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1.
Miki Yoshida Makio Saeki Hiroshi Egusa Yasuyuki Irie Yuya Kamano Shinya Uraguchi Maki Sotozono Hitoshi Niwa Yoshinori Kamisaki 《Biochemical and biophysical research communications》2013,430(1):320-324
We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex. 相似文献
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Julien Henri Marie-Eve Chagot Maxime Bourguet Yoann Abel Guillaume Terral Chloé Maurizy Christelle Aigueperse Florian Georgescauld Franck Vandermoere Rénette Saint-Fort Isabelle Behm-Ansmant Bruno Charpentier Bérengère Pradet-Balade Céline Verheggen Edouard Bertrand Philippe Meyer Sarah Cianférani Xavier Manival Marc Quinternet 《Structure (London, England : 1993)》2018,26(9):1196-1209.e8
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Huang Huang Wang Yuhan Huang Tianmiao Wang Lingyan Liu Yangzhi Wu Qiong Yu Ang Shi Meiyun Wang Xiaoyu Li Wenli Zhang Jianing Liu Yubo 《Journal of physiology and biochemistry》2021,77(4):625-638
Journal of Physiology and Biochemistry - Apoptosis plays an important role in both carcinogenesis and cancer treatment. Understanding the mechanisms through which resistance to apoptosis occurs in... 相似文献
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The two closely related AAA+family ATPases Rvb1 and Rvb2 are part of several critical multiprotein complexes, and, thus, are involved in a wide range of cellular processes including chromatin remodelling, telomerase assembly, and snoRNP biogenesis. It was found that Rvb1 and Rvb2 form a tight functional complex with Pih1 (Protein interacting with Hsp90) and Tah1 (TPR-containing protein associated with Hsp90), which are two Hsp90 interactors. We named the complex R2TP. The complex was originally isolated from Saccharomyces cerevisiae and was, subsequently, identified in mammalian cells. R2TP was found to be required for box C/D snoRNP biogenesis in yeast and mammalian cells. More recently, several studies revealed that the complex is also involved in multiple biological processes including apoptosis, phosphatidylinositol-3 kinase-related protein kinase (PIKK) signalling, and RNA polymerase II assembly. In this review, we describe the discovery of the complex and discuss the emerging critical roles that R2TP plays in distinct cellular processes. 相似文献
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Assembly principles of the human R2TP chaperone complex reveal the presence of R2T and R2P complexes
《Structure (London, England : 1993)》2022,30(1):156-171.e12
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7.
Background
Box C/D snoRNPs, which are typically composed of box C/D snoRNA and the four core protein components Nop1, Nop56, Nop58, and Snu13, play an essential role in the modification and processing of pre-ribosomal RNA. The highly conserved R2TP complex, comprising the proteins Rvb1, Rvb2, Tah1, and Pih1, has been shown to be required for box C/D snoRNP biogenesis and assembly; however, the molecular basis of R2TP chaperone-like activity is not yet known.Results
Here, we describe an unexpected finding in which the activity of the R2TP complex is required for Nop58 protein stability and is controlled by the dynamic subcellular redistribution of the complex in response to growth conditions and nutrient availability. In growing cells, the complex localizes to the nucleus and interacts with box C/D snoRNPs. This interaction is significantly reduced in poorly growing cells as R2TP predominantly relocalizes to the cytoplasm. The R2TP-snoRNP interaction is mainly mediated by Pih1.Conclusions
The R2TP complex exerts a novel regulation on box C/D snoRNP biogenesis that affects their assembly and consequently pre-rRNA maturation in response to different growth conditions.Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0404-4) contains supplementary material, which is available to authorized users. 相似文献8.
