Background:Ossification of the posterior longitudinal ligament(OPLL)is a prevalent condition in orthopedics.While death-associated protein kinase 2(DAPK2)is known to play roles in cellular apoptosis and autophagy,its ...Background:Ossification of the posterior longitudinal ligament(OPLL)is a prevalent condition in orthopedics.While death-associated protein kinase 2(DAPK2)is known to play roles in cellular apoptosis and autophagy,its specific contributions to the advancement of OPLL are not well understood.Methods:Ligament fibroblasts were harvested from patients diagnosed with OPLL.Techniques such as real-time reverse transcriptasepolymerase chain reaction(RT-qPCR)and Western blot analysis were employed to assess DAPK2 levels in both ligament tissues and cultured fibroblasts.The extent of osteogenic differentiation in these cells was evaluated using an alizarin red S(ARS)staining.Additionally,the expression of ossification markers and autophagy markers was quantified.The autophagic activity was further analyzed through LC3 immunofluorescence and transmission electron microscopy(TEM).An in vivo heterotopic bone formation assay was conducted in mice to assess the role of DAPK2 in ossification.Results:Elevated DAPK2 expression was confirmed in both OPLL patient tissues and derived fibroblasts,in contrast to non-OPLL controls.Silencing of DAPK2 significantly curtailed osteogenic differentiation and autophagy in these fibroblasts,evidenced by decreased levels of LC3,and Beclin1,and reduced autophagosome formation.Additionally,DAPK2 was found to inhibit the mechanistic target of the rapamycin complex 1(mTORC1)complex’s activity.In vivo studies demonstrated that DAPK2 facilitates ossification,and this effect could be counteracted by the mTORC1 inhibitor rapamycin.Conclusion:DAPK2 enhances autophagy and osteogenic processes in OPLL through modulation of the mTORC1 pathway.展开更多
目的探讨MAL2(myelin and lymphocyte protein 2)在肺癌中的功能及作用机制。方法分析TCGA数据中MAL2在肺癌组织中的表达情况,在H1299和A549细胞中用克隆形成实验检测MAL2基因敲减后对肺癌细胞增殖的影响,探讨MAL2在肺腺癌细胞增殖中的...目的探讨MAL2(myelin and lymphocyte protein 2)在肺癌中的功能及作用机制。方法分析TCGA数据中MAL2在肺癌组织中的表达情况,在H1299和A549细胞中用克隆形成实验检测MAL2基因敲减后对肺癌细胞增殖的影响,探讨MAL2在肺腺癌细胞增殖中的作用,并用CCK8等细胞增殖、转移、凋亡实验进一步验证其功能;将MAL2干扰后,采用Western blot实验检测下游通路基因表达情况以分析MAL2基因可能的作用机制。结果MAL2基因在肺癌组织与癌旁组织相比表达显著升高(P<0.05),且MAL2敲减后抑制细胞增殖和转移、促进细胞凋亡(P<0.05);MAL2可能通过调控SGK1通路发挥作用。结论MAL2基因在肺癌的恶性进展中起重要的作用,该作用通过调控SGK1机制进行,MAL2基因是肺癌的一个新的潜在的分子诊断指标及治疗靶点。展开更多
Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complex...Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.展开更多
Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to have consensus sequence of phosphorylation site R-x-R-x-x-(S/T)-Φ, where Φ is any hydrophobic amino acid and arginine residues are conserved at positions...Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to have consensus sequence of phosphorylation site R-x-R-x-x-(S/T)-Φ, where Φ is any hydrophobic amino acid and arginine residues are conserved at positions −5 and −3 relative to positions of Ser/Thr residues that are phosphorylated in the presence of SGK1. UNC-21-like kinase 2 (ULK2) also harbors putative SGK1 phosphorylation sites at both Ser507 (502RsRnsSG508) and Ser750 (745RtRttSV751) residues. Thus, the objective of this study was to determine whether Ser507 and Ser750 residues of ULK2 could be phosphorylation sites of SGK1 as one of its authentic substrate proteins. Using ULK2 507 and 750 serine residue un- or phosphorylation analog (S507AS750A or 507DS750D), we observed that modification of Ser507 or Ser750 residue was required to activate the kinase activity of ULK2 and sensitize ULK2 to stress or starvation while simultaneously enhancing its active state and autophagy characteristics, suggesting that phosphorylation at Ser750 or Ser507 residue could modulate its subcellular localization and protein interaction with AMPK1α to activate ULK2. We also observed that ULK2 autophagy activity was enhanced by GSK650394 (an SGK1 inhibitor) to compensate survival capacity through increasing its association with LC3 and phosphorylation. When SGK1 known to be associated with cell survival was inhibited by GSK650394, ULK2 autophagy pathway was activated to avoid cell death alternatively. Thus, our observations indicate that phosphorylation of ULK2 by SGK1 can regulate cell survival as an alternative modulation of ULK2 functions.展开更多
