Protein Hsp Research Articles

Azaphilones produced by Penicillium maximae with their cell death-inducing activity on Adriamycin-treated cancer cell

BackgroundHeat shock proteins (Hsps) are overexpressed in several tumors and contribute to cell proliferation, metastasis, and anticancer drug resistance. Therefore, Hsp inhibitors have enhanced cytotoxicity as chemotherapeutic agents and may be effective with a reduced dosage for tumor therapy to avoid side effects.ResultsFour new azaphilones, maximazaphilones I–IV (1–4), and three known compounds (5–7) have been isolated from the airborne-derived fungus Penicillium maximae. Inhibitory effects of isolated compounds against induction of Hsp105 were evaluated by the luciferase assay system using Hsp105 promoter. In this assay, 2–4, 6, and 7 significantly inhibited hsp105 promoter activity without cytotoxicity. In addition, all isolated compounds except for 5 significantly induced the death of Adriamycin (ADR)-treated HeLa cells. Interestingly, 1–4, 6, and 7 didn’t show anti-proliferative and cell death-inducing activity without ADR.ConclusionThis study revealed the chemical structures of maximazaphilones I–IV (1–4) and the potency of azaphilones may be useful for cancer treatment and reducing the dose of anticancer agents. In addition, one of the mechanisms of cell death-inducing activity for 2–4, 6, and 7 was suggested to be inhibitory effects of Hsp105 expression.

Temperature alters the oxidative and metabolic biomarkers and expression of environmental stress-related genes in chocolate mahseer (Neolissochilus hexagonolepis).

Long-term acclimation temperature effects on biomarkers of oxidative stress, metabolic stress, expression of heat shock proteins (Hsps), and warm-temperature acclimation related 65-kDa protein (Wap65) were evaluated in the threatened chocolate mahseer (Neolissochilus hexagonolepis). Fifteen-day-old larvae were acclimated to different water temperatures (15, 19, 23-control group, 27, and 31°C) for 60days prior to the sampling for quantification of mRNA, enzyme, nitric oxide, and malondialdehyde (MDA) content. Acclimation to 31°C increased the basal mRNA level of glutathione S-transferase alpha 1 (GSTa1), and activities of catalase (CAT), glutathione reductase (GR), and GST enzymes and but downregulated the expression of superoxide dismutase 1 (SOD1) in the whole-body homogenate. Other antioxidant genes, i.e., CAT and GPx1a, were unaffected at 31°C, and nitric oxide (NO) concentration was significantly lower. In contrast, fish acclimated to 15°C showed an upregulated transcript level of all the antioxidant genes and no significant difference in the CAT, GR, and GST enzymes. Activities of the metabolic enzymes, aspartate transaminase (AST) and alanine transaminase (ALT), were significantly lower at 15°C. The expression of Hsp47 was upregulated at both 15 and 31°C groups, whereas Hsp70 was elevated at 27 and 31°C groups. Wap65-1 transcription did not show significant variation in treatment groups compared to control. Fish in the high (31°C) and low-temperature (15°C) acclimation groups were capable of maintaining oxidative stress by modulating their antioxidant transcripts, enzymes, and Hsps.

Comparative transcriptome analysis of Callosobruchus chinensis (L.) (Coleoptera: Chrysomelidae-Bruchinae) after heat and cold stress exposure

Callosobruchus chinensis is regarded as one of the cosmopolitan pests of legume crops and can cause tremendous losses to a variety of beans. This study focused on comparative transcriptome analyses of C. chinensis exposed to 45 °C (heat stress), 27 °C (ambient temperature) and −3 °C (cold stress) for 3 h to investigate the gene differences and underlying molecular mechanisms. There were 402 and 111 differentially expressed genes (DEGs) identified in the heat and cold stress treatments, respectively. “cell process”, “cell” and “binding” were the main enriched functions and biological processes revealed by gene ontology (GO) analysis. The clusters of orthologous genes (COG) showed that DEGs were assigned to the categories: “posttranslational modification, protein turnover, chaperones”, “lipid transport and metabolism”, and “general function prediction only”. With respect to the Kyoto Encyclopedia of Genes and Genomes (KEGG), the “longevity regulating pathway-multiple species”, “carbon metabolism”, “peroxisome”, “protein processing in endoplasmic”, “glyoxylate and dicarboxylate metabolism” pathways were significantly enriched. The annotation and enrichment analysis revealed that genes encoding heat shock proteins (Hsps) and cuticular proteins were significantly upregulated under high and low-temperature stresses, respectively. In addition, some DEGs encoding “Protein lethal essential for life”, “Reverse transcriptase”, “DnaJ domain”, “Cytochrome” and “Zinc finger protein” were also upregulated to varying degrees. Transcriptomic data were validated using qRT‒PCR, which confirmed that they were consistent. In this paper, the temperature tolerance of C. chinensis adults was evaluated and the results showed that female adults were more sensitive to heat and cold stress than males, and the upregulation of heat shock protein and epidermal protein was the largest in DEGs after heat and cold stress, respectively. These findings provide a reference for further understanding the biological characteristics of C. chinensis adults and the molecular mechanisms underlying the response to low and high temperatures.

