ABSTRACT
Acquired middle ear cholesteatoma leads to hearing loss, ear discharge, ear pain, and more serious intracranial complications. However, there is still no effective treatment other than surgery. TFIIB-related factor 2 (BRF2) acted as a redox sensor overexpressing in oxidative stress which linked endoplasmic reticulum (ER) stress, while glucose-regulated protein 78 (GRP78) was a biomarker of ER stress in cancer, atherosclerosis and inflammation. In our study, we investigated the roles of BRF2 and GRP78 in acquired middle ear cholesteatoma. Our results revealed that the expression of BRF2 was significant increased in acquired middle ear cholesteatoma, and which was positively correlated with the expression of GRP78. In addition, BRF2 and GRP78 showed colocalization in epithelium of acquired middle ear cholesteatomas and HaCaT cells. Prolongation of LPS stimulation in HaCaT cells escalated the expression of BRF2 and GRP78. To confirm the role of BRF2 and GRP78, we transfected si-BRF2 into HaCaT cells. All results indicated that BRF2 expression positively regulates the expression of GRP78 and may participate in the pathogenesis of acquire middle ear cholesteatoma.
Subject(s)
Cholesteatoma, Middle Ear/metabolism , Heat-Shock Proteins/metabolism , Transcription Factor TFIIIB/metabolism , Cell Line, Tumor , Endoplasmic Reticulum Chaperone BiP , Gene Knockdown Techniques , Heat-Shock Proteins/deficiency , Humans , Lipopolysaccharides/immunology , Transcription Factor TFIIIB/deficiency , Up-RegulationABSTRACT
Flocculation is an aggregation phenomenon of microbial cells in which they form flocs or flakes. In this study, it was found that addition of glycerol to a complex glucose medium promoted spontaneous floc formation by an Escherichia coli degP-deficient mutant strain (ΔdegP) in a dose-dependent manner. In the presence of 10% (v/v) glycerol, the amount of floc formation (quantified as floc protein) reached its maximum value (230 mg/L), five times that in its absence. 10% (v/v) glycerol was the limit concentration that does not inhibit cell growth of ΔdegP strain. Glycerol was not consumed by ΔdegP cells during floc formation. To provide media having nearly the same viscosity as that containing 10% (v/v) glycerol, carboxymethyl cellulose (CMC) or polyvinylpyrrolidone (PVP) were added to medium as viscosifying agents. Floc formation was not promoted by increasing the medium viscosity with CMC or PVP. However, addition of ethylene glycol also significantly promoted floc formation in the same manner as glycerol. Addition of short-chain polyols decreased the number of viable ΔdegP cells in the floc structure and enhanced outer membrane vesicle (OMV) production by ΔdegP cells; polyols-induced damage on the outer membrane of ΔdegP cells may contribute to the promoted floc formation.
Subject(s)
Escherichia coli/cytology , Escherichia coli/drug effects , Glycerol/pharmacology , Heat-Shock Proteins/deficiency , Serine Endopeptidases/deficiency , Flocculation/drug effects , Periplasmic ProteinsABSTRACT
UC is a chronic inflammatory disease of the colonic mucosa and lacks effective treatments because of unclear pathogenesis. Excessive apoptosis of IECs damages the intestinal epithelial barrier and is involved in the progression of UC, but the mechanism is unknown. HSPs are important in maintaining homeostasis and regulate apoptosis through the mitochondrial pathway. In our previous studies, HSF2, an important regulator of HSPs, was highly expressed in UC patients and negatively correlated with inflammation in mice and IECs. Therefore, we hypothesized that HSF2 may protect against intestinal mucositis by regulating the apoptosis of IECs. In this study, a DSS-induced colitis model of hsf2-/- mice was used to explore the relationship between HSF2 and apoptosis in IECs for the first time. The expression of HSF2 increased in the WT + DSS group compared with that in the WT + H2O group. Moreover, the extent of apoptosis was more severe in the KO + DSS group than in the WT + DSS group. The results showed that HSF2 was negatively correlated with apoptosis in vivo. The expression of HSF2 in Caco-2 cells was changed by lentiviral transfection, and the expression of Bax, cytoplasmic Cyto-C, Cleaved Caspase-9 and Cleaved Caspase-3 were negatively correlated with the different levels of HSF2. These results suggest that HSF2 negatively regulates apoptosis of IECs through the mitochondrial pathway. This may be one of the potential mechanisms to explain the protective role of HSF2 in UC.