哺乳动物雷帕霉素靶蛋白复合体(mammalian target of rapamycin complex,mTORC)是细胞生长、存活、代谢的重要调控中心,它对维持生命有机体的正常生理活动和内稳态的平衡有着重要作用。mTORC根据其蛋白组份可分为mTORC1和mTORC2。mTORC...哺乳动物雷帕霉素靶蛋白复合体(mammalian target of rapamycin complex,mTORC)是细胞生长、存活、代谢的重要调控中心,它对维持生命有机体的正常生理活动和内稳态的平衡有着重要作用。mTORC根据其蛋白组份可分为mTORC1和mTORC2。mTORC2的主要组成蛋白有mTOR、Rictor、mLST8、Deptor、mSin1、Protor和Hsp70。mTORC2通过作用于Akt,PKCα和SGK1等来调控多项生命活动,如胚胎发育,细胞骨架重建,细胞迁移,蛋白质翻译和修饰等。mTOR信号通路异常已被证实与肿瘤相关,同时发现多种肿瘤发生与mTORC2及其异常调节信号通路相关。因此,对mTORC2组成、功能以及参与的信号通路的研究,可能为进一步研制其相关的靶向抑制药物乃至肿瘤治疗提供新思路。本综述将介绍mTORC2的组成结构、功能、参与的信号通路,及其在血液肿瘤中作用的研究进展。展开更多
Akt/mTOR/p70S6K1 signaling pathway plays an important role in the pathogenesis of cancer and diabetes.Macrophages and lymphocytes are involved in the pathogenesis of diabetes,diabetic atherosclerosis,formation of insu...Akt/mTOR/p70S6K1 signaling pathway plays an important role in the pathogenesis of cancer and diabetes.Macrophages and lymphocytes are involved in the pathogenesis of diabetes,diabetic atherosclerosis,formation of insulin resistance as well as immune response to cancer and tumor maintenance.The aim of the study was to determine the Akt activation by mTORC2 in peripheral blood mononuclear cell(PBMC)of patients with type 2 diabetes and cancer.The following groups were studied:control group,patients with type 2 diabetes,cancer patients and patients with both cancer and diabetes.The amounts of phospho-Akt(р-S473)and phospho-p70S6K1(p-T389)were determined using ELISA kits.The amount of phosphorylated Akt significantly increases in PBMC of patients with cancer.There was no effect in PBMC from patients with type 2 diabetes and significant decrease in the amount of phospho-Akt in PBMC of the patients group both with cancer and diabetes.p70S6K1 activation was observed in PBMC of the groups 2 and 3 patients.Thus,chronic diseases such as type 2 diabetes and cancer can affect the signaling mechanisms in blood cells.The state of Akt phosphorylation in leukocytes can indicate the activity of mTORC1 and its substrates,which may be important for the evaluation of the pathological process and the efficacy of the drugs.展开更多
There are many fuctions with the representative autophagy associated protein p62,such as cell signal transduction,cell proliferation,apoptosis,inflammation,tumorigenesis and oxidative stress.p62 over express will lead...There are many fuctions with the representative autophagy associated protein p62,such as cell signal transduction,cell proliferation,apoptosis,inflammation,tumorigenesis and oxidative stress.p62 over express will lead to autophagy disorder by controlling keap1-Nrf2mTORC1,NF-κB signaling pathways,thus promoting the development of the tumor.The overexpression of p62 in tissues can lead to the occurrence of the gynecological malignant tumors,such as ceicival cancer,ovarian carcinoma and Endometrial carcinoma.And the level of p62 is correlated with the tumor progression,the tumor grade and the survival rates of patients.p62 and its upstream and downstream molecules are expected to be the markers and therapeutic targets for predicting the clinical outcome of the gynecological malignant cancer patients.展开更多
基金This research received funding from the Natural Science Foundation of Shanghai(Grant No.20ZR1457600)the School-Level Basic Medical Project of Naval Medical University(Grant No.2021MS13).