Transcription dynamics of heat-shock proteins (Hsps) and endosymbiont titres in response to thermal stress in whitefly, Bemisia tabaci (Asia-I).

The sweet potato whitefly, Bemisia tabaci (Gennadius), is one of the several species complexes of whitefly that are currently significant agricultural pests. Bemisia tabaci infests more than 600 plant species and thrives under a wide range of temperature conditions. In addition to the direct damage caused by sucking plant sap, it vectors several plant viruses. Heat-shock proteins play a pivotal role in enabling the insect to extend its geographical location, survival, and reproduction under different stress conditions. B. tabaci harbours several endosymbionts under the genera Portiera, Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium, and Fritschea that directly or indirectly affect its fitness. By accelerating cuticle biosynthesis and sclerotisation, symbiotic microbes can reduce or enhance tolerance to extreme temperatures and detoxify heavy metals. Thus, symbionts or microbial communities can expand or constrain the abiotic niche space of their host and affect its ability to adapt to changing conditions. The present study delineates the effect of thermal stress on the expression of heat-shock genes and endosymbionts in B. tabaci. Studies of the expression level of heat-shock proteins with the help of quantitative real-time polymerase chain reaction (qRT-PCR) showed that heat- and cold-shock treatment fuels the increased expression of heat-shock proteins (Hsp40 and Hsp70). However, Hsp90 was not induced by a heat- and cold-shock treatment. A significant decrease in the relative titre of secondary endosymbionts, such as Rickettsia, Arsenophonus, and Wolbachia, were recorded in B. tabaci upon heat treatment. However, the titre of the primary symbiont, C. Portiera, was relatively unaffected by both cold and heat treatments. These results are indicative of the fact that Hsp genes and endosymbionts in B. tabaci are modulated in response to thermal stress, and this might be responsible for the adaptation of whitefly under changing climatic scenario.

Prolonged heat waves reduce the condition index and alter the molecular parameters in the pacific oyster Crassostrea gigas

The entire shellfish farming sector is negatively affected by heat waves. Predictive models show that while heat waves are not predicted to exceed 28 °C in the northern Adriatic Sea over the coming decades, their duration will increase to periods of up to 30 days. Knowledge regarding the effects of heat waves on bivalves at physiological and molecular level is still limited. This study attempted to simulate what will happen in the future in Pacific oysters exposed to prolonged heat waves, assessing morphometric and physiological indices, and investigating the expression level of a number of genes, including the chaperone heat shock proteins HSP70, HSP72 and HSP90, and the factor P53. A state of stress in the heat wave-exposed animals was found, with loss of body weight and energy resources: despite showing a higher clearance rate, these animals were unable to absorb the nutrients required to maintain homeostasis, as well as demonstrating an alteration in hemolymphatic AST activity, total calcium and magnesium concentration. mRNA levels of all examined genes increased in response to thermal stress, with long-term overexpression, activating cell stress defense mechanisms and modulating the cycle cell. The results of this study indicate that heat waves affect oyster welfare, with consequences for the productivity of the sector due to the lack of salable products.

Hsp27, Hsp60, Hsp70, or Hsp90 depletion enhances the antitumor effects of resveratrol via oxidative and ER stress response in human glioblastoma cells

