Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK-dependent manner

The mechanisms by which β1 integrins regulate chemoresistance of cancer cells are still poorly understood. In this study, we report that collagen/β1 integrin signaling inhibits doxorubicin-induced apoptosis of Jurkat and HSB2 leukemic T-cells by up-regulating the expression and function of the ATP-binding cassette C 1 (ABCC1) transporter, also known as multidrug resistance-associated protein 1. We find that collagen but not fibronectin reduces intracellular doxorubicin content and up-regulates the expression levels of ABCC1. Inhibition and knockdown studies show that up-regulation of ABCC1 is necessary for collagen-mediated reduction of intracellular doxorubicin content and collagen-mediated inhibition of doxorubicin-induced apoptosis. We also demonstrate that activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway is involved in collagen-induced reduction of intracellular doxorubicin accumulation, collagen-induced up-regulation of ABCC1 expression levels, and collagen-mediated cell survival. Finally, collagen-mediated up-regulation of ABCC1 expression and function also requires actin polymerization. Taken together, our results indicate for the first time that collagen/β1 integrin/ERK signaling up-regulates the expression and function of ABCC1 and suggest that its activation could represent an important pathway in cancer chemoresistance. Thus simultaneous targeting of collagen/β1 integrin and ABCC1 may be more efficient in preventing drug resistance than targeting each pathway alone.     

Ammonium Increases TRPC1 Expression Via Cav-1/PTEN/AKT/GSK3β Pathway

Hyperammonemia occurring following acute liver failure is the primary cause of hepatic encephalopathy. In the brain, ammonium is catabolised by glutamine synthetase expressed exclusively in astroglia; ammonium overload impairs astroglial homeostatic systems. Previously, we had reported that chronic treatment with 3 mM ammonia increased expression of transient receptor potential canonic 1 (TRPC1) channels and Ca²⁺ release from intracellular Ca²⁺ stores (Liang et al. in Neurochem Res 39:2127–2135, 2014). Glycogen synthase kinase 3β (GSK-3β) has a key role in several astroglial signalling pathways and is known to be affected in various CNS diseases. We have studied the involvement of Cav-1/PTEN/AKT/GSK-3β signalling system in regulation of TRPC1 gene expression by ammonium. Effects of chronic (1–5 days) treatment with ammonium chloride (ammonium), at pathologically relevant concentrations of 1–5 mM were investigated on primary cultures of mouse cerebral astrocytes. We quantified expression of caveolin-1 (Cav-1), membrane content of phosphatase and tensin homologue (PTEN), phosphorylation of AKT and GSK-3β, and expression of TRPC1 channels. Ammonium significantly increased expression of Cav-1 mRNA and protein, mRNA of TRPC1 as well as membrane content of PTEN; conversely phosphorylation of AKT and GSK-3β were significantly decreased. These changes were abolished following astrocytes treatment with siRNA specific to Cav-1, indicating the involvement of Cav-1/PTEN/PI3K/AKT pathway. Similar results were found in the brains of adult mice subjected to intraperitoneal injection of urease (a model for hyperammoniemia) for 1–5 days. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers (used for fluorescence-activated cell sorting of astrocytes vs. neurones) and treated with intraperitoneal injections of urease for 3 days, the Cav-1 gene mRNA expression was up-regulated in astrocytes, but not in neurones. The up-regulation of TRPC1 gene expression by ammonium was suppressed by GSK-3β inhibitors, lithium salt and AR-A014418, suggesting that increase of GSK-3β activity may play a role in ammonium-related pathologies.     

