Sirt1-deficiency causes defective protein quality control. Tomita, T; Hamazaki, J; Hirayama, S; McBurney, MW; Yashiroda, H; Murata, S Scientific reports
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12613
2015
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Protein quality control is an important mechanism to maintain cellular homeostasis. Damaged proteins have to be restored or eliminated by degradation, which is mainly achieved by molecular chaperones and the ubiquitin-proteasome system. The NAD(+)-dependent deacetylase Sirt1 has been reported to play positive roles in the regulation of cellular homeostasis in response to various stresses. However, its contribution to protein quality control remains unexplored. Here we show that Sirt1 is involved in protein quality control in both an Hsp70-dependent and an Hsp70-independent manner. Loss of Sirt1 led to the accumulation of ubiquitinated proteins in cells and tissues, especially upon heat stress, without affecting proteasome activities. This was partly due to decreased basal expression of Hsp70. However, this accumulation was only partially alleviated by overexpression of Hsp70 or induction of Hsp70 upon heat shock in Sirt1-deficient cells and tissues. These results suggest that Sirt1 mediates both Hsp70-dependent and Hsp70-independent protein quality control. Our findings cast new light on understanding the role of Sirt1 in maintaining cellular homeostasis. | | | 26219988
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Redundant Roles of Rpn10 and Rpn13 in Recognition of Ubiquitinated Proteins and Cellular Homeostasis. Hamazaki, J; Hirayama, S; Murata, S PLoS genetics
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e1005401
2015
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Intracellular proteins tagged with ubiquitin chains are targeted to the 26S proteasome for degradation. The two subunits, Rpn10 and Rpn13, function as ubiquitin receptors of the proteasome. However, differences in roles between Rpn10 and Rpn13 in mammals remains to be understood. We analyzed mice deficient for Rpn13 and Rpn10. Liver-specific deletion of either Rpn10 or Rpn13 showed only modest impairment, but simultaneous loss of both caused severe liver injury accompanied by massive accumulation of ubiquitin conjugates, which was recovered by re-expression of either Rpn10 or Rpn13. We also found that mHR23B and ubiquilin/Plic-1 and -4 failed to bind to the proteasome in the absence of both Rpn10 and Rpn13, suggesting that these two subunits are the main receptors for these UBL-UBA proteins that deliver ubiquitinated proteins to the proteasome. Our results indicate that Rpn13 mostly plays a redundant role with Rpn10 in recognition of ubiquitinated proteins and maintaining homeostasis in Mus musculus. | | | 26222436
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Sqstm1-GFP knock-in mice reveal dynamic actions of Sqstm1 during autophagy and under stress conditions in living cells. Eino, A; Kageyama, S; Uemura, T; Annoh, H; Saito, T; Narita, I; Waguri, S; Komatsu, M Journal of cell science
128
4453-61
2015
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Sqstm1 serves as a signaling hub and receptor for selective autophagy. Consequently, dysregulation of Sqstm1 causes imbalances in signaling pathways and disrupts proteostasis, thereby contributing to the development of human diseases. Environmental stresses influence the level of Sqstm1 by altering its expression and/or autophagic degradation, and also changes the localization of Sqstm1, making it difficult to elucidate the actions and roles of this protein. In this study, we developed knock-in mice expressing Sqstm1 fused to GFP (Sqstm1-GFP(KI/+)). Using these Sqstm1-GFP(KI/+) mice, we revealed for the first time the dynamics of endogenous Sqstm1 in living cells. Sqstm1-GFP was translocated to a restricted area of LC3-positive structures, which primarily correspond to the inside of autophagosomes, and then degraded. Moreover, exposure to arsenite induced expression of Sqstm1-GFP, followed by accumulation of the fusion protein in large aggregates that were degraded by autophagy. Furthermore, suppression of autophagy in Sqstm1-GFP(KI/+) mouse livers caused accumulation of Sqstm1-GFP and formation of GFP-positive aggregate structures, leading to severe hepatic failure. These results indicate that Sqstm1-GFP(KI/+) mice are a useful tool for analyzing Sqstm1 in living cells and intact animals. | | | 26483381
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Phytometabolite Dehydroleucodine Induces Cell Cycle Arrest, Apoptosis, and DNA Damage in Human Astrocytoma Cells through p73/p53 Regulation. Bailon-Moscoso, N; González-Arévalo, G; Velásquez-Rojas, G; Malagon, O; Vidari, G; Zentella-Dehesa, A; Ratovitski, EA; Ostrosky-Wegman, P PloS one
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e0136527
2015