Subject(s)
Apoptosis , Colitis, Ulcerative/metabolism , Epithelial Cells/metabolism , Heat-Shock Proteins/metabolism , Intestinal Mucosa/metabolism , Mitochondria/metabolism , Transcription Factors/metabolism , Animals , Apoptosis/drug effects , Caco-2 Cells , Cells, Cultured , Colitis, Ulcerative/pathology , Dose-Response Relationship, Drug , Epithelial Cells/pathology , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Humans , Intestinal Mucosa/pathology , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Structure , Structure-Activity Relationship , Transcription Factors/deficiency , Transcription Factors/geneticsABSTRACT
Intestinal macrophages participate in the pathogenesis of inflammatory bowel diseases (IBDs) through secreting pro-inflammatory and tissue-damaging factors as well as inducing the differentiation of T helper 1 (Th1) and T helper 17 (Th17) cells. Elucidating the regulatory mechanisms of intestinal macrophage activity in IBDs is important for developing new therapeutic approaches. In the current study, the expression of Sestrins in myeloid cells and lymphocytes in colonic lamina propria (LP) was evaluated in a murine acute colitis model. We found that Sestrin3 was significantly up-regulated in LP macrophages by the colonic LP microenvironment. In the in vitro experiments, lentivirus-mediated Sestrin3 knockdown significantly reduced the production of IL-12 and IL-23 in activated macrophages, in addition to decreasing the expression of classical pro-inflammatory cytokines such as IL-1ß, IL-6 and TNF-α. Additionally, Sestrin3 knockdown impaired macrophage-mediated generation of Th1 and Th17 cells from CD4+ T cells, probably through up-regulating the phosphorylation of mechanistic target of rapamycin complex 1 (mTORC1) in macrophages. In the in vivo experiments, adoptive transfer of Sestrin3-deficient macrophages alleviated the generation of Th1 and Th17 cells in the colonic LP and mesenteric lymph nodes. Furthermore, the adoptive transfer mitigated the severity of colitis, as demonstrated by lower production of pro-inflammatory cytokines and fewer tissue lesions in the colon. Our study suggests that Sestrin3 might be crucial for macrophage-mediated generation of pathogenic Th1 and Th17 cells in IBDs.
Subject(s)
Colitis/immunology , Heat-Shock Proteins/immunology , Macrophages/immunology , Th1 Cells/immunology , Th17 Cells/immunology , Animals , Cell Differentiation/immunology , Cells, Cultured , Colitis/chemically induced , Colitis/pathology , Dextran Sulfate/administration & dosage , Disease Models, Animal , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Th1 Cells/pathology , Th17 Cells/pathologyABSTRACT
Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749.
Subject(s)
Aging/metabolism , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/deficiency , Hippocampus/metabolism , Molecular Chaperones/metabolism , Adaptation, Physiological/physiology , Aging/genetics , Animals , Embryonic Stem Cells/metabolism , Gene Knockout Techniques/methods , HSP70 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Hippocampus/cytology , Humans , Male , Mice , Mice, Inbred C57BL , Molecular Chaperones/genetics , Neurons/metabolismABSTRACT
Intrahepatic cholestasis of pregnancy (ICP) is a liver disorder occurred in pregnant women, and the mechanism for such disease is still unclear. The bioinformatics analysis of our previous study has revealed the abnormal expression of endoplasmic reticulum protein 29 (ERp29) in placental tissue of ICP patients. In this study, the function of ERp29 was further explored using in vitro model of ICP. The results showed that up-regulation of ERp29 occurred in TCA (taurocholic acid)-treated human trophoblast HTR-8/SVeno cells, and ERp29 inhibition reversed TCA toxicity via attenuating G2/M arrest and cell apoptosis. Mechanical study revealed ERp29 inhibition suppressed phosphorylation and kinase activity of p38, thus subsequently affecting expression and phosphorylation of p53 (ser18) as well as the transcriptional activity of p53. The conduction of this study might confirm the important role of ERp29 in ICP and which would be helpful for the development of target therapeutic method for ICP.