文摘Background:Ossification of the posterior longitudinal ligament(OPLL)is a prevalent condition in orthopedics.While death-associated protein kinase 2(DAPK2)is known to play roles in cellular apoptosis and autophagy,its specific contributions to the advancement of OPLL are not well understood.Methods:Ligament fibroblasts were harvested from patients diagnosed with OPLL.Techniques such as real-time reverse transcriptasepolymerase chain reaction(RT-qPCR)and Western blot analysis were employed to assess DAPK2 levels in both ligament tissues and cultured fibroblasts.The extent of osteogenic differentiation in these cells was evaluated using an alizarin red S(ARS)staining.Additionally,the expression of ossification markers and autophagy markers was quantified.The autophagic activity was further analyzed through LC3 immunofluorescence and transmission electron microscopy(TEM).An in vivo heterotopic bone formation assay was conducted in mice to assess the role of DAPK2 in ossification.Results:Elevated DAPK2 expression was confirmed in both OPLL patient tissues and derived fibroblasts,in contrast to non-OPLL controls.Silencing of DAPK2 significantly curtailed osteogenic differentiation and autophagy in these fibroblasts,evidenced by decreased levels of LC3,and Beclin1,and reduced autophagosome formation.Additionally,DAPK2 was found to inhibit the mechanistic target of the rapamycin complex 1(mTORC1)complex’s activity.In vivo studies demonstrated that DAPK2 facilitates ossification,and this effect could be counteracted by the mTORC1 inhibitor rapamycin.Conclusion:DAPK2 enhances autophagy and osteogenic processes in OPLL through modulation of the mTORC1 pathway.
文摘目的探讨MAL2(myelin and lymphocyte protein 2)在肺癌中的功能及作用机制。方法分析TCGA数据中MAL2在肺癌组织中的表达情况,在H1299和A549细胞中用克隆形成实验检测MAL2基因敲减后对肺癌细胞增殖的影响,探讨MAL2在肺腺癌细胞增殖中的作用,并用CCK8等细胞增殖、转移、凋亡实验进一步验证其功能;将MAL2干扰后,采用Western blot实验检测下游通路基因表达情况以分析MAL2基因可能的作用机制。结果MAL2基因在肺癌组织与癌旁组织相比表达显著升高(P<0.05),且MAL2敲减后抑制细胞增殖和转移、促进细胞凋亡(P<0.05);MAL2可能通过调控SGK1通路发挥作用。结论MAL2基因在肺癌的恶性进展中起重要的作用,该作用通过调控SGK1机制进行,MAL2基因是肺癌的一个新的潜在的分子诊断指标及治疗靶点。
文摘Heat shock proteins (HSPs) serve to correct proteins’ conformation, send the damaged proteins for degradation (quality control function). Heat shock factors (HSFs) are their transcription factors. The protein complexes mTOR1 and 2 (with the same core mTOR), the phosphoinositide-dependent protein kinase-1 (PDK1), the seine/threonine-specific protein kinase (Akt), HSF1, plus their associated proteins form a network participating in protein synthesis, bio-energy generation, signaling for apoptosis with the help of HSPs. A cancer cell synthesizes proteins at fast rate and needs more HSPs to work on quality control. Shutting down this network would lead to cell death. Thus inhibitors of mTOR (mTORI) and inhibitors of HSPs (HSPI) could drive cancer cell to apoptosis—a “passive approach”. On the other hand, HSPs form complexes with polypeptides characteristic of the cancer cells;on excretion from the cell, they becomes antigens for the immunity cells, eventually leading to maturation of the cytotoxic T cells, forming the basic principle of preparing cancer-specific, person-specific vaccine. Recent finding shows that HSP70 can penetrate cancer cell and expel its analog to extracellular region, giving the hope to prepare a non-person-specific vaccine covering a variety of cancers. Activation of anti-cancer immunity is the “active approach”. On the other hand, mild hyperthermia, with increase of intracellular HSPs, has been found to activate the immunity response, and demonstrate anti-cancer effects. There are certain “mysteries” behind the mechanisms of the active and passive approaches. We analyze the mechanisms involved and provide explanations to some mysteries. We also suggest future research to improve our understanding of these two approaches, in which HSPs play many roles.