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Accumulating evidence supports the idea that secondary metabolites obtained from medicinal plants (phytometabolites) may be important contributors in the development of new chemotherapeutic agents to reduce the occurrence or recurrence of cancer. Our study focused on Dehydroleucodine (DhL), a sesquiterpene found in the provinces of Loja and Zamora-Chinchipe. In this study, we showed that DhL displayed cytostatic and cytotoxic activities on the human cerebral astrocytoma D384 cell line. With lactone isolated from Gynoxys verrucosa Wedd, a medicinal plant from Ecuador, we found that DhL induced cell death in D384 cells by triggering cell cycle arrest and inducing apoptosis and DNA damage. We further found that the cell death resulted in the increased expression of CDKN1A and BAX proteins. A marked induction of the levels of total TP73 and phosphorylated TP53, TP73, and γ-H2AX proteins was observed in D384 cells exposed to DhL, but no increase in total TP53 levels was detected. Overall these studies demonstrated the marked effect of DhL on the diminished survival of human astrocytoma cells through the induced expression of TP73 and phosphorylation of TP73 and TP53, suggesting their key roles in the tumor cell response to DhL treatment. | Western Blotting | | 26309132
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Neurodegeneration and unfolded-protein response in mice expressing a membrane-tethered flexible tail of PrP. Dametto, P; Lakkaraju, AK; Bridel, C; Villiger, L; O'Connor, T; Herrmann, US; Pelczar, P; Rülicke, T; McHugh, D; Adili, A; Aguzzi, A PloS one
10
e0117412
2015
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The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23-128) hinged to a membrane-anchored globular domain (GD, aa 129-231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrPΔ141-225, or "FTgpi"). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection. | Western Blotting | | 25658480
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The unexpected role of polyubiquitin chains in the formation of fibrillar aggregates. Morimoto, D; Walinda, E; Fukada, H; Sou, YS; Kageyama, S; Hoshino, M; Fujii, T; Tsuchiya, H; Saeki, Y; Arita, K; Ariyoshi, M; Tochio, H; Iwai, K; Namba, K; Komatsu, M; Tanaka, K; Shirakawa, M Nature communications
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6116
2015
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Ubiquitin is known to be one of the most soluble and stably folded intracellular proteins, but it is often found in inclusion bodies associated with various diseases including neurodegenerative disorders and cancer. To gain insight into this contradictory behaviour, we have examined the physicochemical properties of ubiquitin and its polymeric chains that lead to aggregate formation. We find that the folding stability of ubiquitin chains unexpectedly decreases with increasing chain length, resulting in the formation of amyloid-like fibrils. Furthermore, when expressed in cells, polyubiquitin chains covalently linked to EGFP also form aggregates depending on chain length. Notably, these aggregates are selectively degraded by autophagy. We propose a novel model in which the physical and chemical instability of polyubiquitin chains drives the formation of fibrils, which then serve as an initiation signal for autophagy. | | | 25600778
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Wilms' tumor gene 1 regulates p63 and promotes cell proliferation in squamous cell carcinoma of the head and neck. Li, X; Ottosson, S; Wang, S; Jernberg, E; Boldrup, L; Gu, X; Nylander, K; Li, A BMC cancer
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342
2015
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Wilms' tumor gene 1 (WT1) can act as a suppressor or activator of tumourigenesis in different types of human malignancies. The role of WT1 in squamous cell carcinoma of the head and neck (SCCHN) is not clear. Overexpression of WT1 has been reported in SCCHN, suggesting a possible oncogenic role for WT1. In the present study we aimed at investigating the function of WT1 and its previously identified protein partners p63 and p53 in the SCCHN cell line FaDu.Silencing RNA (siRNA) technology was applied to knockdown of WT1, p63 and p53 in FaDu cells. Cell proliferation was detected using MTT assay. Chromatin immunoprecipitation (ChIP)/PCR analysis was performed to confirm the effect of WT1 on the p63 promoter. Protein co-immunoprecipitation (co-IP) was used to find protein interaction between WT1 and p53/p63. Microarray analysis was used to identify changes of gene expression in response to knockdown of either WT1 or p63. WT1 RNA level was detected using real-time quantitative PCR (RT-qPCR) in patients with SCCHN.We found that WT1 and p63 promoted cell proliferation, while mutant p53 (R248L) possessed the ability to suppress cell proliferation. We reported a novel positive correlation between WT1 and p63 expression. Subsequently, p63 was identified as a WT1 target gene. Furthermore, expression of 18 genes involved in cell proliferation, cell cycle regulation and DNA replication was significantly altered by downregulation of WT1 and p63 expression. Several known WT1 and p63 target genes were affected by WT1 knockdown. Protein interaction was demonstrated between WT1 and p53 but not between WT1 and p63. Additionally, high WT1 mRNA levels were detected in SCCHN patient samples.Our findings suggest that WT1 and p63 act as oncogenes in SCCHN, affecting multiple genes involved in cancer cell growth. | | | 25929687
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Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum. Staudacher, JJ; Naarmann-de Vries, IS; Ujvari, SJ; Klinger, B; Kasim, M; Benko, E; Ostareck-Lederer, A; Ostareck, DH; Bondke Persson, A; Lorenzen, S; Meier, JC; Blüthgen, N; Persson, PB; Henrion-Caude, A; Mrowka, R; Fähling, M Nucleic acids research
43
3219-36
2015