Subject(s)
Gene Knockdown Techniques , Heat-Shock Proteins/genetics , Taurocholic Acid/toxicity , Trophoblasts/cytology , Trophoblasts/metabolism , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Apoptosis/drug effects , Apoptosis/genetics , Cell Line , Cholestasis, Intrahepatic/genetics , Cholestasis, Intrahepatic/metabolism , Cholestasis, Intrahepatic/pathology , Female , G2 Phase Cell Cycle Checkpoints/drug effects , G2 Phase Cell Cycle Checkpoints/genetics , Heat-Shock Proteins/deficiency , Humans , M Phase Cell Cycle Checkpoints/drug effects , M Phase Cell Cycle Checkpoints/genetics , Phosphorylation/drug effects , Phosphorylation/genetics , Pregnancy , Pregnancy Complications/genetics , Pregnancy Complications/metabolism , Pregnancy Complications/pathology , Trophoblasts/drug effectsABSTRACT
The host presents an array of environments which induce bacterial stress including changes in pH, antimicrobial compounds and reactive oxygen species. The bacterial envelope sits at the interface between the intracellular and extracellular environment and its maintenance is essential for Salmonella cell viability under a range of conditions, including during infection. In this study, we aimed to understand the contribution of the σH- and σE-regulated small heat shock proteins IbpA, IbpB, and AgsA and the putative σE-regulated stress response protein STM1250 to the Salmonella envelope stress response. Due to shared sequence identity, regulatory overlap, and the specificity of STM1250 and AgsA to Salmonella sp., we hypothesized that functional overlap exists between these four stress response proteins, which might afford a selective advantage during Salmonella exposure to stress. We present here new roles for three small heat shock proteins and a putative stress response protein in Salmonella that are not limited to heat shock. We have shown that, compared to WT, a quadruple mutant is significantly more sensitive to hydrogen peroxide, has a lower minimum bactericidal concentration to the cationic antimicrobial peptide polymyxin B, and is attenuated in macrophages.
Subject(s)
Bacterial Proteins/metabolism , Host-Pathogen Interactions , Macrophages/immunology , Microbial Viability , Oxidative Stress , Salmonella typhimurium/immunology , Stress, Physiological , Animals , Bacterial Proteins/genetics , Gene Deletion , Gene Regulatory Networks , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/metabolism , Macrophages/microbiology , Mice , Models, Biological , RAW 264.7 Cells , Salmonella Infections/immunology , Salmonella Infections/microbiology , Salmonella typhimurium/physiology , Sigma Factor/deficiency , Sigma Factor/metabolism , VirulenceABSTRACT
The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81-176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains - ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.
