103、45×103、31×103。结论:在293细胞中表达了Flag-Plk1(K82R)、Flag-Plk1KD、Flag-Plk1PBD蛋白,有助于进一步探究Plk1对底物的功能。     

Impaired Mitochondrial Dynamics and Nrf2 Signaling Contribute to Compromised Responses to Oxidative Stress in Striatal Cells Expressing Full-Length Mutant Huntingtin

Huntington disease (HD) is an inherited neurodegenerative disease resulting from an abnormal expansion of polyglutamine in huntingtin (Htt). Compromised oxidative stress defense systems have emerged as a contributing factor to the pathogenesis of HD. Indeed activation of the Nrf2 pathway, which plays a prominent role in mediating antioxidant responses, has been considered as a therapeutic strategy for the treatment of HD. Given the fact that there is an interrelationship between impairments in mitochondrial dynamics and increased oxidative stress, in this present study we examined the effect of mutant Htt (mHtt) on these two parameters. STHdh^Q111/Q111 cells, striatal cells expressing mHtt, display more fragmented mitochondria compared to STHdh^Q7/Q7 cells, striatal cells expressing wild type Htt, concurrent with alterations in the expression levels of Drp1 and Opa1, key regulators of mitochondrial fission and fusion, respectively. Studies of mitochondrial dynamics using cell fusion and mitochondrial targeted photo-switchable Dendra revealed that mitochondrial fusion is significantly decreased in STHdh^Q111/Q111 cells. Oxidative stress leads to dramatic increases in the number of STHdh^Q111/Q111 cells containing swollen mitochondria, while STHdh^Q7/Q7 cells just show increases in the number of fragmented mitochondria. mHtt expression results in reduced activity of Nrf2, and activation of the Nrf2 pathway by the oxidant tBHQ is significantly impaired in STHdh^Q111/Q111 cells. Nrf2 expression does not differ between the two cell types, but STHdh^Q111/Q111 cells show reduced expression of Keap1 and p62, key modulators of Nrf2 signaling. In addition, STHdh^Q111/Q111 cells exhibit increases in autophagy, whereas the basal level of autophagy activation is low in STHdh^Q7/Q7 cells. These results suggest that mHtt disrupts Nrf2 signaling which contributes to impaired mitochondrial dynamics and may enhance susceptibility to oxidative stress in STHdh^Q111/Q111 cells.     

Thioredoxin Reductase Deficiency Potentiates Oxidative Stress,Mitochondrial Dysfunction and Cell Death in Dopaminergic Cells

Mitochondria are considered major generators of cellular reactive oxygen species (ROS) which are implicated in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease (PD). We have recently shown that isolated mitochondria consume hydrogen peroxide (H₂O₂) in a substrate- and respiration-dependent manner predominantly via the thioredoxin/peroxiredoxin (Trx/Prx) system. The goal of this study was to determine the role of Trx/Prx system in dopaminergic cell death. We asked if pharmacological and lentiviral inhibition of the Trx/Prx system sensitized dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂ levels and death in response to toxicants implicated in PD. Incubation of N27 dopaminergic cells or primary rat mesencephalic cultures with the Trx reductase (TrxR) inhibitor auranofin in the presence of sub-toxic concentrations of parkinsonian toxicants paraquat; PQ or 6-hydroxydopamine; 6OHDA (for N27 cells) resulted in a synergistic increase in H₂O₂ levels and subsequent cell death. shRNA targeting the mitochondrial thioredoxin reductase (TrxR2) in N27 cells confirmed the effects of pharmacological inhibition. A synergistic decrease in maximal and reserve respiratory capacity was observed in auranofin treated cells and TrxR2 deficient cells following incubation with PQ or 6OHDA. Additionally, TrxR2 deficient cells showed decreased basal mitochondrial oxygen consumption rates. These data demonstrate that inhibition of the mitochondrial Trx/Prx system sensitizes dopaminergic cells to mitochondrial dysfunction, increased steady-state H₂O₂, and cell death. Therefore, in addition to their role in the production of cellular H₂O₂ the mitochondrial Trx/Prx system serve as a major sink for cellular H₂O₂ and its disruption may contribute to dopaminergic pathology associated with PD.     

Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.     