Therapeutic resistance of gliomas is still a crucial issue and closely related to induced heat shock response (HSR). Resveratrol (RSV) is a promising experimental agent for glioblastoma (GB) therapy. However, the role of heat shock protein (Hsp)27, Hsp60, Hsp70, and Hsp90 on the therapeutic efficacy of RSV remains unclear in gliomas. Herein, small interfering (si)RNA transfection was performed to block Hsp expressions. RSV treatments reduced glioma cells' viability dose- and time-dependent while keeping HEK-293 normal cells alive. Furthermore, a low dose of RSV (15 µM/48 h) offered protection against oxidative stress and apoptosis due to Hsp depletion in healthy cells. On the contrary, in glioma cells, RSV (15 µM/48 h) increased ROS (reactive oxygen species) production, led to autophagy and induced endoplasmic reticulum (ER) stress and apoptosis, and reduced 2D- and 3D-clonogenic survival. Hsp27, Hsp60, Hsp70, or Hsp90 depletion also resulted in cell death through ER stress response and ROS burst. Remarkably, the heat shock response (increased HSF1 levels) due to Hsp depletion was attenuated by RSV in glioma cells. Collectively, our data show that these Hsp silencings make glioma cells more sensitive to RSV treatment, indicating that these Hsps are potential therapeutic targets for GB treatment.

The relationship between SOD1 and Hsp70 expression in broiler ileum throughout post-hatching development

Heat shock proteins (Hsps) are molecular chaperones that play critical functions in the survival and development of cells. Hsps influence adaptive and innate immune responses and may promote cross-talk between the two systems. Superoxide dismutases (SODs) are metalloenzymes that play an essential role in the body's defense against oxidative stress by efficiently removing excess reactive oxygen species. This study is an experimental study that was conducted to determine the relationship between SOD1 and Hsp70 expression in the ileum during the post-hatching development of the broiler. In the study, samples were taken from ileum tissue of 0-, 21- and 42-day-old broilers were used as material. While the Hsp70 immunoreactivity observed in the epithelial cells was specific to a few cells on day 0, it was detected in more villus epithelial cells on days 21 and 42. The Hsp70 expression in the ileum increased from the age 0 to up to day 42, especially in villus epithelial cells. In sections stained by SOD1, the ileum's villus epithelial cells and smooth muscle cells showed an intracytoplasmic reaction. From day 21 to day 42, a regular increase in SOD1 expression was detected in the crypt and villus epithelial cells. As a remarkable finding, a more intense intracytoplasmic staining was detected in villus epithelial cells located at the apex of intestinal villi. In conclusion, it was observed that SOD1 and Hsp70 expression increased in the ileum tissue throughout post-hatching development in broilers with a positive correlation with age. Based on the histological findings, it can be concluded that SOD1 and Hsp70 play a critical protective role in the small intestine after hatching and contribute to the rapid development of the intestine.

Diagnostic and Prognostic Risk Assessment of Heat Shock Protein HSPA1B rs2763979 Gene Variant in Asthma.

Given the significant role the heat shock protein Hsp70 plays in modulating cellular homeostasis in several chronic inflammatory disorders, the genetic variation of the inducible HSP70 (HSPA1B) gene may impact protein expression and disease phenotype. The HSPA1B rs2763979 variant has been associated with multiple inflammatory scenarios, but no previous studies have explored its association with asthma. In this sense, this cross-sectional study enrolled 90 children with asthma and 218 age-/sex-matched healthy volunteers for rs2763979 variant genotyping by TaqMan allelic discrimination analysis. The results were investigated under several genetic models and associated with disease susceptibility and clinicolaboratory data. Overall analysis, including the 308 participants, revealed a higher C allele frequency among patients relative to controls (43.0% vs. 33%, p = 0.006). Furthermore, patients with the C variant initially had a higher risk of asthma under heterozygous (OR = 2.75, 95%CI = 1.46-5.18, p = 0.003), homozygous (OR = 3.35, 95%CI = 1.19-9.39, p = 0.008), dominant (OR = 2.83, 95%CI = 1.52-5.25, p < 0.001), and overdominant (OR = 2.12, 95%CI = 1.20-3.74, p = 0.008) models. However, after employing a 1:1 nearest propensity matching analysis, the studied variant showed only borderline significance with asthma under the dominant model in 71 matched cohorts. Interestingly, patients who carry the rs2763979 CC genotype showed favorable spirometric parameters in terms of better (mean ± SD) forced vital capacity (86.3 ± 7.4 vs. 77.7 ± 6.1 and 75.7 ± 7.2 for CT and TT, respectively, p = 0.021), forced expiratory volume in one second before bronchodilation (60.7 ± 12.9 vs. 54.9 ± 7.6 and 56.1 ± 7.5 for CT and TT, respectively, p = 0.021), and an improvement in peak expiratory flow rate after inhaled salbutamol bronchodilator (p = 0.044) relative to the counterpart genotypes. In conclusion, the HSPA1B rs2763979 variant might have prognostic utility as a genetic marker for asthma in our population. Further larger studies on different ethnicities are recommended to validate the results.