Subject(s)
Bacterial Proteins/analysis , Campylobacter jejuni/chemistry , Campylobacter jejuni/drug effects , Extracellular Vesicles/chemistry , Gene Deletion , Proteome/analysis , Stress, Physiological , Anti-Bacterial Agents/pharmacology , Campylobacter jejuni/genetics , Heat-Shock Proteins/deficiency , Oxidoreductases/deficiency , Oxygen/toxicityABSTRACT
In Escherichia coli, the periplasmic protease DegP plays a critical role in degrading misfolded outer membrane proteins (OMPs). Consequently, mutants lacking DegP display a temperature-sensitive growth defect, presumably due to the toxic accumulation of misfolded OMPs. The Tol-Pal complex plays a poorly defined but an important role in envelope biogenesis, since mutants defective in this complex display a classical periplasmic leakage phenotype. Double mutants lacking DegP and an intact Tol-Pal complex display exaggerated temperature-sensitive growth defects and the leaky phenotype. Two revertants that overcome the temperature-sensitive growth phenotype carry missense mutations in the degS gene, resulting in D102V and D320A substitutions. D320 and E317 of the PDZ domain of DegS make salt bridges with R178 of DegS's protease domain to keep the protease in the inactive state. However, weakening of the tripartite interactions by D320A increases DegS's basal protease activity. Although the D102V substitution is as effective as D320A in suppressing the temperature-sensitive growth phenotype, the molecular mechanism behind its effect on DegS's protease activity is unclear. Our data suggest that the two DegS variants modestly activate RseA-controlled, σE-mediated envelope stress response pathway and elevate periplasmic protease activity to restore envelope homeostasis. Based on the release of a cytoplasmic enzyme in the culture supernatant, we conclude that the conditional lethal phenotype of ΔtolB ΔdegP mutants stems from a grossly destabilized envelope structure that causes excessive cell lysis. Together, the data point to a critical role for periplasmic proteases when the Tol-Pal complex-mediated envelope structure and/or functions are compromised.IMPORTANCE The Tol-Pal complex plays a poorly defined role in envelope biogenesis. The data presented here show that DegP's periplasmic protease activity becomes crucial in mutants lacking the intact Tol-Pal complex, but this requirement can be circumvented by suppressor mutations that activate the basal protease activity of a regulatory protease, DegS. These observations point to a critical role for periplasmic proteases when Tol-Pal-mediated envelope structure and/or functions are perturbed.
Subject(s)
Adaptation, Physiological/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/genetics , Periplasmic Proteins/genetics , Serine Endopeptidases/genetics , Amino Acid Substitution , Cell Wall/genetics , Cell Wall/metabolism , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Heat-Shock Proteins/deficiency , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Mutation , Periplasmic Proteins/metabolism , Phenotype , Protein Binding , Protein Structure, Secondary , Serine Endopeptidases/deficiency , Sigma Factor/genetics , Sigma Factor/metabolism , Stress, Physiological , Temperature , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The Gram-negative outer membrane (OM) is a selectively permeable asymmetric bilayer that allows vital nutrients to diffuse into the cell but prevents toxins and hydrophobic molecules from entering. Functionally and structurally diverse ß-barrel outer membrane proteins (OMPs) build and maintain the permeability barrier, making the assembly of OMPs crucial for cell viability. In this work, we characterize an assembly-defective mutant of the maltoporin LamB, LamBG439D We show that the folding defect of LamBG439D results in an accumulation of unfolded substrate that is toxic to the cell when the periplasmic protease DegP is removed. Selection for suppressors of this toxicity identified the novel mutant degSA323E allele. The mutant DegSA323E protein contains an amino acid substitution at the PDZ/protease domain interface that results in a partially activated conformation of this protein. This activation increases basal levels of downstream σE stress response signaling. Furthermore, the enhanced σE activity of DegSA323E suppresses a number of other assembly-defective conditions without exhibiting the toxicity associated with high levels of σE activity. We propose that the increased basal levels of σE signaling primes the cell to respond to envelope stress before OMP assembly defects threaten cell viability. This finding addresses the importance of envelope stress responses in monitoring the OMP assembly process and underpins the critical balance between envelope defects and stress response activation.IMPORTANCE Gram-negative bacteria, such as Escherichia coli, inhabit a natural environment that is prone to flux. In order to cope with shifting growth conditions and the changing availability of nutrients, cells must be capable of quickly responding to stress. Stress response pathways allow cells to rapidly shift gene expression profiles to ensure survival in this unpredictable environment. Here we describe a mutant that partially activates the σE stress response pathway. The elevated basal level of this stress response allows the cell to quickly respond to overwhelming stress to ensure cell survival.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Escherichia coli Proteins/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Proteins/genetics , Periplasmic Proteins/genetics , Porins/genetics , Receptors, Virus/genetics , Serine Endopeptidases/genetics , Sigma Factor/genetics , Adaptation, Physiological/genetics , Amino Acid Substitution , Bacterial Outer Membrane Proteins/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Heat-Shock Proteins/deficiency , Microbial Viability , Models, Molecular , Mutation , Periplasm/genetics , Periplasm/metabolism , Porins/chemistry , Porins/deficiency , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Receptors, Virus/chemistry , Receptors, Virus/deficiency , Serine Endopeptidases/deficiency , Sigma Factor/metabolism , Signal Transduction , Stress, PhysiologicalABSTRACT
Aberrant sperm flagella impair sperm motility and cause male infertility, yet the genes which have been identified in multiple morphological abnormalities of the flagella (MMAF) can only explain the pathogenic mechanisms of MMAF in a small number of cases. Here, we identify and functionally characterize homozygous loss-of-function mutations of QRICH2 in two infertile males with MMAF from two consanguineous families. Remarkably, Qrich2 knock-out (KO) male mice constructed by CRISPR-Cas9 technology present MMAF phenotypes and sterility. To elucidate the mechanisms of Qrich2 functioning in sperm flagellar formation, we perform proteomic analysis on the testes of KO and wild-type mice. Furthermore, in vitro experiments indicate that QRICH2 is involved in sperm flagellar development through stabilizing and enhancing the expression of proteins related to flagellar development. Our findings strongly suggest that the genetic mutations of human QRICH2 can lead to male infertility with MMAF and that QRICH2 is essential for sperm flagellar formation.
