Ras and Calcium Signaling Pathways Converge at Raf1 via the Shoc2 Scaffold Protein

Situated downstream of Ras is a key signaling molecule, Raf1. Increase in Ca²⁺ concentration has been shown to modulate the Ras-dependent activation of Raf1; however, the mechanism underlying this effect remains elusive. Here, to characterize the role of Ca²⁺ in Ras signaling to Raf1, we used a synthetic guanine nucleotide exchange factor (GEF) for Ras, eGRF. In HeLa cells expressing eGRF, Ras was activated by the cAMP analogue 007 as efficiently as by epidermal growth factor (EGF), whereas the activation of Raf1, MEK, and ERK by 007 was about half of that by EGF. Using a biosensor based on fluorescence resonance energy transfer, it was found that activation of Raf1 at the plasma membrane required not only Ras activation but also an increase in Ca²⁺ concentration or inhibition of calmodulin. Furthermore, the Ca²⁺-dependent activation of Raf1 was found to be abrogated by knockdown of Shoc2, a scaffold protein that binds both Ras and Raf1. These observations indicated that the Shoc2 scaffold protein modulates Ras-dependent Raf1 activation in a Ca²⁺- and calmodulin-dependent manner.     

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.     

The Proteasomal and Autophagic Pathways Converge on Lipid Droplets

Apolipoprotein B (apoB) is the primary protein of very low-density lipoproteins (VLDL). We found that apoB accumulated on the surface of cytoplasmic lipid droplets (LDs) of hepatocytes when the proteasomal or autophagic processes were suppressed. ApoB associated with LDs was poly-ubiquitinated and surrounded by autophagic vacuoles. Moreover, proteasomal subunits were concentrated around LDs. Our data suggest that apoB to be degraded remains adhered to with LDs until being processed by the proteasomal and autophagic pathways. We speculate that the LD surface serves as a platform to prevent hydrophobic apoB from forming aggregates, and that LDs may play a similar role for other aggregation-prone hydrophobic proteins.     

Parvin Overexpression Uncovers Tissue-Specific Genetic Pathways and Disrupts F-Actin to Induce Apoptosis in the Developing Epithelia in Drosophila

Parvin is a putative F-actin binding protein important for integrin-mediated cell adhesion. Here we used overexpression of Drosophila Parvin to uncover its functions in different tissues in vivo. Parvin overexpression caused major defects reminiscent of metastatic cancer cells in developing epithelia, including apoptosis, alterations in cell shape, basal extrusion and invasion. These defects were closely correlated with abnormalities in the organization of F-actin at the basal epithelial surface and of integrin-matrix adhesion sites. In wing epithelium, overexpressed Parvin triggered increased Rho1 protein levels, predominantly at the basal side, whereas in the developing eye it caused a rough eye phenotype and severely disrupted F-actin filaments at the retina floor of pigment cells. We identified genes that suppressed these Parvin-induced dominant effects, depending on the cell type. Co-expression of both ILK and the apoptosis inhibitor DIAP1 blocked Parvin-induced lethality and apoptosis and partially ameliorated cell delamination in epithelia, but did not rescue the elevated Rho1 levels, the abnormal organization of F-actin in the wing and the assembly of integrin-matrix adhesion sites. The rough eye phenotype was suppressed by coexpression of either PTEN or Wech, or by knock-down of Xrp1. Two main conclusions can be drawn from our studies: (1), high levels of cytoplasmic Parvin are toxic in epithelial cells; (2) Parvin in a dose dependent manner affects the organization of actin cytoskeleton in both wing and eye epithelia, independently of its role as a structural component of the ILK-PINCH-Parvin complex that mediates the integrin-actin link. Thus, distinct genetic interactions of Parvin occur in different cell types and second site modifier screens are required to uncover such genetic circuits.     

