Cell Stress and Chaperones最新文献

Studies on chromatin structure and function have gained a revived popularity. Histone chaperones are significant players in chromatin organization. They play a significant role in vital nuclear functions like transcription, DNA replication, DNA repair, DNA recombination, and epigenetic regulation, primarily by aiding processes such as histone shuttling and nucleosome assembly/disassembly. Like the other eukaryotes, plants also have a highly orchestrated and dynamic chromatin organization. Plants seem to have more isoforms within the same family of histone chaperones, as compared with other organisms. As some of these are specific to plants, they must have evolved to perform functions unique to plants. However, it appears that only little effort has gone into understanding the structural features of plant histone chaperones and their structure-function relationships. Studies on plant histone chaperones are essential for understanding their role in plant chromatin organization and how plants respond during stress conditions. This review is on the structural and functional aspects of plant histone chaperone families, specifically those which bind to H2A-H2B, viz nucleosome assembly protein (NAP), nucleoplasmin (NPM), and facilitates chromatin transcription (FACT). Here, we also present comparative analyses of these plant histone chaperones with available histone chaperone structures. The review hopes to incite interest among researchers to pursue further research in the area of plant chromatin and the associated histone chaperones.

It has come to authors' attention that an inadvertent mistake was made in the construction of Figure 4 and Figure 6 of the original publication.

The eukaryotic cytosolic chaperonin, t-complex polypeptide 1 (TCP-1) ring complex or TRiC, is responsible for folding a tenth of the proteins in the cell. TRiC is a double-ringed barrel with each ring composed of eight different CCT (chaperonin containing TCP-1) subunits. In order for the subunits to assemble together into mature TRiC, which is believed to contain one and only one of each of these subunits per ring, they must be translated from different chromosomes, correctly folded and assembled. When expressed alone in Escherichia coli, the subunits CCT4 and CCT5, interestingly, form TRiC-like homo-oligomeric rings. To explore potential subunit-subunit interactions, we co-expressed these homo-oligomerizing CCT4 and CCT5 subunits or the archaeal chaperonin Mm-Cpn (Methanococcus maripaludis chaperonin) with CCT1-8, one at a time. We found that CCT5 shifted all of the CCT subunits, with the exception of CCT6, into double-barrel TRiC-like complexes, while CCT4 only interacted with CCT5 and CCT8 to form chaperonin rings. We hypothesize that these specific interactions may be due to the formation of hetero-oligomers in E. coli, although more work is needed for validation. We also observed the interaction of CCT5 and Mm-Cpn with smaller fragments of the CCT subunits, confirming their intrinsic chaperone activity. Based on this hetero-oligomer data, we propose that TRiC assembly relies on subunit exchange with some stable homo-oligomers, possibly CCT5, as base assembly units. Eventually, analysis of CCT arrangement in various tissues and at different developmental times is anticipated to provide additional insight on TRiC assembly and CCT subunit composition.

Previously, we demonstrated that species of the Stratiomyidae family exhibit higher tolerance to thermal stress in comparison with that of many representatives of Diptera, including Drosophila species. We hypothesized that species of this group inherited the specific structures of their chaperones from an ancestor of the Stratiomyidae family, and this enabled the descendants to colonize various extreme habitats. To explore this possibility, we cloned and expressed in Escherichia coli copies of the Hsp70 genes from Stratiomys singularior, a typical eurythermal species, and Drosophila melanogaster, for comparison. To investigate the thermal sensitivity of the chaperone function of the inducible 70-kDa heat shock proteins from these species, we used an in vitro refolding luciferase assay. We demonstrated that under conditions of elevated temperature, S. singularior Hsp70 exhibited higher reactivation activity in comparison with D. melanogaster Hsp70 and even human Hsp70. Similarly, S. singularior Hsp70 was significantly more thermostable and showed in vitro refolding activity after preheatment at higher temperatures than D. melanogaster paralog. Thermally induced unfolding experiments using differential scanning calorimetry indicated that Hsp70 from both Diptera species is formed by two domains with different thermal stabilities and that the ATP-binding domain of S. singularior is stable at temperatures 4 degrees higher than that of the D. melanogaster paralog. To the best of our knowledge, this study represents the first report that provides direct experimental data indicating that the evolutionary history of a species may result in adaptive changes in the structures of chaperones to enable them to elicit protective functions at extreme environments.