Label-free relative quantitative proteomics reveals extracellular vesicles as a vehicle for Salmonella effector protein delivery.

Extracellular vesicles are small vesicles with a diameter of 30-150 nm that are actively secreted by eukaryotic cells and play important roles in intercellular communication, immune responses, and tumorigenesis. Previous studies have shown that extracellular vesicles are involved in the process of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. However, changes in the protein content of extracellular vesicles elicited by S. Typhimurium infection have not been determined. Here, we extracted the extracellular vesicles with high purity from S. Typhimurium-infected Henle-407 cells, a kind of human intestinal epithelial cells, by ultracentrifugation combined with an extracellular vesicles purification kit, and analyzed their protein composition using label-free relative quantitative proteomics. The extracted extracellular vesicles exhibited an oval vesicular structure under electron microscopy, with a mean diameter of 140.4 ± 32.4 nm. The exosomal marker proteins CD9, CD63, and HSP70 were specifically detected. Compared with the uninfected group, nearly 1,234 specifically loaded proteins were uncovered in S. Typhimurium-infected Henle-407 cells. Among them were 409 S. Typhimurium-derived specific proteins, indicating a significant alteration in protein composition of extracellular vesicles by S. Typhimurium infection. Notably, these proteins included 75 secretory proteins and over 300 non-secretory proteins of S. Typhimurium, implicating novel pathways for bacterial protein delivery, although it remains unclear if their loading into extracellular vesicles is active or passive. To investigate the roles of these extracellular proteins, we exemplified the function of SopB, a well-known T3SS effector protein, and showed that the extracellular SopB could be taken up by RAW264.7 macrophages, activating the phosphorylation of Akt. This study provides new insights into the mechanism of Salmonella infection through extracellular vesicles that transport virulence proteins to uninfected neighboring cells to facilitate further infection.

Targeting heat shock proteins 90 and 70: A promising remedy for both autoimmune bullous diseases and COVID-19.

Heat shock proteins (Hsps), including Hsp90 and Hsp70, are intra- and extracellular molecules implicated in cellular homeostasis and immune processes and are induced by cell stress such as inflammation and infection. Autoimmune bullous disorders (AIBDs) and COVID-19 represent potentially life-threatening inflammatory and infectious diseases, respectively. A significant portion of AIBDs remain refractory to currently available immunosuppressive therapies, which may represent a risk factor for COVID-19, and suffer from treatment side-effects. Despite advances in vaccination, there is still a need to develop new therapeutic approaches targeting SARS-CoV-2, especially considering vaccine hesitancy, logistical distribution challenges, and breakthrough infections. In this mini review, we briefly summarize the role of targeting Hsp90/70 as a promising double-edged sword in the therapy of AIBDs and COVID-19.

Transcriptome Analysis of the Differentially Expressed Heat-resistant Genes between Calliptamus italicus and Gomphocerus sibiricus.

Calliptamus italicus and Gomphocerus sibiricus are indicator species in Xinjiang's low-altitude (700-1,900 m) and high-altitude (2,000-3,400 m) grasslands, respectively. C. italicus is tolerant to high-temperature stress, with its semilethal temperature (LT50) being 10.5°C higher than that of G. sibiricus. The two locust species were subjected to high-temperature stress to explore the molecular mechanisms and differences in high temperature tolerance between the two locust species. Next, the next generation sequencing (NGS) data were mapped to reference transcripts obtained using single molecule real Time (SMRT) sequencing to construct a nonparameter transcriptome. The transcriptomic response of these two locust species displayed different patterns. C. italicus had 126 differentially expressed genes (DEGs), with 59 and 67 being significantly up-regulated and down-regulated, respectively. The heat shock protein (Hsp) genes were highly expressed upon two locust species exposure to high-temperature stress, with Hsp70 being expressed the most. G. sibiricus had 86 DEGs, of which 45 were significantly up-regulated and 41 significantly down-regulated. In addition, the expression of the key enzyme encoding gene Myo-inositol oxygenase (MIOX) in inositol degradation was the highest in G. sibiricus. In the KEGG pathway, the biological processes and metabolic pathways were the most enriched pathways in C. italicus and G. sibiricus, respectively. Moreover, the quantitative fluorescence results were consistent with the transcriptome results, implying that the transcriptome results were accurate. The findings in this study provide valuable information for future research exploring the evolution mechanisms of heat resistance in C. italicus and G. sibiricus.