Akt1K64/276与SUMO1结合激活ERK1/2-Elk1-BDNF信号通路

本研究主要从蛋白质结构分析Akt1 SUMO化的位点及位点的突变对其结构与功能的影响。采用多种软件分析Akt1 SUMO化位点和Akt1野生型(Akt1wt)及Akt1K64/276R的理化性质、亲/疏水性及二/三级结构;分析结果显示,Akt1K64/276R较Akt1wt,亲/疏水性未改变,α-螺旋和β-折叠都有少量的不同。三级结构分析显示,与野生型组相比,Akt1K64R氢键增多。以Myc-Akt1wt-pcDNA3.1为模板,采用PCR定点突变技术扩增出Myc-Akt1K64/276R。DNA序列分析结果显示,Myc-Akt1K64/276R基因序列编码赖氨酸(K)的密码子AAG被成功突变为精氨酸(R)密码子AGG。免疫沉淀和免疫印迹结果显示,不共转PIAS3,Akt1 也能与SUMO1结合;Myc-Akt1wt和Myc-Akt1K64/276R均可在HEK293细胞中高效表达;转染Myc-Akt1K64/276R组SUMO化水平降低了70%左右(P<0.05)。免疫印迹结果显示,在小鼠海马神经细胞HT22中,Myc-Akt1wt 组ERK1/2磷酸化水平及BDNF蛋白水平是突变组的约1.5倍(P<0.05);野生型组p-Elk1是突变体组的2倍(P<0.05),而mTOR、P70S6K、4E-BP1的表达及磷酸化均无显著改变。以上结果表明,Akt1中K64/276的突变对蛋白质结构和表达未见影响,仅引起Akt1 SUMO化降低及下游ERK1/2-Elk1-BDNF信号通路的抑制。     

低氧经mTOR/4E-BP1和FoxO1/Atrogin-1通路影响骨骼肌蛋白质积累

低氧暴露对骨骼肌蛋白质合成/分解的影响受到广泛关注,但该过程中相关调控通路的研究仍十分有限。本研究拟通过蛋白质相对积累量来研究合成和分解通路的变化。将骨骼肌细胞置于低氧环境中培养,分别在0 h、6 h、12 h和24 h收集细胞,并进行检测。免疫荧光观察肌球蛋白(myosin),翻译表面感应检测蛋白质合成,Western印迹法测试蛋白质合成相关基因(ERK1/2、p-ERK1/2、mTOR、p-mTOR、4E-BP1、p-4E-BP1)、蛋白质分解相关基因(泛素、FoxO1、p-FoxO1、MuRF1和Atrogin-1)表达量。结果发现,随着低氧干预时间延长,肌纤维直径和骨骼肌细胞中蛋白质相对积累量随时间逐渐减小(P<0.01)。与0 h相比,6 h p-4E-BP1/4E-BP1和Atrogin-1的表达显著上调(P<0.05),p-mTOR表达显著高于0 h(P<0.01);6 h和24 h p-mTOR/mTOR的比值显著大于0 h(P<0.05),而p-FoxO1/FoxO1的比值随时间逐渐减小(P<0.01)。上述结果表明,低氧干预能够使骨骼肌细胞直径减少、骨骼肌细胞蛋白质积累减少,并且低氧打破骨骼肌细胞蛋白质合成和分解的平衡,可能是通过调节mTOR/4E-BP1通路活性和FoxO1/Atrogin-1通路的活性实现的。     

全球PD-1/PD-L1单克隆抗体市场竞争格局

目的:从产品开发角度分析PD-1/PD-L1单克隆抗体的发展现状和未来趋势。方法:检索科睿唯安(Clarivate Analytics)的Cortellis数据库的数据,利用定量分析法和对比分析法对检索结果进行分析。结果:目前已有5种PD-1/PD-L1单克隆抗体上市、4种PD-1/PD-L1单克隆抗体处于预注册及6种PD-1/PD-L1单克隆抗体处于临床Ⅲ期,未来市场上的PD-1/PD-L1单克隆抗体将呈现快速增长趋势。此外,PD-1/PD-L1单克隆抗体的商业交易也越来越多,目前共发生包括药物开发及商业化许可、专利资产出售及早期药物研发合作等10余起交易,其中药物开发及商业化许可是最主要的交易模式。结论:虽然PD-1/PD-L1单克隆抗体市场尚处于起步阶段,但随着未来技术的不断发展改进,相信未来有更多的PD-1/PD-L1单克隆抗体上市,为癌症及其他疾病的治疗提供新的契机。     

Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.     