Subject(s)
Infertility, Male/genetics , Loss of Function Mutation , Microtubule Proteins/genetics , Sperm Tail/metabolism , A Kinase Anchor Proteins/deficiency , A Kinase Anchor Proteins/genetics , Adult , Animals , Calcium-Binding Proteins/deficiency , Calcium-Binding Proteins/genetics , Consanguinity , Gene Expression , Gene Expression Profiling , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Humans , Infertility, Male/metabolism , Infertility, Male/pathology , Male , Mice , Mice, Knockout , Pedigree , Phosphoproteins/deficiency , Phosphoproteins/genetics , Sperm Motility , Sperm Tail/pathology , Sperm Tail/ultrastructure , Testis/chemistry , Testis/metabolism , Whole Genome SequencingABSTRACT
Embryos of the crustacean Artemia franciscana may arrest as gastrulae, forming cysts that enter diapause, which is a state of reduced metabolism and enhanced stress tolerance. Diapausing cysts survive physiological stresses for years due, in part, to molecular chaperones. p26, a small heat-shock protein, is an abundant diapause-specific molecular chaperone in cysts, and it affects embryo development and stress tolerance. p26 is therefore thought to influence many proteins in cysts, and this study was undertaken to determine how the loss of p26 by RNA interference (RNAi) affects the diapause proteome of A. franciscana. The proteome was analyzed by shot-gun proteomics coupled to differential isotopic labeling and tandem mass spectrometry. Proteins in the diapause proteome included metabolic enzymes, antioxidants, binding proteins, structural proteins, transporters, translation factors, receptors, and signal transducers. Proteins within the diapause proteome either disappeared or were reduced in amount when p26 was knocked down, or conversely, proteins appeared or increased in amount. Those proteins that disappeared may be p26 substrates, whereas the synthesis of those proteins that appeared or increased may be regulated by p26. This study provides the first global characterization of the diapause proteome of A. franciscana and demonstrates that the sHsp p26 influences proteome composition.