Upper gut heat shock proteins HSP70 and GRP78 promote insulin resistance, hyperglycemia, and non-alcoholic steatohepatitis

A high-fat diet increases the risk of insulin resistance, type-2 diabetes, and non-alcoholic steato-hepatitis. Here we identified two heat-shock proteins, Heat-Shock-Protein70 and Glucose-Regulated Protein78, which are increased in the jejunum of rats on a high-fat diet. We demonstrated a causal link between these proteins and hepatic and whole-body insulin-resistance, as well as the metabolic response to bariatric/metabolic surgery. Long-term continuous infusion of Heat-Shock-Protein70 and Glucose-Regulated Protein78 caused insulin-resistance, hyperglycemia, and non-alcoholic steato-hepatitis in rats on a chow diet, while in rats on a high-fat diet continuous infusion of monoclonal antibodies reversed these phenotypes, mimicking metabolic surgery. Infusion of these proteins or their antibodies was also associated with shifts in fecal microbiota composition. Serum levels of Heat-Shock-Protein70 and Glucose-Regulated Protein78were elevated in patients with non-alcoholic steato-hepatitis, but decreased following metabolic surgery. Understanding the intestinal regulation of metabolism may provide options to reverse metabolic diseases.

Moringa oleifera alleviated oxidative stress, physiological and molecular disruption induced by acute thermal stress in grass carp, Ctenopharyngodon idella.

The present study was designed to investigate the protective effect of dietary Moringa oleifera leaf meal (MLM) supplementation against high temperature-induced stress in grass carp (Ctenopharyngodon idella). A total of 180 apparent healthy juvenile grass carp (15.48g ± 0.054) were divided into three groups in triplicate (20 fish in each replicate). Fish were fed with diets containing 0, 1, and 5% MLM for 60days and then subjected to a high-temperature challenge for 48h (32-33°C). The results revealed that feeding fish with 1 and 5% MLM resulted in a significant increase in weight gain and specific growth rate compared to the control. In addition, feed conversion ratio was significantly reduced in groups fed with MLM. No significant difference was reported in the serum cortisol level among different experimental groups before heat stress while serum glucose level significantly decreased in fish fed with 5% MLM. Serum alanine transaminase, aspartate transaminase, and alkaline phosphatase significantly decreased in fish fed with 1 and 5% MLM before and after heat stress. Hepatic lipid peroxidation significantly decreased in fish fed with MLM for 60days. A non-significant increase in hepatic reduced glutathione level was reported in fish fed with 1 and 5% MLM before heat stress. Catalase and superoxide dismutase activities increased significantly in the liver of fish fed with 5% MLM. No significant change was observed in the expression profile of heat shock protein (hsp) 70 and 90 before heat stress. Meanwhile, after heat stress, up to a fivefold increase was recorded in mRNA level of hsp 70 and fourfold increase in the expression level of hsp 90 in the liver of the control fish which were not fed with MLM-supplemented diets. Fish fed with 1 and 5% MLM showed a significant decrease in the expression of hsp 70 and a non-significant decrease in the expression of hsp 90. Results of the present study suggest that supplementing the diet of grass carp with 5% MLM could improve growth and physiological performance and provide resistance against high temperature-induced stress.

Роль ендокринних факторів та маркерів клітинного стресу у прогнозуванні ефективності лікування клімактеричного синдрому в період перименопаузи