Increased oxidative stress is a frequent feature in Duchenne muscular dystrophy (DMD). High reactive oxygen species (ROS) levels, associated with altered enzyme antioxidant activity, have been reported in dystrophic patients and mdx mice, an experimental model of DMD. In this study, we investigated the effects of coenzyme Q10 (CoQ10) on oxidative stress marker levels and calcium concentration in primary cultures of dystrophic muscle cells from mdx mice. Primary cultures of skeletal muscle cells from C57BL/10 and mdx mice were treated with coenzyme Q10 (5 μM) for 24 h. The untreated mdx and C57BL/10 muscle cells were used as controls. The MTT and live/dead cell assays showed that CoQ10 presented no cytotoxic effect on normal and dystrophic muscle cells. Intracellular calcium concentration, H₂O₂ production, 4-HNE, and SOD-2 levels were higher in mdx muscle cells. No significant difference in the catalase, GPx, and Gr levels was found between experimental groups. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H₂O₂, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. This study demonstrated that CoQ10 treatment was able to reduce levels of oxidative stress markers, such as H₂O₂, acting as an antioxidant, as well as decreasing abnormal intracellular calcium influx in dystrophic muscles cells. Our findings also suggest that the decrease of oxidative stress reduces the need for upregulation of antioxidant pathways, such as SOD and GSH.

This study was aimed to genetic profiling of heat shock protein 70 (Hsp70) gene in Murrah buffalo investigating 50 unrelated adult animals at ICAR-Research Complex for Eastern Region, Patna (India) in winter, spring, and summer. PCR ready genomic DNA samples and season-wise total RNA samples were prepared. The PCR products of Hsp70 eluted from agarose gel were sequenced and analyzed. The first-strand cDNA was synthesized and concentration was equalized to 25 ng/μl. Expression kinetics of mRNA transcripts in different seasons was studied using Brilliant SYBR Green QPCR technique and the data retrieved was analyzed by least-squares ANOVA. DNA sequencing by primer walking revealed four allelic variants of Hsp70 gene. Alignment study revealed one substitution in 5'UTR, six substitutions in coding region, and one addition in 3'UTR. The highest percent identity and negligible phylogenetic distance were found among the alleles and reference bovine sequences. The relative mRNA expression was significantly higher in summer when THI ≥ 84 than the spring and winter; fold change increased by 4.5 times in summer than the spring whereas found nearly half in winter. These findings can be useful for heat stress management in buffaloes and help in understanding the mechanism of thermo-regulation well.

As an extension of their orchestration of intracellular pathways, secretion of extracellular heat shock proteins (HSPs) is an emerging paradigm of homeostasis imperative to multicellular organization. Extracellular HSP is axiomatic to the survival of cells during tumorigenesis; proportional representation of specific HSP family members is indicative of invasive potential and prognosis. Further significance has been added by the knowledge that all cancer-derived exosomes have surface-exposed HSPs that reflect the membrane topology of cells that secrete them. Extracellular HSPs are also characteristic of chronic inflammation and sepsis. Accordingly, interrogation of extracellular HSPs secreted from cell culture models may represent a facile means of identifying translational biomarker signatures for targeting in situ. In the current study, we evaluated a simple peptide-based multivalent HSP affinity approach using the Vn96 peptide for low speed pelleting of HSP complexes from bioreactor cultures of cell lines with varying invasive phenotype in xenotransplant models: U87 (glioblastoma multiforme; invasive); HELA (choriocarcinoma; minimally invasive); HEK293T (virally transformed immortalized; embryonic). Proteomic profiling by bottom-up mass spectrometry revealed a comprehensive range of candidate biomarkers including primary HSP ligands. HSP complexes were associated with additional chaperones of prognostic significance such as protein disulfide isomerases, as well as pleiotropic metabolic enzymes, established as proportionally reflective of invasive phenotype. Biomarkers of inflammatory and mechanotransductive phenotype were restricted to the most invasive cell model U87, including chitinase CHI3L1, lamin C, amyloid derivatives, and histone isoforms.