Stobadine inhibits doxorubicin-induced apoptosis through a caspase-9 dependent pathway in P815 mastocytoma cells 总被引:3,自引:0,他引:3
Doxorubicin (DOXO), a widely used chemotherapeutic agent, induces apoptosis in transformed and non-transformed cells. The apoptotic effect of DOXO has been linked to the generation of reactive oxygen species (ROS). Antioxidants may be effective in the prevention of DOX-induced apoptosis. In the present study we investigated the effects of stobadine, a pyridoindole antioxidant in a DOXO-induced apoptosis model of P815 cells by flow cytometric analyses and by measuring caspase-3 and caspase-9 activities. Pretreating cells with stobadine significantly increased cell viability and decreased apoptosis rate. Inhibition in apoptosis was observed at maximum levels following treatment of cells with 10(-7)M stobadine as evident from flow cytometric analyses. The antiapoptotic effect of stobadine was further confirmed by inhibition of caspase-3 and caspase-9 activities. We found that the antioxidative effects of stobadine were comparable to the effects of a well known antioxidant, N-acetyl l-cysteine (NAC). 相似文献
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In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability. 相似文献
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Strushkevich N Usanov SA Plotnikov AN Jones G Park HW 《Journal of molecular biology》2008,380(1):95-106
The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D3. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D3 is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1. 相似文献
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Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level. 相似文献
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Li G Yang Q Krishnan S Alexander EA Borkan SC Schwartz JH 《Cell death and differentiation》2006,13(12):2109-2117
The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis. 相似文献
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Assembly of dynein arms requires cytoplasmic processes which are mediated by dynein preassembly factors (DNAAFs). CFAP298, which is conserved in organisms with motile cilia, is required for assembly of dynein arms but with obscure mechanisms. Here, we show that FBB18, a Chlamydomonas homologue of CFAP298, localizes to the cytoplasm and functions in folding/stabilization of almost all axonemal dyneins at the early steps of dynein preassembly. Mutation of FBB18 causes no or short cilia accompanied with partial loss of both outer and inner dynein arms. Comparative proteomics using 15N labeling suggests partial degradation of almost all axonemal dynein heavy chains (DHCs). A mutant mimicking a patient variant induces particular loss of DHCα. FBB18 associates with 9 DNAAFs and 14 out of 15 dynein HCs but not with IC1/IC2. FBB18 interacts with RuvBL1/2, components of the HSP90 co-chaperone R2TP complex but not the holo-R2TP complex. Further analysis suggests simultaneous formation of multiple DNAAF complexes involves dynein folding/stability and thus provides new insights into axonemal dynein preassembly. 相似文献
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Reactive oxygen formation plays a mechanistic role in the cardiotoxicity of doxorubicin, a chemotherapeutic agent that remains an important component of treatment programs for breast cancer and hematopoietic malignancies. To examine the role of doxorubicin-induced reactive oxygen species (ROS) in drug-related cardiac apoptosis, murine embryonic fibroblast cell lines were derived from the hearts of glutathione peroxidase 1 (Gpx-1) knockout mice. Cells from homozygous Gpx-1 knockout mice and parental animals were propagated