Women of menopausal age make up 10% of the world's population, and the problem of pathological menopause remains relevant today. Estrogen deficiency is considered to be the cause of menopause, so the use of replacement therapy is a pathogenetically sound method. It is possible that the immune system also plays a role in shaping the clinical picture. There is a certain relationship between estrogen levels and markers of intracellular stress. Studying the level of heat shock proteins as proven factors of cellular stress, along with endocrine factors, can become a reliable marker for the prognosis, course and compensation of disorders of the neurovegetative state of women in this period of life. Purpose - to study the role of levels of antimullerian, follicle-stimulating, luteinizing, thyroid-stimulating hormones, levels of antibodies (IgG) to human heat shock protein (Hsp60) and its bacterial homologue (Groel) in the prognosis, diagnosis and treatment of psychopathological manifestations in perimenopausal women. Materials and methods. The study examined 158 patients with perimenopausal menopausal syndrome between October 2019 and March 2022. The patients were divided into two groups. The 1st (main) group included 80 patients who agreed to receive treatment according to the standard regimen (combined treatment in a cyclic regimen (estrogens + gestagens), and the 2nd (comparative) group included 78 women who refused the proposed treatment Results. A decrease (p&lt;0.001) in the risk of not achieving a high treatment effect was found for the main group, OR=0.12 (95% CI: 0.06-0.26) compared with the control group. It was found that a higher level of antibodies to GroEl, OR=0.989 (95% CI: 0.984-0.996), revealed a lower (p=0.001) risk of not achieving a high treatment effect. A higher body mass index (BMI) of a woman increases the risk of not achieving a high treatment effect (p&lt;0.001), OR=1.19 (95% CI: 1.10-1.30) for every 1 kg/m2. When standardized by BMI, an integral indicator of psychopathological manifestations, taking into account the method of treatment and the level of antimullerian hormone, a decrease (p=0.039) in the risk of failure to achieve a high treatment effect was found for the main group, OR=0.16 (95% CI: 0.07-0.39) compared with the control group. Conclusions. It has been established that the most informative indicator for predicting the effectiveness of treatment of women with psychopathological manifestations is antimüllerian hormone. The data also show that the determination of the level of antibodies to GroEl correlates with the success of treatment of women with psychopathological manifestations of menopausal syndrome. The study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Local Ethics Committee of the institutions mentioned in the paper. Informed consent of women was obtained for the study. No conflict of interests was declared by the authors.

KBTBD11, encoding a novel PPARγ target gene, is involved in NFATc1 proteolysis by interacting with HSC70 and HSP60

We previously revealed that Kbtbd11 mRNA levels increase during 3T3-L1 differentiation and Kbtbd11 knockdown suppresses whereas its overexpression promotes adipogenesis. However, how Kbtbd11 mRNA is regulated during adipocyte differentiation and how the KBTBD11 protein functions in adipocytes remain elusive. This study aimed to examine the transcriptional regulatory mechanism of Kbtbd11 during adipocyte differentiation, KBTBD11-interacting protein functions, and elucidate the role of KBTBD11 in adipocytes. First, we identified the PPRE consensus sequences in the Kbtbd11 exon 1- and intron 1-containing region and demonstrated that PPARγ acts on this region to regulate Kbtbd11 expression. Next, we purified the KBTBD11 protein complex from 3T3-L1 adipocytes and identified heat shock proteins HSC70 and HSP60 as novel KBTBD11-interacting proteins. HSC70 and HSP60 inhibition increased KBTBD11 protein levels that promoted NFATc1 ubiquitination. These data suggest that HSC70 and HSP60 are involved in KBTBD11 stabilization and are responsible for NFATc1 regulation on the protein level. In summary, this study describes first the protein regulatory mechanism of NFATc1 through the HSC70/HSP60-KBTBD11 interaction that could provide a potential new target for the differentiation and proliferation of various cells, including adipocytes and tumors.

Conformational dynamics of the Hsp70 chaperone throughout key steps of its ATPase cycle

The 70 kDa heat shock proteins (Hsp70s) are highly versatile molecular chaperones that assist in a wide variety of protein-folding processes. They exert their functions by continuously cycling between states of low and high affinity for client polypeptides, driven by ATP-binding and hydrolysis. This cycling is tuned by cochaperones and clients. Although structures for the high and low client affinity conformations of Hsp70 and Hsp70 domains in complex with various cochaperones and peptide clients are available, it is unclear how structural rearrangements in the presence of cochaperones and clients are orchestrated in space and time. Here, we report insights into the conformational dynamics of the prokaryotic model Hsp70 DnaK throughout its adenosine-5'-triphosphate hydrolysis (ATPase) cycle using proximity-induced fluorescence quenching. Our data suggest that ATP and cochaperone-induced structural rearrangements in DnaK occur in a sequential manner and resolve hitherto unpredicted cochaperone and client-induced structural rearrangements. Peptides induce large conformational changes in DnaK·ATP prior to ATP hydrolysis, whereas a protein client induces significantly smaller changes but is much more effective in stimulating ATP hydrolysis. Analysis of the enthalpies of activation for the ATP-induced opening of the DnaK lid in the presence of clients indicates that the lid does not exert an enthalpic pulling force onto bound clients, suggesting entropic pulling as a major mechanism for client unfolding. Our data reveal important insights into the mechanics, allostery, and dynamics of Hsp70 chaperones. We established a methodology for understanding the link between dynamics and function, Hsp70 diversity, and activity modulation.