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Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. | Western Blotting | | 25753659
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Docosahexaenoic acid (DHA): a modulator of microglia activity and dendritic spine morphology. Chang, PK; Khatchadourian, A; McKinney, RA; Maysinger, D Journal of neuroinflammation
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34
2015
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Recent studies have revealed that excessive activation of microglia and inflammation-mediated neurotoxicity are implicated in the progression of several neurological disorders. In particular, chronic inflammation in vivo and exposure of cultured brain cells to lipopolysaccharide (LPS) in vitro can adversely change microglial morphology and function. This can have both direct and indirect effects on synaptic structures and functions. The integrity of dendritic spines, the postsynaptic component of excitatory synapses, dictates synaptic efficacy. Interestingly, dysgenesis of dendritic spines has been found in many neurological diseases associated with ω-3 polyunsaturated fatty acid (PUFA) deficiency and cognitive decline. In contrast, supplemented ω-3 PUFAs, such as docosahexaenoic acid (DHA), can partly correct spine defects. Hence, we hypothesize that DHA directly affects synaptic integrity and indirectly through neuron-glia interaction. Strong activation of microglia by LPS is accompanied by marked release of nitric oxide and formation of lipid bodies (LBs), both dynamic biomarkers of inflammation. Here we investigated direct effects of DHA on synaptic integrity and its indirect effects via microglia in the hippocampal CA1 region.Microglia (N9) and organotypic hippocampal slice cultures were exposed to the proinflammagen LPS (100 ng/ml) for 24 h. Biochemical and morphological markers of inflammation were investigated in microglia and CA1 regions of hippocampal slices. As biomarkers of hyperactive microglia, mitochondrial function, nitric oxide release and LBs (number, size, LB surface-associated proteins) were assessed. Changes in synaptic transmission of CA1 pyramidal cells were determined following LPS and DHA (25-50 μM) treatments by recording spontaneous AMPA-mediated miniature excitatory postsynaptic currents (mEPSCs).Microglia responded to LPS stimulation with a significant decrease of mitochondrial function, increased nitric oxide production and an increase in the formation of large LBs. LPS treatment led to a significant reduction of dendritic spine densities and an increase in the AMPA-mediated mEPSC inter-event interval (IEI). DHA normalized the LPS-induced abnormalities in both neurons and microglia, as revealed by the restoration of synaptic structures and functions in hippocampal CA1 pyramidal neurons.Our findings indicate that DHA can prevent LPS-induced abnormalities (neuroinflammation) by reducing inflammatory biomarkers, thereby normalizing microglia activity and their effect on synaptic function. | | | 25889069
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Human Cytomegalovirus miR-UL112-3p Targets TLR2 and Modulates the TLR2/IRAK1/NFκB Signaling Pathway. Landais, I; Pelton, C; Streblow, D; DeFilippis, V; McWeeney, S; Nelson, JA PLoS pathogens
11
e1004881
2015
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Human Cytomegalovirus (HCMV) encodes multiple microRNAs (miRNAs) whose functions are just beginning to be uncovered. Using in silico approaches, we identified the Toll-Like Receptor (TLR) innate immunity pathway as a possible target of HCMV miRNAs. Luciferase reporter assay screens further identified TLR2 as a target of HCMV miR-UL112-3p. TLR2 plays a major role in innate immune response by detecting both bacterial and viral ligands, including HCMV envelope proteins gB and gH. TLR2 activates a variety of signal transduction routes including the NFκB pathway. Furthermore, TLR2 plays an important role in controlling CMV infection both in humans and in mice. Immunoblot analysis of cells transfected with a miR-UL112-3p mimic revealed that endogenous TLR2 is down-regulated by miR-UL112-3p with similar efficiency as a TLR2-targeting siRNA (siTLR2). We next found that TLR2 protein level decreases at late times during HCMV infection and correlates with miR-UL112-3p accumulation in fibroblasts and monocytic THP1 cells. Confirming direct miR-UL112-3p targeting, down-regulation of endogenous TLR2 was not observed in cells infected with HCMV mutants deficient in miR-UL112-3p expression, but transfection of miR-UL112-3p in these cells restored TLR2 down-regulation. Using a NFκB reporter cell line, we found that miR-UL112-3p transfection significantly inhibited NFκB-dependent luciferase activity with similar efficiency as siTLR2. Consistent with this observation, miR-UL112-3p transfection significantly reduced the expression of multiple cytokines (IL-1β, IL-6 and IL-8) upon stimulation with a TLR2 agonist. Finally, miR-UL112-3p transfection significantly inhibited the TLR2-induced post-translational activation of IRAK1, a kinase located in the upstream section of the TLR2/NFκB signaling axis. To our knowledge, this is the first identified mechanism of TLR2 modulation by HCMV and is the first report of functional targeting of TLR2 by a viral miRNA. These results provide a novel mechanism through which a HCMV miRNA regulates the innate immune response by down-regulating TLR-2 expression. | | | 25955717
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