检测不同毒力结核分枝杆菌感染对巨噬细胞凋亡的影响及其Caspase-3和Bcl-2的表达

该文为探讨不同毒力的结核分枝杆菌感染对巨噬细胞凋亡的调控作用及其机制。实验用结核分枝杆菌国际标准强毒株H37Rv株和卡介苗BCG分别感染巨噬细胞RAW264.7株,同时设空白对照组,在感染后1,6,12,24 h,用流式细胞技术检测各组巨噬细胞的凋亡率,应用Western blot检测细胞Caspase-3和Bcl-2蛋白表达。结果发现,结核分枝杆菌感染组的凋亡率显著高于对照组,差异具有统计学意义(P<0.05);BCG感染组凋亡率高于H37Rv感染组,在感染后1,12,24 h凋亡率显著升高,差异具有统计学意义(P<0.05)。巨噬细胞感染结核分枝杆菌后其Caspase-3蛋白表达增高,结核分枝杆菌感染组的Caspase-3蛋白表达高于对照组:对照组相似文献   

An Oligopeptide Transporter of Mycobacterium tuberculosis Regulates Cytokine Release and Apoptosis of Infected Macrophages

Background

The Mycobacterium tuberculosis genome encodes two peptide transporters encoded by Rv3665c-Rv3662c and Rv1280c-Rv1283c. Both belong to the family of ABC transporters containing two nucleotide-binding subunits, two integral membrane proteins and one substrate-binding polypeptide. However, little is known about their functions in M. tuberculosis. Here we report functional characterization of the Rv1280c-Rv1283c-encoded transporter and its substrate-binding polypeptide OppA_MTB.

Methodology/Principal Findings

OppA_MTB was capable of binding the tripeptide glutathione and the nonapeptide bradykinin, indicative of a somewhat broad substrate specificity. Amino acid residues G109, N110, N230, D494 and F496, situated at the interface between domains I and III of OppA, were required for optimal peptide binding. Complementaton of an oppA knockout mutant of M. smegmatis with OppA_MTB confirmed the role of this transporter in importing glutathione and the importance of the aforesaid amino acid residues in peptide transport. Interestingly, this transporter regulated the ability of M. tuberculosis to lower glutathione levels in infected compared to uninfected macrophages. This ability was partly offset by inactivation of oppD. Concomitantly, inactivation of oppD was associated with lowered levels of methyl glyoxal in infected macrophages and reduced apoptosis-inducing ability of the mutant. The ability to induce the production of the cytokines IL-1β, IL-6 and TNF-α was also compromised after inactivation of oppD.

Conclusions

Taken together, these studies uncover the novel observations that this peptide transporter modulates the innate immune response of macrophages infected with M. tuberculosis.     

The Autophagic and Endocytic Pathways Converge at the Nascent Autophagic Vacuoles

We used an improved cryosectioning technique in combination with immunogold cytochemistry and morphometric analysis to study the convergence of the autophagic and endocytic pathways in isolated rat hepatocytes. The endocytic pathway was traced by continuous uptake of gold tracer for various time periods, up to 45 min, while the cells were incubated in serum-free medium to induce autophagy. Endocytic structures involved in fusion with autophagic vacuoles (AV) were categorized into multivesicular endosomes (MVE) and vesicular endosomes (VE). Three types of AV—initial (AVi), intermediate (AVi/d), and degradative (AVd)—were defined by morphological criteria and immunogold labeling characteristics of marker enzymes.

The entry of tracer into AV, manifested as either tracer-containing AV profiles (AV⁺) or fusion profiles (FP⁺) between AV and tracer-positive endosomal vesicles/vacuoles, was detected as early as 10 min after endocytosis. The number of AV⁺ exhibited an exponential increase with time. FP⁺ between MVE or VE and all three types of AV were observed. Among the 112 FP⁺ scored, 36% involved VE. Of the AV types, AVi and AVi/d were found five to six times more likely to be involved in fusions than AVd. These fusion patterns did not significantly change during the period of endocytosis (15–45 min). We conclude that the autophagic and endocytic pathways converge in a multistage fashion starting within 10 min of endocytosis. The nascent AV is the most upstream and preferred fusion partner for endosomes.