Subject(s)
Artemia/metabolism , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/metabolism , Proteome/metabolism , RNA Interference , Animals , Computational Biology , Female , Heat-Shock Proteins/isolation & purificationABSTRACT
Aims: Inadequate healing after myocardial infarction (MI) leads to heart failure and fatal ventricular rupture, while optimal healing requires timely induction and resolution of inflammation. This study tested the hypothesis that heat shock protein B1 (HSPB1), which limits myocardial inflammation during endotoxemia, modulates wound healing after MI. Methods and results: To test this hypothesis, cardiomyocyte-specific HSPB1 knockout (Hspb1-/-) mice were generated using the Cre-LoxP recombination system. MI was induced by ligation of the left anterior descending coronary artery in Hspb1-/- and wild-type (WT) littermates. HSPB1 was up-regulated in cardiomyocytes of WT animals in response to MI, and deficiency of cardiomyocyte HSPB1 increased MI-induced cardiac rupture and mortality within 21 days after MI. Serial echocardiography showed more aggravated remodelling and cardiac dysfunction in Hspb1-/- mice than in WT mice at 1, 3, and 7 days after MI. Decreased collagen deposition and angiogenesis, as well as increased MMP2 and MMP9 activity, were also observed in Hspb1-/- mice compared with WT controls after MI, using immunofluorescence, polarized light microscopy, and zymographic analyses. Notably, Hspb1-/- hearts exhibited enhanced and prolonged leucocyte infiltration, enhanced expression of inflammatory cytokines, and enhanced TLR4/MyD88/NFκB activation compared with WT controls after MI. In-depth molecular analyses in both mice and primary cardiomyocytes demonstrated that cardiomyocyte-specific knockout of HSPB1 increased nuclear factor-κB (NFκB) activation, which promoted the expression of proinflammatory mediators. This led to increased leucocyte recruitment, thereby to excessive inflammation, ultimately resulting in adverse remodelling, cardiac dysfunction, and cardiac rupture following MI. Conclusion: These data suggest that HSPB1 acts as a negative regulator of NFκB-mediated leucocyte recruitment and the subsequent inflammation in cardiomyocytes. Cardiomyocyte HSPB1 is required for wound healing after MI and could be a target for myocardial repair in MI patients.
Subject(s)
Chemotaxis, Leukocyte , Heart Rupture, Post-Infarction/metabolism , Heat-Shock Proteins/deficiency , Leukocytes/metabolism , Myocardial Infarction/metabolism , Myocardial Reperfusion Injury/metabolism , Myocytes, Cardiac/metabolism , NF-kappa B/metabolism , Ventricular Remodeling , Animals , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , HSP27 Heat-Shock Proteins/deficiency , HSP27 Heat-Shock Proteins/genetics , Heart Rupture, Post-Infarction/immunology , Heart Rupture, Post-Infarction/pathology , Heart Rupture, Post-Infarction/physiopathology , Heat-Shock Proteins/genetics , Leukocytes/immunology , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones/genetics , Myocardial Infarction/immunology , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardial Reperfusion Injury/immunology , Myocardial Reperfusion Injury/pathology , Myocardial Reperfusion Injury/physiopathology , Myocytes, Cardiac/immunology , Myocytes, Cardiac/pathology , Neovascularization, Physiologic , Rats, Sprague-Dawley , Signal Transduction , Wound HealingABSTRACT
Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.
Subject(s)
Heat-Shock Proteins/metabolism , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Animals , Cell Survival , Cells, Cultured , Echocardiography , Endoplasmic Reticulum Chaperone BiP , Heat-Shock Proteins/deficiency , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Sprague-DawleyABSTRACT
Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2 Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X ). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.
Subject(s)
Enhancer of Zeste Homolog 2 Protein/metabolism , Epigenesis, Genetic , Heat-Shock Proteins/genetics , Lymphoma, Follicular/genetics , Mechanistic Target of Rapamycin Complex 1/metabolism , Animals , Chromosome Deletion , Chromosomes, Human, Pair 6/genetics , Enhancer of Zeste Homolog 2 Protein/antagonists & inhibitors , Gene Silencing , Genetic Testing , Genome, Human , Heat-Shock Proteins/deficiency , Humans , Mice , Mutation/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismSubject(s)
Haploinsufficiency , Heat-Shock Proteins/genetics , Muscular Diseases/genetics , Protein Serine-Threonine Kinases/genetics , Adult , Disease Progression , Family , Female , Heat-Shock Proteins/deficiency , Humans , Male , Middle Aged , Molecular Chaperones , Muscular Diseases/diagnostic imaging , Muscular Diseases/pathology , Muscular Diseases/physiopathology , Phenotype , Protein Serine-Threonine Kinases/deficiencyABSTRACT
The plant-specific RING-domain finger proteins play important roles in plant development and stress responses. We recently identified and functionally characterized a stress-induced gene OsSRFP1 (Oryza sativa Stress-related RING Finger Protein 1) from rice. We showed evidences of the biotechnological potential of the suppression of OsSRFP1 expression in conferring cold tolerance. The increased cold tolerance of OsSRFP1 knock-down plants was associated with higher amounts of free proline and activities of antioxidant enzymes. In vitro ubiquitination assays showed that OsSRFP1 possessed E3 ubiquitin ligase activity. Some predicted interacting partners of OsSRFP1 might be the substrates for OsSRFP1-mediated protein degradation. Interestingly, OsSRFP1 had trans-activation activity, suggesting the dual roles of OsSRFP1 in post-translational and transcriptional regulations in stress responses.
