PKC? counteracts oxidative stress by regulating Hsc70 in an esophageal cancer cell line.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang BS
PROVIDER: S-EPMC3631091 | BioStudies | 2013-01-01T00:00:00Z
REPOSITORIES: biostudies
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Project description:Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.
2014-01-01 | S-EPMC4011886 | BioStudies
Project description:The carboxyl terminus of constitutive heat shock cognate 70 (HSC70)-interacting protein (CHIP, also known as Stub1) is a U box-containing E3 ubiquitin ligase that is important for protein quality control. The role of CHIP in innate immunity is not known. Here, we report that CHIP knockdown inhibits Toll-like receptor (TLR) 4- and TLR9-driven signaling, but not TLR3-driven signaling; proinflammatory cytokine and type 1 interferon (IFN) production; and maturation of antigen-presenting cells, including macrophages and dendritic cells. We demonstrate that CHIP can recruit the tyrosine kinase Src and atypical protein kinase C ? (PKC?) to the TLR complex, thereby leading to activation of IL-1 receptor-associated kinase 1, TANK-binding kinase 1, and IFN regulatory factors 3 and 7. CHIP acts as an E3 ligase for Src and PKC? during TLR signaling. CHIP-mediated enhancement of TLR signaling is inhibited by IFNAR deficiency or expression of ubiquitination resistant mutant forms of Src or PKC?. These findings suggest that CHIP facilitates the formation of a TLR signaling complex by recruiting, ubiquitinating, and activating Src and PKC?.
2011-01-01 | S-EPMC3182058 | BioStudies
Project description:Harmful oxidation of proteins, lipids and nucleic acids is observed when reactive oxygen species (ROS) are produced excessively and/or the antioxidant capacity is reduced, causing 'oxidative stress'. Nuclear poly-ADP-ribose (PAR) formation is thought to be induced in response to oxidative DNA damage and to promote cell death under sustained oxidative stress conditions. However, what exactly triggers PAR induction in response to oxidative stress is incompletely understood. Using reverse phase protein array (RPPA) and in-depth analysis of key stress signaling components, we observed that PAR formation induced by H2O2 was mediated by the PLC/IP3R/Ca(2+)/PKC? signaling axis. Mechanistically, H2O2-induced PAR formation correlated with Ca(2+)-dependent DNA damage, which, however, was PKC?-independent. In contrast, PAR formation was completely lost upon knockdown of PKC?, suggesting that DNA damage alone was not sufficient for inducing PAR formation, but required a PKC?-dependent process. Intriguingly, the loss of PAR formation observed upon PKC? depletion was overcome when the chromatin structure-modifying protein HMGB1 was co-depleted with PKC?, suggesting that activation and nuclear translocation of PKC? releases the inhibitory effect of HMGB1 on PAR formation. Together, these results identify PKC? and HMGB1 as important co-regulators involved in H2O2-induced PAR formation, a finding that may have important relevance for oxidative stress-associated pathophysiological conditions.
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Project description:Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-d protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-d associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-d became tyrosine phosphorylated, dissociated from trophinin, and entered the nucleus. In PKC-d knockdown endometrial cells, GWRQ did not induce apoptosis.
2011-01-01 | S-EPMC3048080 | BioStudies
Project description:NLRC4 [nucleotide-binding domain and leucine-rich repeat (NLR) family, caspase recruitment domain (CARD) containing 4] is an innate immune receptor, which, upon detection of certain pathogens or internal distress signals, initiates caspase-1-mediated interleukin-1? maturation and an inflammatory response. A gain-of-function mutation, H443P in NLRC4, causes familial cold autoinflammatory syndrome (FCAS) characterized by cold-induced hyperactivation of caspase-1, enhanced interleukin-1? maturation, and inflammation. Although the H443P mutant shows constitutive activity, the mechanism involved in hyperactivation of caspase-1 by NLRC4-H443P upon exposure of cells to lower temperature is not known. Here, we show that heat shock cognate protein 70 (HSC70) complexes with NLRC4 and negatively regulates caspase-1 activation by NLRC4-H443P in human cells. Compared with NLRC4, the structurally altered NLRC4-H443P shows enhanced interaction with HSC70. Nucleotide binding- and leucine-rich repeat domains of NLRC4, but not its CARD, can engage in complex formation with HSC70. Knockdown of HSC70 enhances apoptosis-associated speck-like protein containing a CARD (ASC)-speck formation and caspase-1 activation by NLRC4-H443P. Exposure to subnormal temperature results in reduced interaction of NLRC4-H443P with HSC70, and an increase in its ability to form ASC specks and activate caspase-1. Unlike the NLRC4-H443P mutant, another constitutively active mutant (NLRC4-V341A) associated with autoinflammatory diseases, but not FCAS, showed neither enhanced interaction with HSC70 nor an increase in inflammasome formation upon exposure to subnormal temperature. Our results identify HSC70 as a negative regulator of caspase-1 activation by the temperature-sensitive NLRC4-H443P mutant. We also show that low-temperature-induced hyperactivation of caspase-1 by NLRC4-H443P is due to loss of inhibition by HSC70.