     

TLR Ligands Induce Antiviral Responses in Chicken Macrophages

Chicken macrophages express several receptors for recognition of pathogens, including Toll-like receptors (TLRs). TLRs bind to pathogen-associated molecular patterns (PAMPs) derived from bacterial or viral pathogens leading to the activation of macrophages. Macrophages play a critical role in immunity against viruses, including influenza viruses. The present study was designed to test the hypothesis that treatment of chicken macrophages with TLR ligands reduces avian influenza replication. Furthermore, we sought to study the expression of some of the key mediators involved in the TLR-mediated antiviral responses of macrophages. Chicken macrophages were treated with the TLR2, 3, 4, 7 and 21 ligands, Pam3CSK4, poly(I:C), LPS, R848 and CpG ODN, respectively, at different doses and time points pre- and post-H4N6 avian influenza virus (AIV) infection. The results revealed that pre-treatment of macrophages with Pam3CSK4, LPS and CpG ODN reduced the replication of AIV in chicken macrophages. In addition, the relative expression of genes involved in inflammatory and antiviral responses were quantified at 3, 8 and 18 hours post-treatment with the TLR2, 4 and 21 ligands. Pam3CSK4, LPS and CpG ODN increased the expression of interleukin (IL)-1β, interferon (IFN)-γ, IFN-β and interferon regulatory factor (IFR) 7. The expression of these genes correlated with the reduction of viral replication in macrophages. These results shed light on the process of immunity to AIV in chickens.     

Ca^2＋与细胞凋亡

Ｃａ＾２＋在某些因素诱导的细胞凋亡中起着重要信使作用。细胞内Ｃａ＾２＋浓度上升可来源于胞外Ｃａ＾２＋内汉、内库钙动员或者二者兼之。     

钙离子信号与细胞凋亡

细胞凋亡的分子机制是什么?这个问题当前引起人们广泛的研究兴趣。作为重要的第二信使,钙信号在许多生理和细胞活动中都起到了十分重要的作用。钙信号是否也在凋亡的调控中起作用呢?虽然在过去十多年中,许多研究证据都表明钙信号参与凋亡的调控,但是,钙信号如何作用于凋亡过程的具体机理仍然是众说纷纭。事实上,许多研究结果仍存有争议。文章总结了近几年来大量关于钙信号与凋亡研究的成果,集中讨论了两个问题：1)在凋亡前期“决定阶段”有没有钙离子信号的参与?2)钙离子信号与哪些凋亡调控因子(包括Bcl-2族蛋白)相互作用及如何作用?这问题还牵涉到亚细胞结构中钙库的作用(包括细胞质、内质网和线粒体)。根据作者自己的实验结果,文章对这些文献中不同的说法作了一些具体的评估。最后,文章还提出了一个钙离子信号参与调控细胞凋亡的可能模型。     

Amyloid-Like Aggregates of the Yeast Prion Protein Ure2 Enter Vertebrate Cells by Specific Endocytotic Pathways and Induce Apoptosis

Background

A number of amyloid diseases involve deposition of extracellular protein aggregates, which are implicated in mechanisms of cell damage and death. However, the mechanisms involved remain poorly understood.

Methodology/Principal Findings

Here we use the yeast prion protein Ure2 as a generic model to investigate how amyloid-like protein aggregates can enter mammalian cells and convey cytotoxicity. The effect of three different states of Ure2 protein (native dimer, protofibrils and mature fibrils) was tested on four mammalian cell lines (SH-SY5Y, MES23.5, HEK-293 and HeLa) when added extracellularly to the medium. Immunofluorescence using a polyclonal antibody against Ure2 showed that all three protein states could enter the four cell lines. In each case, protofibrils significantly inhibited the growth of the cells in a dose-dependent manner, fibrils showed less toxicity than protofibrils, while the native state had no effect on cell growth. This suggests that the structural differences between the three protein states lead to their different effects upon cells. Protofibrils of Ure2 increased membrane conductivity, altered calcium homeostasis, and ultimately induced apoptosis. The use of standard inhibitors suggested uptake into mammalian cells might occur via receptor-mediated endocytosis. In order to investigate this further, we used the chicken DT40 B cell line DKOR, which allows conditional expression of clathrin. Uptake into the DKOR cell-line was reduced when clathrin expression was repressed suggesting similarities between the mechanism of PrP uptake and the mechanism observed here for Ure2.