Ubiquitin ligase SYVN1/HRD1 facilitates degradation of the SERPINA1 Z variant/α-1-antitrypsin Z variant via SQSTM1/p62-dependent selective autophagy

SERPINA1/AAT/α-1-antitrypsin (serpin family A member 1) deficiency (SERPINA1/ AAT-D) is an autosomal recessive disorder characterized by the retention of misfolded SERPINA1/AAT in the endoplasmic reticulum (ER) of hepatocytes and a significant reduction of serum SERPINA1/AAT level. The Z variant of SERPINA1/AAT, containing a Glu342Lys (E342K) mutation (SERPINA1^E342K/ATZ), the most common form of SERPINA1/AAT-D, is prone to misfolding and polymerization, which retains it in the ER of hepatocytes and leads to liver injury. Both proteasome and macroautophagy/autophagy pathways are responsible for disposal of SERPINA1^E342K/ATZ after it accumulates in the ER. However, the mechanisms by which SERPINA1^E342K/ATZ is selectively degraded by autophagy remain unknown. Here, we showed that ER membrane-spanning ubiquitin ligase (E3) SYVN1/HRD1 enhances the degradation of SERPINA1^E342K/ATZ through the autophagy-lysosome pathway. We found that SYVN1 promoted SERPINA1^E342K/ATZ, especially Triton X 100-insoluble SERPINA1^E342K/ATZ clearance. However, the effect of SYVN1 in SERPINA1^E342K/ATZ clearance was impaired after autophagy inhibition, as well as in autophagy-related 5 (atg5) knockout cells. On the contrary, autophagy induction enhanced SYVN1-mediated SERPINA1^E342K/ATZ degradation. Further study showed that SYVN1 mediated SERPINA1^E342K/ATZ ubiquitination, which is required for autophagic degradation of SERPINA1^E342K/ATZ by promoting the interaction between SERPINA1^E342K/ATZ and SQSTM1/p62 for formation of the autophagy complex. Interestingly, SYVN1-mediated lysine 48 (K48)-linked polyubiquitin chains that conjugated onto SERPINA1^E342K/ATZ might predominantly bind to the ubiquitin-associated (UBA) domain of SQSTM1 and couple the ubiquitinated SERPINA1^E342K/ATZ to the lysosome for degradation. In addition, autophagy inhibition attenuated the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ cytotoxicity, and the autophagy inducer rapamycin enhanced the suppressive effect of SYVN1 on SERPINA1^E342K/ATZ-induced cell apoptosis. Therefore, this study proved that SYVN1 enhances SERPINA1^E342K/ATZ degradation through SQSTM1-dependent autophagy and attenuates SERPINA1^E342K/ATZ cytotoxicity.     

1/4世纪的求索

中国生理学会第18届理事会第二次会议中强调,生理学的发展必须吸收各方面的新技术,但更重要的是要有好的思路,才能做出高水平的工作。1992年1月8日《生理科学进展》在京编委会上,委员们一致同意在本刊开辟一个“科研思路”新栏目,鼓励目前正在科研第一线拼搏的中青年科研工作者谈谈他们的设想,中老年科学工作者则可对既住的工作进行阶段性的回顾,并引出对今后的展望。为了尽速体现编委会决议,本刊在短期内组织了两篇稿件,希望起到抛砖引玉的作用,争取更多的生理科学工作者参加这项交流活动,以期把刊物办得更加活跃和更有生气。     

A/swine/H1N1流感病毒中国分离株血凝素进化分析

2009年3月以来,甲型H1N1病毒已在全球包括中国造成了巨大的危害,所以利用生物信息技术对其进行研究显得十分必要。从NCBI数据库下载中国大陆境内A/swine/H1N1病毒HA基因核酸序列及其编码蛋白序列,利用MEGA4.0软件对核苷酸编码序列构建系统进化树,利用BioEdit软件对蛋白序列进行比对,分析重要抗原位点变异情况,结果显示2010年在广东流行的病毒,从其他地方传播到广东的,而非早期广东流行的的病毒变异而来。2008年福建,山东,北京等地区的病毒传播比较迅速.这些分析结果阐明了中国大陆境内A/swine/H1N1病毒血凝素(HA)基因的进化关系和变异趋向,对于研究A/swine/H1N1病毒具有重要的参考价值。     

SIRT1/PGC-1: a neuroprotective axis?