2019-01-01 | S-EPMC6815140 | BioStudies
Project description:The high mortality associated with pancytopenia and multi-organ failure resulting from hematopoietic disorders of acute radiation syndrome (h-ARS) creates an urgent need for developing more effective treatment strategies. Here, we showed that bone marrow multipotent mesenchymal stromal cells (BMMSCs) effectively regulate oxidative stress following radiative injury, which might be on account of irradiation-induced elevation of protein levels of CR6-interacting factor 1(CRIF1) and nuclear factor E2-related factor 2(NRF2). Crif1-knockdown BMMSCs presented increased oxidative stress and apoptosis after irradiation, which were partially due to a suppressed antioxidant response mediated by decreased NRF2 nuclear translocation. Co-immunoprecipitation (Co-IP) experiments indicated that CRIF1 interacted with protein kinase C-δ (PKC-δ). NRF2 Ser40 phosphorylation was inhibited in Crif1-deficient BMMSCs even in the presence of three kinds of PKC agonists, suggesting that CRIF1 might co-activate PKC-δ to phosphorylate NRF2 Ser40. After radiative injury, the supporting effect of BMMSCs for the colony forming ability of HSCs in vitro was reduced, and the deficiency of CRIF1 aggravated such damage. Thus, CRIF1 plays an essential role in PKC-δ/NRF2 pathway modulation to alleviate oxidative stress in BMMSCs after irradiative injury, and at some level it may maintain the HSCs-supporting effect of BMMSCs after radiative injuries.
2017-01-01 | S-EPMC5558558 | BioStudies
Project description:Accumulation of amyloid ? (A?) and tau represent the two major pathological hallmarks of Alzheimer's disease (AD). Despite the critical importance of A? accumulation as an early event in AD pathogenesis, multiple lines of evidence indicate that tau is required to mediate A?-induced neurotoxic signals in neurons. We have previously shown that the scaffolding protein Ran-binding protein 9 (RanBP9), which is highly elevated in brains of AD and AD mouse models, both enhances A? production and mediates A?-induced neurotoxicity. However, it is unknown whether and how RanBP9 transmits A?-induced neurotoxic signals to tau. Here we show for the first time that overexpression or knockdown of RanBP9 directly enhances and reduces tau levels, respectively, in vitro and in vivo. Such changes in tau levels are associated with the ability of RanBP9 to physically interact with tau and heat shock protein 90/heat shock cognate 70 (Hsp90/Hsc70) complexes. Meanwhile, both RanBP9 and tau levels are simultaneously reduced by Hsp90 or Hsc70 inhibitors, whereas overexpression or knockdown of RanBP9 significantly diminishes the anti-tau potency of Hsp90/Hsc70 inhibitors as well as Hsc70 variants (WT & E175S). Further, RanBP9 increases the capacity for Hsp90 and Hsc70 complexes to bind ATP and enhances their ATPase activities in vitro. These observations in vitro and cell lines are recapitulated in primary neurons and in vivo, as genetic reduction in RanBP9 not only ameliorates tauopathy in Tau-P301S mice but also rescues the deficits in synaptic integrity and plasticity.
2017-01-01 | S-EPMC6075219 | BioStudies
Project description:Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.
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Project description:Steatosis, oxidative stress, and apoptosis underlie the development of nonalcoholic steatohepatitis (NASH). Protein kinase C delta (PKC?) has been implicated in fatty liver disease and is activated in the methionine and choline-deficient (MCD) diet model of NASH, yet its pathophysiological importance towards steatohepatitis progression is uncertain. We therefore addressed the role of PKC? in the development of steatosis, inflammation, oxidative stress, apoptosis, and fibrosis in an animal model of NASH. We fed PKC?(-/-) mice and wildtype littermates a control or MCD diet. PKC?(-/-) primary hepatocytes were used to evaluate the direct effects of fatty acids on hepatocyte lipid metabolism gene expression. A reduction in hepatic steatosis and triglyceride levels were observed between wildtype and PKC?(-/-) mice fed the MCD diet. The hepatic expression of key regulators of ?-oxidation and plasma triglyceride metabolism was significantly reduced in PKC?(-/-) mice and changes in serum triglyceride were blocked in PKC?(-/-) mice. MCD diet-induced hepatic oxidative stress and hepatocyte apoptosis were reduced in PKC?(-/-) mice. MCD diet-induced NADPH oxidase activity and p47(phox) membrane translocation were blunted and blocked, respectively, in PKC?(-/-) mice. Expression of pro-apoptotic genes and caspase 3 and 9 cleavage in the liver of MCD diet fed PKC?(-/-) mice were blunted and blocked, respectively. Surprisingly, no differences in MCD diet-induced fibrosis or pro-fibrotic gene expression were observed in 8 week MCD diet fed PKC?(-/-) mice. Our results suggest that PKC? plays a role in key pathological features of fatty liver disease but not ultimately in fibrosis in the MCD diet model of NASH.
2014-01-01 | S-EPMC3893275 | BioStudies
Project description:Astrocytic dysfunction has been implicated in Parkinson's disease (PD) pathogenesis. While the Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 signaling axis is known to play a role in PD-like neuropathology, the molecular mechanisms that govern this process remain poorly understood. Herein, we show that TWEAK levels are elevated in PD serum compared to controls. Moreover, using both U373 human astrocyte cells and primary mouse astrocytes, we demonstrate that TWEAK induces mitochondrial oxidative stress as well as protein kinase C delta (PKC?) and signal transducer and activator of transcription 3 (STAT3) activation, accompanied by NLRC4 inflammasome activation and upregulation and release of proinflammatory cytokines, including IL-1?, TNF-?, and IL-18. Mechanistically, TWEAK-induced PKC? activation enhances the STAT3/NLRC4 signaling pathway and other proinflammatory mediators through a mitochondrial oxidative stress-dependent mechanism. We further show that PKC? knockdown and mito-apocynin, a mitochondrial antioxidant, suppress TWEAK-induced proinflammatory NLRC4/STAT3 signaling and cellular oxidative stress response. Notably, we validated our in vitro findings in an MPTP mouse model of PD and in mice receiving intrastriatal administration of TWEAK. These results indicate that TWEAK is a key regulator of astroglial reactivity and illustrate a novel mechanism by which mitochondrial oxidative stress may influence dopaminergic neuronal survival in PD.
2020-01-01 | S-EPMC7464730 | BioStudies