Conclusions/Significance

The results provide insight into the mechanisms by which amyloid aggregates may cause pathological effects in prion and amyloid diseases.     

Pre-Exposure of Mycobacterium tuberculosis-Infected Macrophages to Crystalline Silica Impairs Control of Bacterial Growth by Deregulating the Balance between Apoptosis and Necrosis

Inhalation of crystalline silica (CS) particles increases the risk of pulmonary tuberculosis; however, the precise mechanism through which CS exposure facilitates Mycobacterium tuberculosis (Mtb) infection is unclear. We speculate that macrophage exposure to CS deregulates the cell death pathways that could explain, at least in part, the association observed between exposure to CS and pulmonary tuberculosis. We therefore established an in vitro model in which macrophages were exposed to CS and then infected with Mtb. Expression of surface markers was analyzed by flow cytometry, JNK1/2, ASK1, caspase 9, P-p38, Bcl-2 and Mcl-1 were analyzed by Western blot, and cytokines by ELISA. Our results show that exposure to CS limits macrophage ability to control Mtb growth. Moreover, this exposure reduced the expression of TLR2, Bcl-2 and Mcl-1, but increased that of JNK1 and ASK1 molecules in the macrophages. Finally, when the pre-exposed macrophages were infected with Mtb, the concentrations of TNFα, IL-1β and caspase-9 expression increased. This pro-inflammatory profile of the macrophage unbalanced the apoptosis/necrosis pathway. Taken together, these data suggest that macrophages exposed to CS are sensitized to cell death by MAPK kinase-dependent signaling pathway. Secretion of TNF-α and IL-1β by Mtb-infected macrophages promotes necrosis, and this deregulation of cell death pathways may favor the release of viable bacilli, thus leading to the progression of tuberculosis.     

线粒体凋亡途径的研究进展

线粒体凋亡途径是细胞凋亡的主要途径之一。是目前研究凋亡的热点,各种凋亡刺激信号通过BH3（Bcl-2homology domain3)-only蛋白引起Bax(Bcl-2-asslciated proteinX)蛋白移位到线粒体外膜并多聚化,形成膜通道,刺激线粒体释放细胞色素C（CytC)和Smac(second mitochondrial-derived activator of caspase),CytC通过Apaf-1因子的多聚化与胱天蛋白酶（caspases)-9形成凋亡小体,导致下游胱天蛋白酶的级联反应,而凋亡蛋白抑制因子（IAP）和Smac通过抑制和促进胱天蛋白酶的级联反应来调控细胞凋亡。     

The Quantity of Calcium that Appears to Induce Neuronal Death

Abstract: Excessive entry of Ca²⁺ through the NMDA receptor is thought to be the major cause of glutamate toxicity in brain neurons. However, actual quantitation of the calcium overload has not been achieved. Here we show that the absolute amount of ⁴⁵Ca²⁺ taken up via the NMDA receptor correlates quantitatively with the amount of acute cell death in cultured cerebellar granule cells of the rat. Analysis of 9-and 16-day cultures reveals that the NMDA-induced Ca²⁺ uptake is about the same at these ages, whereas the Ca-dependent lethal process is more developed in the older neurons. The calculated lethal concentration of ⁴⁵Ca taken up exceeds by ∼10,000 times the maximal concentration of [Ca²⁺], that can be measured by fluorescence imaging. It is suggested that the Ca²⁺ taken up induces the lethal process in a subcellular structure in which it has been segregated.