Mitochondrial DNA Mutation-Elicited Oxidative Stress, Oxidative Damage, and Altered Gene Expression in Cultured Cells of Patients with MERRF Syndrome

Myoclonic epilepsy and ragged-red fibers (MERRF) syndrome is a rare disorder characterized by myoclonus, muscle weakness, cerebellar ataxia, heart conduction block, and dementia. It has been documented that 80–90% of the patients with MERRF syndrome are caused by the A8344G mutation in the tRNA^Lys gene of mitochondrial DNA (mtDNA). We and other investigators have reported that the mtDNA mutation results in not only inefficient generation of adenosine triphosphate but also increased production of reactive oxygen species (ROS) in cultured cells harboring A8344G mutation of mtDNA. In addition, we found an imbalance in the gene expression of antioxidant enzymes in the skin fibroblasts of MERRF patients. The mRNA, protein, and enzyme activity levels of manganese-superoxide dismutase were increased, but those of Cu,Zn-SOD, catalase, and glutathione peroxidase did not show significant changes. Recently, we showed that the excess ROS could damage voltage-dependent anion channel, prohibitin, Lon protease, and aconitase in the MERRF cells. Moreover, there was a dramatic increase in the gene expression and activity of matrix metalloproteinase 1, which may contribute to the cytoskeleton remodeling involved in the weakness and atrophy of muscle commonly seen in MERRF patients. Taken together, we suggest that mtDNA mutation-elicited oxidative stress, oxidative damage, and altered gene expression are involved in the pathogenesis and progression of MERRF syndrome.     

镉通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡

镉（cadmium,Cd）是一种生物累积性的有毒重金属元素,能够在肾组织大量蓄积并引起肾发生病变和功能损伤。前期研究证实,Cd处理能够引起猪肾PK-15细胞的活性氧（reactive oxygen species,ROS）水平升高和细胞死亡,但详细机制有待进一步研究。本研究以PK-15细胞为研究对象,通过CCK-8检测、透射电镜观察、DCFH-DA标记、JC-1染色、彗星实验和流式细胞术等研究手段,分别检测Cd处理后的细胞活性、形态变化、ROS生成、线粒体膜电位Δψm、DNA损伤及细胞凋亡情况。CCK-8实验结果显示,CdCl2处理后PK-15细胞活性下降,且呈时间和剂量依赖性;形态学观察发现,CdCl2处理引起PK-15细胞皱缩、变圆,细胞核固缩、染色质凝聚,线粒体肿胀、线粒体嵴减少或消失;荧光染色和流式细胞术检测结果显示,CdCl2处理引起PK-15细胞内ROS水平升高、线粒体膜电位Δψm下降和DNA损伤,最终导致细胞凋亡。Western印迹结果显示,CdCl2处理组中促凋亡蛋白质Bax表达量上调,抑凋亡蛋白质Bcl-2表达量下调,并且CdCl2处理组检测到了活化状态的裂解胱天蛋白酶3(cleaved caspase 3)。此外,ROS清除剂N-乙酰基-L-半胱氨酸（N-acetyl-L-cysteine,NAC）缓解了CdCl2引起的线粒体损伤、DNA损伤和细胞凋亡。综上所述,Cd通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡。     

C-Phycocyanin Confers Protection against Oxalate-Mediated Oxidative Stress and Mitochondrial Dysfunctions in MDCK Cells

Oxalate toxicity is mediated through generation of reactive oxygen species (ROS) via a process that is partly dependent on mitochondrial dysfunction. Here, we investigated whether C-phycocyanin (CP) could protect against oxidative stress-mediated intracellular damage triggered by oxalate in MDCK cells. DCFDA, a fluorescence-based probe and hexanoyl-lysine adduct (HEL), an oxidative stress marker were used to investigate the effect of CP on oxalate-induced ROS production and membrane lipid peroxidation (LPO). The role of CP against oxalate-induced oxidative stress was studied by the evaluation of mitochondrial membrane potential by JC1 fluorescein staining, quantification of ATP synthesis and stress-induced MAP kinases (JNK/SAPK and ERK1/2). Our results revealed that oxalate-induced cells show markedly increased ROS levels and HEL protein expression that were significantly decreased following pre-treatment with CP. Further, JC1 staining showed that CP pre-treatment conferred significant protection from mitochondrial membrane permeability and increased ATP production in CP-treated cells than oxalate-alone-treated cells. In addition, CP treated cells significantly decreased the expression of phosphorylated JNK/SAPK and ERK1/2 as compared to oxalate-alone-treated cells. We concluded that CP could be used as a potential free radical-scavenging therapeutic strategy against oxidative stress-associated diseases including urolithiasis.     