Apoptosis and cellular stress induction in human leukocytes by dental X-rays

In clinical practice, the use of ionizing radiation, such as X-rays, is of importance in diagnostic procedures. The X-ray equipment is operated by the dentist himself before, during, and after an oral cavity procedure in dental offices. The occupationally exposed personnel (POES) receive an exposure dose, causing stochastic damage that is not commonly considered but is cumulative. Due to the above, a relationship between leukopenia and exposure to X-rays has been reported. Analysing the expression of bioindicators that allow determination of the degree of homeostatic tolerance to different exposure doses is one way to diagnose cell damage caused by this. This study aimed to evaluate cell viability, the expression of Hsp70, p53, caspase 8 and 3, and morphological changes related to apoptosis in human leukocytes exposed to X-rays from dental equipment. Whole blood from healthy subjects was used and exposed to different doses of X-rays from dental diagnostic equipment (2.28 ± 0.05, 4.56 ± 0.09, 9.12 ± 0.18, and 13.69 ± 0.27 mGy). Following this, morphology was assessed by Wright's stained blood smear of whole blood leukocytes. Subsequently, leukocytes were isolated by Ficoll Histopaque gradients from whole blood, and cell viability was measured. Finally, the Western blot technique identified the leukocyte proteins Hsp70, p53, and caspases. In the observed results, a decrease in cell viability from 96.6% to 45.4% was noticed; viability decreased as the dose of X-ray exposure increased. The expression of Hsp70, p53, Caspase 8, and 3 gradually increased up to the third dose and decreased in the last dose. Likewise, morphological changes characteristic of apoptotic cell death were observed from the first dose of exposure and became more significant at higher doses. In conclusion, there is cell damage in leukocytes exposed to low doses of X-rays; this information is relevant to implementing protection mechanisms for POES that regularly use this equipment for diagnosis in the field of dentistry.

Mucilage-induced necrosis reveals cellular oxidative stress in the Mediterranean gorgonian Paramuricea clavata

The red gorgonian Paramuricea clavata is a fundamental ecosystem engineer of coralligenous biocenosis in the Mediterranean Sea, where it plays crucial ecological functions and enhances the habitat biodiversity. However, this species is strongly endangered due to climate change-related stressors such as mucilage events, which have increased in recent decades causing extensive damage and mortality, but whose impacts at the molecular and cellular levels are still unexplored. In this study, different cellular stress biomarkers were analyzed in mucilage-colonized P. clavata specimens living in the Portofino Marine Protected Area (Ligurian Sea, Italy), in order to elucidate the cellular pathways affected by mucilage and the cellular defense mechanisms adopted. Within these colonies, portions of healthy tissue located at different distances from mucilage-induced necrosis were considered. Our results showed that mucilage affected the cellular oxidative status and impaired lipid cell membranes of the whole P. clavata colony. Indeed, an increase in the superoxide dismutase (SOD) and glutathione reductase (GR) enzymatic activity and in the level of lipid peroxidation (LPO) was detected in colonies showing mucilage-induced necrotic tissues compared to healthy ones. In particular, fragments sampled just next to the necrotic tissue displayed the highest levels of SOD and LPO, indicating that harmful oxidative damage was in progress, especially in portions neighboring the mucilage colonization. In this context, the up-regulation of the antioxidant mechanisms might represent a defense and a barrier from hypoxic or anoxic conditions created by mucilage overgrowth or by the invasion of toxic organisms embedded in the mucilaginous aggregates. On the contrary, the cellular protein homeostasis, analyzed by the expression of the 60-kDa Heat shock protein (Hsp60), was not altered by the mucilage. This analysis contributes to provide new insights into the cellular and molecular response of gorgonians to a stress source whose effects are likely to worsen in a climate change scenario.

Evaluation of Taraxacum mongolicum flavonoids in diets for Channa argus based on growth performance, immune responses, apoptosis and antioxidant defense system under lipopolysaccharide stress