The purpose of the current study was to demonstrate the neuroprotective effect of protodioscin (Prot) in an in vitro model of ischemia/reperfusion (I/R) and investigate the underlying molecular mechanism. After PC12 cells were exposed to oxygen and glucose deprivation (OGD) reperfusion, PI staining by flow cytometry was used to quantify the rate of apoptosis. The levels of hypoxia-inducible factor 1-alpha (HIF-1α), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were determined using commercially available kits. Intracellular reactive oxygen species (ROS) level was detected using the 20,70-dichlorodihy-drofluorescein diacetate (DCFH-DA) fluorescence assay. The expression levels of heat-shock proteins (HSP), PI3K, AKT, Nrf2, and miR-124 were tested by western blot or quantitative PCR. Prot significantly attenuated oxygen-glucose deprivation/reperfusion (OGD/R)-induced apoptotic death. Prot also reduced the oxidative stress as revealed by increasing the activities of SOD and GSH-Px, decreasing the levels of ROS and MDA. Moreover, mechanism investigations suggested that Prot prevented the decrease of HSP70, HSP32 (hemeoxygenase-1, HO-1), and PI3K protein expression, phosphorylation of AKT, and the accumulation of nuclear Nrf2. The level of miR-124 was decreased in PC12 cells, which was also effectively reversed by Prot treatment. Prot protected PC12 cells against OGD/R-induced injury through inhibiting oxidative stress and apoptosis, which could be associated with increasing HSP proteins expression via activating PI3K/AKT/Nrf2 pathway and miR-124 modulation.

Heat stress (HS) seriously affects animal performance. In view of global warming, it is essential to understand the regulatory mechanisms by which animals adapt to heat stress. In this study, our aim was to explore the genes and pathways involved in heat stress in sheep. To this end, we used transcriptome analysis to understand the molecular responses to heat stress and thereby identify means to protect sheep from heat shock. To obtain an overview of the effects of heat stress on sheep, we used the hypothalamus for transcriptome sequencing and identified differentially expressed genes (DEGs; false discovery rate (FDR) < 0.01; fold change > 2) during heat stress. A total of 1423 DEGs (1122 upregulated and 301 downregulated) were identified and classified into Gene Ontology (GO) categories and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Heat stress triggered dramatic and complex alterations in gene expression in the hypothalamus. We hypothesized that heat stress induced apoptosis and dysfunction in cells and vital organs and affected growth, development, reproduction, and circadian entrainment via the calcium signaling pathway, which influences ribosome assembly and function. Real-time PCR was used to evaluate the expression of the genes regulating important biological functions or whose expression profiles were significantly changed after acute heat stress (FDR < 0.01; fold change > 4), and the results showed that the expression patterns of these genes were consistent with the results of transcriptome sequencing, indicating that the credibility of the sequencing results. Our data indicated that heat stress induced calcium dyshomeostasis, blocked biogenesis, caused ROS accumulation, impaired the antioxidant system and innate defense, and induced apoptosis through the P53 signaling pathway activated by PEG3, decreased growth and development, and enhanced organ damage. These data is very important and helpful to elucidate the molecular mechanism of heat stress and finally to find ways to deal with heat stress damage in sheep.

Ubiquitin-fold modifier 1 (UFM1)-specific ligase 1 (UFL1) is an important component of the UFM1 conjugation system, which is required for various cellular processes including protein translation, apoptosis, autophagy, and signal transduction. However, both, the expression of UFL1 in mammary cells and its role in endoplasmic reticulum (ER) stress in bovine mammary epithelial cells (BMECs) remain to be fully elucidated. Here, we characterized the potential roles of UFL1 in BMECs. Amino acid sequence comparison indicated that bovine UFL1 shares a high level of sequence identity with the UFL1 of other ruminant species. Notably, UFL1 expression in BMECs was increased by endoplasmic reticulum (ER) stress induced by treatment with tunicamycin (TM). ER stress-related gene expression was further increased in UFL1 knockdown cells upon TM treatment. Moreover, UFL1 overexpression inhibited TM-stimulated ER stress and alleviated ER stress-induced autophagy. Together, our results indicated that UFL1 is a novel ER stress-responsive protein in BMECs. Thus, our study provides a basis for further research into ER stress-related processes in bovine mammary tissues and potential targets for alleviating ER stress in these cells.