Biased Suppression of Hematopoiesis and Multiple Developmental Defects in Chimeric Mice Containing Shp-2 Mutant Cells

Shp-2 is a cytoplasmic tyrosine phosphatase that contains two Src homology 2 (SH2) domains at the N terminus. Biochemical data suggests that Shp-2 acts downstream of a variety of receptor and cytoplasmic tyrosine kinases. A targeted deletion mutation in the N-terminal SH2 (SH2-N) domain results in embryonic lethality of homozygous mutant mice at midgestation. In vitro embryonic stem (ES) cell differentiation assays suggest that Shp-2 might play an important role in hematopoiesis. By aggregating homozygous mutant (Shp-2^−/−) ES cells and wild-type (WT) embryos, we created Shp-2^−/−-WT chimeric animals. We report here an essential role of Shp-2 in the control of blood cell development. Despite the widespread contribution of mutant cells to various tissues, no Shp-2^−/− progenitors for erythroid or myeloid cells were detected in the fetal liver and bone marrow of chimeric animals by using the in vitro CFU assay. Furthermore, hematopoiesis was defective in Shp-2^−/− yolk sacs. In addition, the Shp-2 mutation caused multiple developmental defects in chimeric mice, characterized by short hind legs, aberrant limb features, split lumbar vertebrae, abnormal rib patterning, and pathological changes in the lungs, intestines, and skin. These results demonstrate a functional involvement of Shp-2 in the differentiation of multiple tissue-specific cells and in body organization. More importantly, the requirement for Shp-2 is more stringent in hematopoiesis than in other systems.     

Role of Oxidative Stress,Apoptosis, and Intracellular Homeostasis in Primary Cultures of Rat Proximal Tubular Cells Exposed to Cadmium

Cadmium (Cd) is a known nephrotoxic element. In this study, the primary cultures of rat proximal tubular (rPT) cells were treated with low doses of cadmium acetate (2.5 and 5 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability, together with a significant increase in the number of apoptotic and necrotic cells, were seen in the experiment. Simultaneously, elevation of intracellular [Ca²⁺]i and reactive oxygen species (ROS) levels, significant depletion of mitochondrial membrane potential(Δ Ψ) and cellular glutathione (GSH), intracellular acidification, and inhibition of Na⁺, K⁺-ATPase and Ca²⁺-ATPase activities were revealed in a dose-dependent manner during the exposure, while the cellular death and the apoptosis could be markedly reversed by N-acetyl-l-cysteine (NAC). Also, the calcium overload and GSH depletion were significantly affected by NAC. In conclusion, exposure of rPT cells to low-dose cadmium led to cellular death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death might be the chief mechanism, which may be mediated by oxidative stress. Also, a disorder of intracellular homeostasis induced by oxidative stress and mitochondrial dysfunction is a trigger of apoptosis in rPT cells.     

Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes

Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction.     

Expression of the Maize Mnsod (Sod3) Gene in Mnsod-Deficient Yeast Rescues the Mutant Yeast under Oxidative Stress

Superoxide dismutases (SOD) are ubiquitous in aerobic organisms and are believed to play a significant role in protecting cells against the toxic, often lethal, effect of oxygen free radicals. However, direct evidence that SOD does in fact participate in such a protective role is scant. The MnSOD-deficient yeast strain (Sod2d) offered an opportunity to test the functional role of one of several SOD isozymes from the higher plant maize in hopes of establishing a functional bioassay for other SODs. Herein, we present evidence that MnSOD functions to protect cells from oxidative stress and that this function is conserved between species. The maize Sod3 gene was introduced into the yeast strain Sod2d where it was properly expressed and its product processed into the yeast mitochondrial matrix and assembled into the functional homotetramer. Most significantly, expression of the maize Sod3 transgene in yeast rendered the transformed yeast cells resistant to paraquat-induced oxidative stress by complementing the MnSOD deficiency. Furthermore, analyses with various deletion mutants of the maize SOD-3 transit peptide in the MnSOD-deficient yeast strain indicate that the initial portion (about 8 amino acids) of the maize transit peptide is required to direct the protein into the yeast mitochondrial matrix in vivo to function properly. These findings indicate that the functional role of maize MnSOD is conserved and dependent on its proper subcellular location in the mitochondria of a heterologous system.     