文摘Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to have consensus sequence of phosphorylation site R-x-R-x-x-(S/T)-Φ, where Φ is any hydrophobic amino acid and arginine residues are conserved at positions −5 and −3 relative to positions of Ser/Thr residues that are phosphorylated in the presence of SGK1. UNC-21-like kinase 2 (ULK2) also harbors putative SGK1 phosphorylation sites at both Ser507 (502RsRnsSG508) and Ser750 (745RtRttSV751) residues. Thus, the objective of this study was to determine whether Ser507 and Ser750 residues of ULK2 could be phosphorylation sites of SGK1 as one of its authentic substrate proteins. Using ULK2 507 and 750 serine residue un- or phosphorylation analog (S507AS750A or 507DS750D), we observed that modification of Ser507 or Ser750 residue was required to activate the kinase activity of ULK2 and sensitize ULK2 to stress or starvation while simultaneously enhancing its active state and autophagy characteristics, suggesting that phosphorylation at Ser750 or Ser507 residue could modulate its subcellular localization and protein interaction with AMPK1α to activate ULK2. We also observed that ULK2 autophagy activity was enhanced by GSK650394 (an SGK1 inhibitor) to compensate survival capacity through increasing its association with LC3 and phosphorylation. When SGK1 known to be associated with cell survival was inhibited by GSK650394, ULK2 autophagy pathway was activated to avoid cell death alternatively. Thus, our observations indicate that phosphorylation of ULK2 by SGK1 can regulate cell survival as an alternative modulation of ULK2 functions.
文摘哺乳动物雷帕霉素靶蛋白复合体(mammalian target of rapamycin complex,mTORC)是细胞生长、存活、代谢的重要调控中心,它对维持生命有机体的正常生理活动和内稳态的平衡有着重要作用。mTORC根据其蛋白组份可分为mTORC1和mTORC2。mTORC2的主要组成蛋白有mTOR、Rictor、mLST8、Deptor、mSin1、Protor和Hsp70。mTORC2通过作用于Akt,PKCα和SGK1等来调控多项生命活动,如胚胎发育,细胞骨架重建,细胞迁移,蛋白质翻译和修饰等。mTOR信号通路异常已被证实与肿瘤相关,同时发现多种肿瘤发生与mTORC2及其异常调节信号通路相关。因此,对mTORC2组成、功能以及参与的信号通路的研究,可能为进一步研制其相关的靶向抑制药物乃至肿瘤治疗提供新思路。本综述将介绍mTORC2的组成结构、功能、参与的信号通路,及其在血液肿瘤中作用的研究进展。
文摘Akt/mTOR/p70S6K1 signaling pathway plays an important role in the pathogenesis of cancer and diabetes.Macrophages and lymphocytes are involved in the pathogenesis of diabetes,diabetic atherosclerosis,formation of insulin resistance as well as immune response to cancer and tumor maintenance.The aim of the study was to determine the Akt activation by mTORC2 in peripheral blood mononuclear cell(PBMC)of patients with type 2 diabetes and cancer.The following groups were studied:control group,patients with type 2 diabetes,cancer patients and patients with both cancer and diabetes.The amounts of phospho-Akt(р-S473)and phospho-p70S6K1(p-T389)were determined using ELISA kits.The amount of phosphorylated Akt significantly increases in PBMC of patients with cancer.There was no effect in PBMC from patients with type 2 diabetes and significant decrease in the amount of phospho-Akt in PBMC of the patients group both with cancer and diabetes.p70S6K1 activation was observed in PBMC of the groups 2 and 3 patients.Thus,chronic diseases such as type 2 diabetes and cancer can affect the signaling mechanisms in blood cells.The state of Akt phosphorylation in leukocytes can indicate the activity of mTORC1 and its substrates,which may be important for the evaluation of the pathological process and the efficacy of the drugs.
基金Key Research and Development program of Shanxi Province(No.201903D421065)Shanxi Health Commission(No.201601046)PhD Foundation of Shanxi Medical University Second Affiliated Hospital(No.201701-8)
文摘There are many fuctions with the representative autophagy associated protein p62,such as cell signal transduction,cell proliferation,apoptosis,inflammation,tumorigenesis and oxidative stress.p62 over express will lead to autophagy disorder by controlling keap1-Nrf2mTORC1,NF-κB signaling pathways,thus promoting the development of the tumor.The overexpression of p62 in tissues can lead to the occurrence of the gynecological malignant tumors,such as ceicival cancer,ovarian carcinoma and Endometrial carcinoma.And the level of p62 is correlated with the tumor progression,the tumor grade and the survival rates of patients.p62 and its upstream and downstream molecules are expected to be the markers and therapeutic targets for predicting the clinical outcome of the gynecological malignant cancer patients.