with (Se+) and without (Se-) 100 nM sodium selenite. Activity levels of the peroxide detoxifying selenoprotein glutathione peroxidase (GSHPx) were marginally detectable (<1.6 nmol/min/mg) in fibroblasts from homozygous knockout animals whether or not cells were supplemented with selenium. GSHPx activity in Se- cells from parental murine fibroblasts was also <1.6 nmol/min/mg, whereas GSHPx levels in Se+ parental murine fibroblasts were 12.9 ± 2.7 nmol/min/mg (mean ± SE; P < 0.05). Catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glucose 6-phosphate dehydrogenase, and reduced glutathione activities did not differ amongst the four cell lines. Reactive oxygen production increased from 908 ± 122 (arbitrary units) for untreated control cells to 1668 ± 54 following exposure to 1 μM doxorubicin for 24 h in parental fibroblasts not supplemented with selenium (P < 0.03); reactive oxygen formation in doxorubicin-treated parental fibroblasts propagated in selenium was 996 ± 69 (P = not significant compared to untreated control cells). Reactive oxygen levels in homozygous Gpx-1 knockout fibroblasts, irrespective of selenium supplementation status, were increased and equivalent to that in selenium deficient wild type fibroblasts. When cardiac fibroblasts were exposed to doxorubicin (0.05 μM) for 96 h and examined for cell cycle alterations by flow cytometry, and apoptosis by TUNEL assay, marked G2 arrest and TUNEL positivity were observed in knockout fibroblasts in the presence or absence of supplemental selenium, and in parental fibroblasts propagated without selenium. Parental fibroblasts propagated with selenium and exposed to the same concentration of doxorubicin demonstrated modest TUNEL positivity and substantially diminished amounts of low molecular weight DNA. These results were replicated in cardiac fibroblasts exposed to doxorubicin (1–2 μM) for 2 h (to mimic clinical drug dosing schedules) and examined 96 h following initiation of drug exposure. Doxorubicin uptake in cardiac fibroblasts was similar irrespective of the mRNA expression level or activity of GSHPx. These experiments suggest that the intracellular levels of doxorubicin-induced reactive oxygen species (ROS) are modulated by GSHPx and play an important role in doxorubicin-related apoptosis and altered cell cycle progression in murine cardiac fibroblasts. 相似文献
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目的探讨硫化氢(H2S)对阿霉素(DOX)诱导的H9c2细胞损伤的影响及其作用机制。
方法H2S对DOX心肌毒性保护作用的实验分组为:对照组(Control组),5?μmol/?L DOX处理组(A组),5?μmol/L DOX和400?μmol/L NaHS共同处理组(B组),400?μmol/L NaHS单独处理组(C组),5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组(D组),15?μmol/L Sirtinol单独处理组(E组)。SIRT1是否参与H2S抗DOX心肌毒性作用机制的实验分组为:对照组(Control组),5?μmol/L DOX处理组(F组),5?μmol/L DOX和400?μmol/L NaHS共同处理组(G组),5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组(H组),15?μmol/L Sirtinol单独处理组(I组)。使用MTT法检测细胞活力;Elisa法检测细胞MDA以及SOD水平;DCFH-?DA荧光探针法检测ROS水平;采用Western Blot法检测SIRT1蛋白表达。使用单因素方差分析法进行统计学分析。
结果NaHS预处理可抑制DOX导致的H9c2细胞活力下降:Control组,A组、B组、C组细胞活力分别为100﹪、(54.58±1.58)﹪、(85.05±4.31)﹪、(100.22±4.46)﹪ (F = 134.9,P < 0.001)。NaHS预处理可减弱DOX引起的H9c2细胞ROS、MDA水平的增加以及SOD水平的降低:Control组的ROS、MDA和SOD水平分别是100﹪、(34.18±1.56) μmol/g、(53.69±1.44) U/?mg;A组的ROS、MDA和SOD水平分别是(174.90±12.65)﹪、(72.65±2.66) μmol/g、(31.80±2.05) U/?mg;B组的ROS、MDA和SOD水平分别是(126.08±6.25)﹪、(44.59±1.92) μmol/g、(48.06±1.56) U/mg;C组的ROS、MDA和SOD水平分别是(91.86±1.66)﹪、(32.93±1.56)?μmol/?g、(55.93±1.58)?U/?mg (F?= 83.26,P < 0.001;F = 271.4,P < 0.001;F = 127.0,P < 0.001)。F组(6、12、24?h)H9c2细胞SIRT1蛋白表达水平分别是(0.45±0.03)、(0.27±0.02)、(0.25±0.03),较Control组(1.00±0.00)降低(F = 611.1,P < 0.001)。本研究还发现,NaHS预处理H9c2细胞能阻止DOX引起的SIRT1蛋白表达下调:Control组、F组、G组、H组的SIRT1蛋白表达水平分别是(1.00±0.00)、(0.31±0.03)、(0.60±0.04)、(1.09±0.09)(F = 123.4,P?0.001)。