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.     

Mitochondrial Dysfunction Enhances Susceptibility to Oxidative Stress by Down-Regulation of Thioredoxin in Human Neuroblastoma Cells

Increasing evidence suggests that Alzheimer’s disease is associated with mitochondrial dysfunction and oxidative damage. To develop a cellular model of Alzheimer’s disease, we investigated the effects of thioredoxin (Trx) expression in the response to mitochondrial dysfunction-enhanced oxidative stress in the SH-SY5Y human neuroblastoma cells. Treatment of SH-SY5Y cells with 15 mM of NaN₃, an inhibitor of cytochrome c oxidase (complex IV), led to alteration of mitochondrial membrane potential but no significant changes in cell viability. Therefore, cells were first treated with 15 mM NaN₃ to induce mitochondrial dysfunction, then, exposed to different concentrations of H₂O₂. Cell susceptibility was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay and morphological observation. Expressions of Trx mRNA and protein were determined by RT-PCR; and Western-blot analysis, respectively. It was found that the SH-SY5Y cells with mitochondrial impairment had lower levels of Trx mRNA and protein, and were significantly more vulnerable than the normal cells after exposure to H₂O₂ while no significant changes of Trx mRNA and protein in SH-SY5Y cells exposed to H₂O₂ but without mitochondrial complex IV inhibition. These results, together with our previous study in primary cultured neurons, demonstrated that the increased susceptibility to oxidative stress is induced at least in part by the down-regulation of Trx in SH-SY5Y human neuroblastoma cells with mitochondrial impairment and also suggest the mitochondrial dysfunction-enhanced oxidative stress could be used as a cellular model to study the mechanisms of Alzheimer’s disease and agents for prevention and treatment.     

Curcumin Ameliorates Copper-Induced Neurotoxicity Through Inhibiting Oxidative Stress and Mitochondrial Apoptosis in SH-SY5Y Cells

Neurochemical Research - Impaired homeostasis of copper has been linked to different pathophysiological mechanisms in neurodegenerative diseases and oxidative injury has been proposed as the main...     

Maternal Embryonic Leucine Zipper Kinase (MELK) Reduces Replication Stress in Glioblastoma Cells

Maternal embryonic leucine zipper kinase (MELK) belongs to the subfamily of AMP-activated Ser/Thr protein kinases. The expression of MELK is very high in glioblastoma-type brain tumors, but it is not clear how this contributes to tumor growth. Here we show that the siRNA-mediated loss of MELK in U87 MG glioblastoma cells causes a G₁/S phase cell cycle arrest accompanied by cell death or a senescence-like phenotype that can be rescued by the expression of siRNA-resistant MELK. This cell cycle arrest is mediated by an increased expression of p21^WAF1/CIP1, an inhibitor of cyclin-dependent kinases, and is associated with the hypophosphorylation of the retinoblastoma protein and the down-regulation of E2F target genes. The increased expression of p21 can be explained by the consecutive activation of ATM (ataxia telangiectasia mutated), Chk2, and p53. Intriguingly, the activation of p53 in MELK-deficient cells is not due to an increased stability of p53 but stems from the loss of MDMX (mouse double minute-X), an inhibitor of p53 transactivation. The activation of the ATM-Chk2 pathway in MELK-deficient cells is associated with the accumulation of DNA double-strand breaks during replication, as demonstrated by the appearance of γH2AX foci. Replication stress in these cells is also illustrated by an increased number of stalled replication forks and a reduced fork progression speed. Our data indicate that glioblastoma cells have elevated MELK protein levels to better cope with replication stress during unperturbed S phase. Hence, MELK inhibitors hold great potential for the treatment of glioblastomas as such or in combination with DNA-damaging therapies.