Neurodegenerative diseases are more and more prevalent in our aging societies. A rapid overview of the etiology of many neurodegenerative diseases like Alzheimer, Parkinson, Huntington disease and amyotrophic lateral sclerosis suggests a tight link with mitochondrial dysfunction. Since it has been recently demonstrated that activation of the SIRT1/PGC-1 pathway, in a metabolic context promotes mitochondrial function, we performed a detailed literature review on the implication of this pathway in neurodegeneration. Interestingly, transgenic mice with impaired PGC-1 expression have neurodegenerative lesions and show behavioural abnormalities. As evidenced from independent investigations, enhanced SIRT1 activity has been demonstrated to protect against axonal degeneration and to decrease the accumulation of amyloid beta peptides, the hallmark of Alzheimer disease, in cultured murine embryonic neurons. In addition, several studies suggest that resveratrol, a specific activator of SIRT1, could have protective effects in animal models of neurodegenerative diseases. Taken together, these results strongly suggest that the modulation of the SIRT1/PGC-1 pathway, which has not been well documented in the central nervous system, could become the cornerstone for new therapeutical approaches to combat neurodegeneration.     

p21WAF1/CIP1基因与肿瘤

肿瘤的发生发展是多基因参与的复杂而多步骤的过程.p21WAF1/CIP1基因作为一种重要的细胞周期抑制基因与肿瘤的发生发展密切相关,近年来的研究表明它在不同肿瘤发生发展中有着不同的作用.     

NOR1/OSCP1调节恶性肿瘤进程的研究进展

有机溶质载体伴侣1(NOR1/OSCP1)是2003年中南大学湘雅医学院肿瘤研究所克隆的一个鼻咽癌表达下调新基因,可能参与亚硝胺类化学致癌物在体内的代谢过程。后续研究表明,与癌旁组织相比,NOR1在鼻咽癌、宫颈癌、前列腺癌等一系列癌组织显著低表达,并作为肿瘤抑制因子激活相关信号通路抑制细胞增殖和促进细胞凋亡。本文将系统阐述NOR1与肿瘤的关系及其调节恶性肿瘤进程的可能作用机制,为将来抗肿瘤治疗提供一个新的靶标。     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Human pancreatic cancer invasion and metastasis have been found to correlate with increased levels of active matrix metalloproteinase 2 (MMP-2). The multifunctional cytokine transforming growth factor beta 1 (TGF-β1) has been shown to increase both secretion of MMP-2 and invasion by several pancreatic cancer cell types. In the present study, we investigated the signaling pathway involved in TGF-β1-promoted MMP-2 secretion and invasion by human pancreatic cancer cells SW1990. Using specific inhibitors, we found that stimulation of these tumor cells with TGF-β1 induced secretion and activation of the collagenase MMP-2, which was required for TGF-β1-stimulated invasion. Our results also indicate that signaling events involved in TGF-β1-enhanced SW1990 invasiveness comprehend activation of Rac1 followed by generation of reactive oxygen species through nicotinamide adenine dinucleotide phosphate-oxidase, activation of nuclear factor-kappa beta, release of interleukin-6, and secretion and activation of MMP-2.     

BECN1/Beclin 1 sorts cell-surface APP/amyloid β precursor protein for lysosomal degradation

The regulation of plasma membrane (PM)-localized transmembrane protein/receptor trafficking has critical implications for cell signaling, metabolism and survival. In this study, we investigated the role of BECN1 (Beclin 1) in the degradative trafficking of PM-associated APP (amyloid β precursor protein), whose metabolism to amyloid-β, an essential event in Alzheimer disease, is dependent on divergent PM trafficking pathways. We report a novel interaction between PM-associated APP and BECN1 that recruits macroautophagy/endosomal regulatory proteins PIK3C3 and UVRAG. We found that BECN1 promotes surface APP internalization and sorting predominantly to endosomes and endolysosomes. BECN1 also promotes the targeting of a smaller fraction of internalized APP to LC3-positive phagophores, suggesting a role for BECN1-dependent PM macroautophagy in APP degradation. Furthermore, BECN1 facilitates lysosomal degradation of surface APP and reduces the secretion of APP metabolites (soluble ectodomains, sAPP). The association between APP and BECN1 is dependent on the evolutionarily conserved domain (ECD) of BECN1 (amino acids 267–337). Deletion of a BECN1 ECD subregion (amino acids 285–299) did not impair BECN1- PIK3C3 interaction, PtdIns3K function or macroautophagy, but was sufficient to impair the APP-BECN1 interaction and BECN1's effects on surface APP internalization and degradation, resulting in increased secretion of sAPPs. Interestingly, both the BECN1-APP association and BECN1-dependent APP endocytosis and degradative trafficking were negatively regulated by active AKT. Our results further implicate phosphorylation of the BECN1 Ser295 residue in the inhibition of APP degradation by AKT. Our studies reveal a novel function for BECN1 in the sorting of a plasma membrane protein for endolysosomal and macroautophagic degradation.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.