SIRT1抑制剂Sirtinol预处理能明显逆转H2S对DOX诱导的H9c2细胞活力降低的抑制作用:Control组,F组、G组、H组、I组细胞活力分别为100﹪、(54.58±1.58)﹪、(85.37±3.62)﹪、(71.11±2.11)﹪、(97.53±1.45)﹪ (F = 238.2,P < 0.001)。Sirtinol预处理可明显逆转H2S对DOX导致的H9c2细胞ROS和MDA含量增加及SOD水平降低的抑制作用:Control组的ROS、MDA和SOD水平分别是100﹪、(35.84±2.22)μmol/?g、(53.03±3.16) U/mg;F组的ROS、MDA和SOD水平分别是(184.6±11.33)﹪、(74.78±5.30)μmol/g、(29.26±0.85)U/mg;G组的ROS、MDA和SOD水平分别是(126.5±7.57)﹪、(41.95±3.43)μmol/g、(52.61±2.26)U/mg;H组的ROS、MDA和SOD水平分别是(174.7±5.50)﹪、(67.69±1.52) μmol/g、(35.33±1.95) U/mg,I组的ROS、MDA和SOD水平分别是(98.03±2.86)﹪、(37.66±2.49)μmol/g、51.14 U/mg(F = 112.0,P < 0.001;F = 93.73,P < 0.001;F = 84.92,P < 0.001)。
结论H2S通过调控SIRT1抑制DOX诱导的H9c2细胞损伤。 相似文献
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Zhao R Kakihara Y Gribun A Huen J Yang G Khanna M Costanzo M Brost RL Boone C Hughes TR Yip CM Houry WA 《The Journal of cell biology》2008,180(3):563-578
Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs. 相似文献
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Zhou Z Lu X Zhu P Zhu W Mu X Qu R Li M 《Biochemical and biophysical research communications》2012,420(2):336-342
Vascular endothelial growth factor-correlated chemokine 1 (VCC-1), a recently described chemokine, is hypothesized to be associated with carcinogenesis. However, the molecular mechanisms by which aberrant VCC-1 expression determines poor outcomes of cancers are unknown. In this study, we found that VCC-1 was highly expressed in hepatocellular carcinoma (HCC) tissue. It was also associated with proliferation of HepG2 cells, and inhibition of cisplatin-induced apoptosis of HepG2 cells. Conversely, down-regulation of VCC-1 in HepG2 cells increased cisplatin-induced apoptosis of HepG2 cells. In summary, these results suggest that VCC-1 is involved in cisplatin-induced apoptosis of HepG2 cells, and also provides some evidence for VCC-1 as a potential cellular target for chemotherapy. 相似文献
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The acid-labile subunit (ALS) regulates IGF bioavailability by forming heterotrimeric complexes with IGFs and IGF-binding protein-3 (IGFBP-3). A homozygous missense mutation (D440N) resulting in undetectable circulating levels of ALS with a concomitant reduction in IGF-I and IGFBP-3 has been reported to cause mild growth retardation. To understand how this particular mutation affects ALS circulating levels and IGF-transport function, we expressed recombinant ALS and its variants, D440N-ALS, T442A-ALS, and D440N/T442A-ALS, using adenovirus vectors. Compared with wild-type ALS, the secretion of D440N-ALS was 80% lower. The D440N mutation was proposed to generate an N-glycosylation site additional to the seven existing motifs in ALS. D440N-ALS appeared larger than ALS, attributable to N-linked glycans because deglycosylation with N-glycosidase F reduced both proteins to the same molecular mass. When ALS was incubated with IGF-I and IGFBP-3, 70-80% of IGF-I was detected by gel-filtration chromatography in forms corresponding to the 150-kDa ternary complex. In contrast, when D440N-ALS was tested, less than 30% of IGF-I was found in high molecular mass complexes. Two other ALS variants mutated in the same putative glycosylation site, D440N/T442A-ALS and T442A-ALS, showed similar chromatographic profiles to wild-type ALS. The D440N mutation in ALS generates a hyperglycosylated form with impaired secretion and complex formation, potentially leading to dysregulation of endocrine IGF, thus contributing to the growth retardation observed in the affected patient. This is the first study to explain how a natural mutation, D440N, in ALS impairs its function. 相似文献
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