Bcl-xL Protein Protects from C/EBP Homologous Protein (CHOP)-dependent Apoptosis during Plasma Cell Differentiation

Although it is known that the unfolded protein response (UPR) plays a significant role in the process of plasma cell differentiation, the contribution of the individual sensors of the UPR to this process remains unclear. In this study we examine the death signals and compensatory survival signals activated during B cell activation and the first stages of plasma cell differentiation. During in vitro differentiation of both primary murine B cells and the Bcl1 cell line, we demonstrate that in addition to activation of the physiological UPR, changes in the expression of several Bcl-2 proteins occur, which are consistent with a lowering of the apoptotic threshold of the cell. Specifically, we observed decreased expression of Bcl-2 and Mcl-1 and increased expression of the proapoptotic protein Bim. However, these changes were countered by Bcl-x_L induction, which is necessary to protect differentiating cells both from ER stress-induced death by tunicamycin and from the death signals inherent in differentiation. Consistent with differentiating cells becoming dependent on Bcl-x_L for survival, the addition of ABT-737 resulted in apoptosis in differentiating cells through the inhibition of sequestration of Bim. Confirming this result, differentiation in the context of RNAi-mediated Bcl-x_L knockdown also induced apoptosis. This cell death is C/EBP homologous protein (CHOP)-dependent, connecting these events to the UPR. Thus plasma cell differentiation proceeds through a Bcl-x_L-dependent intermediate.     

C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis

Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop^+/+) and knock-out (Chop^−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop^−/− mice developed greater liver injury, inflammation, and fibrosis than Chop^+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop^+/+ and Chop^−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop^+/+ and Chop^−/− macrophages, Chop^−/− macrophages were resistant to its lipotoxicity. Chop^−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop^−/− and Chop^+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.     

MiR-1-3p通过抑制CAPRIN1调控胰腺癌发生发展

摘要 目的：探讨miR-1-3p在胰腺癌发生发展中的分子机制。方法：以MIA-PaCa-2,SW 1990为研究目标,通过qRT-PCR技术检测miR-1-3p的表达量,利用TargetScan和miRDB数据库预测miR-1-3p的下游靶基因及结合位点,并通过构建双荧光素酶报告基因,进一步确认miR-1-3p与靶基因的结合。利用CCK8细胞增殖实验及平板克隆形成实验检测过表达miR-1-3p及敲低CAPRIN1对细胞增殖的作用;利用流式检测细胞周期;利用蛋白质免疫印迹方法检测miR-1-3p对CAPRIN1及其下游基因的影响;通过流式来确认,过表达miR-1-3p及敲减CAPRIN1基因对细胞周期的影响。结果：miR-1-3p在胰腺癌细胞MIA-PaCa-2,SW 1990中低表达;miR-1-3p直接与CAPRIN1的3''-untranslated region (3''- UTR)结合;过表达miR-1-3p或抑制CAPRIN1基因的表达可明显抑制胰腺癌细胞的增殖能力,同时也产生细胞周期阻滞。结论：miR-1-3p通过抑制CAPRIN1基因表达,而产生细胞周期阻滞进而抑制胰腺癌细胞的增殖能力。     

Acetylation of WRN Protein Regulates Its Stability by Inhibiting Ubiquitination

Background

WRN is a multi-functional protein involving DNA replication, recombination and repair. WRN acetylation has been demonstrated playing an important role in response to DNA damage. We previously found that WRN acetylation can regulate its enzymatic activities and nuclear distribution.

Methodology/Principal Finding

Here, we investigated the factors involved in WRN acetylation and found that CBP and p300 are the only major acetyltransferases for WRN acetylation. We further identified 6 lysine residues in WRN that are subject to acetylation. Interestingly, WRN acetylation can increase its protein stability. SIRT1-mediated deacetylation of WRN reverses this effect. CBP dramatically increases the half-life of wild type WRN, while mutation of these 6 lysine residues (WRN-6KR) abrogates this increase. We further found that WRN stability is regulated by the ubiquitination pathway and WRN acetylation by CBP significantly reduces its ubiquitination. Importantly, we found that WRN is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells stably expressing WRN-6KR, which mimics unacetylated WRN, display significantly higher MMC sensitivity compared with the cells expressing wild-type WRN.