To ascertain the effects of Taraxacum mongolicum flavonoids (TMF) on the growth performance, digestive enzyme activity, immune indices, inflammatory response and antioxidant capacity of Channa argus, 400 C. argus with an average body weight of (8.08 ± 0.21) g were selected and divided randomly into four groups. They were fed with four experimental diets supplemented with TMF of 0 (control), 25, 50 and 100 mg/kg for 56 d, and then challenged with lipopolysaccharide (LPS) for 96 h, afterwards indices were detected. The results manifested that the addition of TMF above 50 mg/kg in the dietary could significantly improve the final body weight, WGR, SGR and PER of C. argus, while decreased FCR (P < 0.05). Similarly, the 50 mg/kg group had the highest activity of digestive enzymes (protease, lipase, amylase) in intestine and hepatopancreas, which were notably higher than those in the control group (P < 0.05). Nevertheless, 100 mg/kg group could effectively inhibit the liver and gut injury caused by LPS and reduce the contents of ALT and AST, LPS and LBP in serum. In the immune (LY, AKP, ACP, IgM, C3) and antioxidant (T-AOC, SOD, CAT, GSH-PX, GR, ASA, MDA) systems, 100 mg/kg groups were the optimal group, which were remarkably higher than those of the control group (P < 0.05). Additionally, the expression of genes revealed that 100 mg/kg group could noteworthy restrain the expression of pro-inflammatory factors (tnf-α, il-1β, il-8) and pro-apoptosis (cas-3,8,9, p53, bax, bcl-2) related genes, up-regulate the expression of anti-inflammatory (il-10, tgf-β) factors, antioxidant-related (nrf2, gpx, gst, cat) genes and heat shock proteins (hsp70, hsp90). Simultaneously, the survival rate of C. argus in the 100 mg/kg TMF-supplemented group was the highest after LPS challenge. Our results elucidate that dietary supplementation TMF protects C. argus from LPS-induced inflammatory injury, to ameliorate digestion, immune response, antioxidant status and apoptosis, implying that TMF could be regarded as an anti-inflammatory and antioxidant agent adding to aquatic animal feed.

HSP27 Promotes Mantle Cell Lymphoma Growth and Mediates Resistance to Venetoclax

Introduction Drug resistance has been a major challenge for the treatment of patients with relapsed or refractory mantle cell lymphoma. Venetoclax is a highly potent, Bcl2-selective inhibitor that is effective in refractory/relapsed MCL patients both as monotherapy or in combination with ibrutinib, but the development of acquired resistance remains inevitable. The heat shock protein HSP27 (encoded by HSPB1) is normally upregulated under conditions of protein folding alteration, such as heat shock. However, it has been demonstrated that HSP27 can promote the growth and metastasis of cancer cells and enable resistance to many anticancer therapies. Accordingly, HSP27 has been targeted for therapy of cancer progression and treatment resistance. We hypothesize that HSP27 can induce therapeutic resistance, and that targeting of HSP27 may overcome venetoclax resistance in MCL. Methods Primary cells from patients' with or without therapeutic resistance as well as ibrutinib or venetoclax-sensitive and -resistant MCL cell lines were used in this study. We established a HSPB1- knockout (KO) human MCL JeKo-1 cell line using the CRISPR-Cas9 knockout system. Cell viability and cell proliferation assays were tested in triplicate for 72 h. The annexin V/propidium iodide-binding assay was utilized to evaluate apoptosis following venetoclax treatment. The underlying resistance mechanisms were verified by immunoblotting. Results HSP27 was highly upregulated in primary cells from patients resistant to BTK inhibitors compared to cells from those sensitive to BTK inhibitors. In addition, HSP27 was significantly increased in the Rec-Ven-R MCL cell line, which has acquired venetoclax resistance, compared to the parental Rec-1 cell line. To further investigate the function of HSP27 in regulating growth and therapeutic resistance, we knocked out the HSPB1 gene in JeKo-1 cells using a CRISPR-Cas9 knockout system. We first evaluated the impact of HSP27 on MCL cell growth. HSP27 knockout in the HSP27-high JeKo-1 cells greatly inhibited their growth and proliferation in vitro. Next, we carried out cell viability assays and found that genetic ablation of HSPB1 enhanced the sensitivity to venetoclax. Furthermore, treatment of HSPB1-knockout JeKo-1 cells with 1 uM venetoclax for 24 h and 48 h induced greater apoptosis than in the parental JeKo-1 cells. Western blot analysis of whole cell lysates showed that knockout of HSPB1 triggered an increase in the pro-apoptotic markers cleaved PARP and cleaved caspase 3 after venetoclax treatment for 6 h. Together, these results suggest that HSP27 potentiates the growth and proliferation of MCL cells and mediates the acquired resistance to venetoclax. Conclusions Targeting HSP27 may overcome venetoclax resistance in MCL cells, which provide impressive insights for therapeutic development to treat refractory/relapsed MCL patients and improve patient prognosis.