Subject(s)
Adaptation, Physiological/genetics , Gene Expression Regulation, Plant , Heat-Shock Proteins/genetics , Oryza/genetics , Plant Proteins/genetics , Trans-Activators/genetics , Cold Temperature , Gene Knockdown Techniques , Heat-Shock Proteins/deficiency , Oryza/metabolism , Plant Proteins/metabolism , Plants, Genetically Modified , Proline/metabolism , Promoter Regions, Genetic , Proteolysis , RING Finger Domains , Stress, Physiological , Trans-Activators/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , UbiquitinationABSTRACT
Stress-inducible phosphoprotein I (STIP1, STI1 or HOP) is a co-chaperone intermediating Hsp70/Hsp90 exchange of client proteins, but it can also be secreted to trigger prion protein-mediated neuronal signaling. Some mothers of children with autism spectrum disorders (ASD) present antibodies against certain brain proteins, including antibodies against STIP1. Maternal antibodies can cross the fetus blood-brain barrier during pregnancy, suggesting the possibility that they can interfere with STIP1 levels and, presumably, functions. However, it is currently unknown whether abnormal levels of STIP1 have any impact in ASD-related behavior. Here, we used mice with reduced (50%) or increased STIP1 levels (fivefold) to test for potential ASD-like phenotypes. We found that increased STIP1 regulates the abundance of Hsp70 and Hsp90, whereas reduced STIP1 does not affect Hsp70, Hsp90 or the prion protein. Interestingly, BAC transgenic mice presenting fivefold more STIP1 show no major phenotype when examined in a series of behavioral tasks, including locomotor activity, elevated plus maze, Morris water maze and five-choice serial reaction time task (5-CSRTT). In contrast, mice with reduced STIP1 levels are hyperactive and have attentional deficits on the 5-CSRTT, but exhibit normal performance for the other tasks. We conclude that reduced STIP1 levels can contribute to phenotypes related to ASD. However, future experiments are needed to define whether it is decreased chaperone capacity or impaired prion protein signaling that contributes to these phenotypes.
Subject(s)
Attention Deficit Disorder with Hyperactivity/metabolism , Behavior, Animal , Heat-Shock Proteins/deficiency , Animals , Attention Deficit Disorder with Hyperactivity/genetics , Attention Deficit Disorder with Hyperactivity/psychology , Disease Models, Animal , Genetic Predisposition to Disease , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/genetics , Male , Maze Learning , Mice, Inbred C57BL , Mice, Knockout , Motor Activity , Phenotype , PrPC Proteins/metabolism , Reaction Time , Swimming , Time FactorsABSTRACT
p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cytoskeletal Proteins/metabolism , Heat-Shock Proteins/metabolism , Imidazoles/pharmacology , NF-E2-Related Factor 2/metabolism , Piperazines/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/deficiency , Adaptor Proteins, Signal Transducing/genetics , Animals , Apoptosis/drug effects , Apoptosis/physiology , Autophagy/drug effects , Autophagy/physiology , Cells, Cultured , Cytoskeletal Proteins/genetics , HeLa Cells , Heat-Shock Proteins/deficiency , Heat-Shock Proteins/genetics , Humans , Kelch-Like ECH-Associated Protein 1 , Mice , Protein Kinase Inhibitors/pharmacology , Proteolysis/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reactive Oxygen Species/metabolism , Sequestosome-1 Protein , Signal Transduction/drug effectsABSTRACT
In Lewy body disease (LBD) such as dementia with LBs and Parkinson's disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin-positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB-like inclusions were observed in transgenic mice that overexpressed α-synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α-synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α-synuclein aggregation.