Conclusion/Significance

Taken together, these data demonstrate that WRN acetylation regulates its stability and has significant implications regarding the role of acetylation on WRN function in response to DNA damage.     

miR-448-3p通过调节KLF5的表达抑制颅内动脉瘤进展

目的:明确mi R-448-3p对颅内动脉瘤发展的影响。方法:我们通过结扎左侧肾动脉和左侧颈总动脉的方法建立大鼠IA模型;qRT-PCR用于检测mi R-448-3p表达;qRT-PCR和western blot用于检测KLF5 m RNA和蛋白表达;qRT-PCR和ELISA法用于检测炎症因子水平。结果:我们发现IA诱导大鼠中mi R-448-3p表达下调,而KLF5表达上调。我们发现并鉴定出KLF5是平滑肌细胞中mi R-448-3p的直接靶点。此外,mi R-448-3p处理使得IA诱导4周后动脉瘤大小和瘤腔截面积变小。mi R-448-3p处理保护了IA诱导后的壁厚比,抑制了巨噬细胞浸润。IAs引起KLF5表达显著增加,被mi R-448-3p处理后表达显著降低。我们还发现mi R-448-3p在脂多糖诱导的RAW 264.7巨噬细胞中具有抗炎作用。脂多糖促进KLF5、MMP2、MMP9的表达水平,但却被mi R-448-3p所抑制。结论:研究结果表明,mi R-448-3p可以抑制IA的进展,其机制可能是通过下调KLF5的介导的炎症反应。     

Cdc7p-Dbf4p Regulates Mitotic Exit by Inhibiting Polo Kinase

Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.     

Angelman Syndrome Protein Ube3a Regulates Synaptic Growth and Endocytosis by Inhibiting BMP Signaling in Drosophila

Altered expression of the E3 ubiquitin ligase UBE3A, which is involved in protein degradation through the proteasome-mediated pathway, is associated with neurodevelopmental and behavioral defects observed in Angelman syndrome (AS) and autism. However, little is known about the neuronal function of UBE3A and the pathogenesis of UBE3A-associated disorders. To understand the in vivo function of UBE3A in the nervous system, we generated multiple mutations of ube3a, the Drosophila ortholog of UBE3A. We found a significantly increased number of total boutons and satellite boutons in conjunction with compromised endocytosis in the neuromuscular junctions (NMJs) of ube3a mutants compared to the wild type. Genetic and biochemical analysis showed upregulation of bone morphogenetic protein (BMP) signaling in the nervous system of ube3a mutants. An immunochemical study revealed a specific increase in the protein level of Thickveins (Tkv), a type I BMP receptor, but not other BMP receptors Wishful thinking (Wit) and Saxophone (Sax), in ube3a mutants. Ube3a was associated with and specifically ubiquitinated lysine 227 within the cytoplasmic tail of Tkv, and promoted its proteasomal degradation in Schneider 2 cells. Negative regulation of Tkv by Ube3a was conserved in mammalian cells. These results reveal a critical role for Ube3a in regulating NMJ synapse development by repressing BMP signaling. This study sheds new light onto the neuronal functions of UBE3A and provides novel perspectives for understanding the pathogenesis of UBE3A-associated disorders.     

Glycogen Synthase Kinase - 3 Phosphorylates and Regulates the Stability of p27kip1 Protein

p27 Kip1 is a critical regulator of the eukaryotic cell cycle. It acts as a check point proteinand regulates cell cycle progression at the G1 and G1/S phase as well as predominantlyblocks cell cycle progression in the absence of growth factors. Intracellular turnover of p27is tightly regulated at the level of translation as well as by posttranslational modification.The mechanism by which p27 protein is rapidly degraded during the G1 and G1/S phasetransition is well characterized. However, the process by which p27 remains extremelystable in the absence of growth factors remains unknown. Here, we report that GSK-3dependent phosphorylation of p27 protein is essential for its enhanced stability. p27 proteinharbours 2 functional GSK-3 phosphorylation sites at the C- terminus, which was found tobe effectively phosphorylated by the cognate enzyme both in vitro and in vivo. Combinedwith earlier observation which shows that it phosphorylates and triggers cyclin Ddegradation; GSK-3 now appears to be a central mediator of the cell-cycle regulatorynetwork, where it acts as a two-way switch, phosphorylating and targeting pro-proliferativefactors for degradation on one hand and simultaneously phosphorylating and stabilizing ananti-proliferative factor on the other hand. This dual mode of activity may doubly ensurethat cell cycle progression is aptly prohibited under conditions of limited growth factoravailability.     

CAAT区/增强子结合蛋白（C/EBP）的结构与功能

C/EBPs是一组耐热的转录调控因子.其作用范围广泛,既参与正常的生理代谢过程,又与多种疾病的发生和发展相关;其作用方式多样,对转录的调控既有正效应又有负作用.C/EBPs的这种功能多样性是与其结构的特征性相联系的,它们属于bZIP蛋白家族.自身或与其他异构体形成蕴含着不同调控信息的同源或异源二聚体,并且能与多种蛋白质因子协同作用,决定C/EBPs发挥作用的方式和细胞特异性.     

Ⅲ型纤连蛋白组件包含蛋白5通过抑制ERK1/2磷酸化抑制C3H10T1/2细胞成脂分化

旨在探究Ⅲ型纤连蛋白组件包含蛋白5(type Ⅲ domain-containing protein5,FNDC5)对C3H10T1/2细胞成脂分化的调控作用.利用qRT-PCR和Western印迹检测FNDC5在C3H10T1/2细胞成脂分化过程中的时序性表达规律;构建慢病毒包被的过表达/干扰FNDC5载体,转染C3...     

Gastrodin Alleviates Cerebral Ischaemia/Reperfusion Injury by Inhibiting Pyroptosis by Regulating the lncRNA NEAT1/miR-22-3p Axis

Cerebral ischaemia/reperfusion (I/R) injury-induced irreversible brain injury is a major cause of mortality and functional impairment in ageing people. Gastrodin (GAS), derived from the traditional Chinese herbal medicine Tianma, has been reported to inhibit the progression of stroke, but the mechanism whereby GAS modulates the progression of cerebral I/R remains unclear. The middle cerebral artery occlusion method was used as a model of I/R in vivo. Rats were pretreated with GAS by intraperitoneal injection 7 days before I/R surgery and were then treated with GAS for 7 days after I/R surgery. Additionally, an oxygen–glucose deprivation/reoxygenation model using neuronal cells was established in vitro to simulate I/R injury. 2,3,5-Triphenyltetrazolium chloride and Nissl staining were used to evaluate infarct size and neuronal damage, respectively. Lactate dehydrogenase release and cell counting kit-8 assays were used to assess neuronal cell viability. Enzyme-linked immunosorbent assay, qPCR, flow cytometry and western blotting were performed to analyse the expression levels of inflammatory factors (IL-1β, IL-18), lncRNA NEAT1, miR-22-3p, NLRP3 and cleaved caspase-1. Luciferase reporter experiments were performed to verify the association between lncRNA NEAT1 and miR-22-3p. The results indicated that GAS could significantly improve the neurological scores of rats and reduce the area of cerebral infarction. Meanwhile, GAS inhibited pyroptosis by downregulating NLRP3, inflammatory factors (IL-1β, IL-18) and cleaved caspase-1. In addition, GAS attenuated I/R-induced inflammation in neuronal cells through the modulation of the lncRNA NEAT1/miR-22-3p axis. GAS significantly attenuated cerebral I/R injury via modulation of the lncRNA NEAT1/miR-22-3p axis. Thus, GAS might serve as a new agent for the treatment of cerebral I/R injury.