Heat Shock Protein 70 Protein Research Articles

Heat shock protein 90 promotes RNA helicase DDX5 accumulation and exacerbates hepatocellular carcinoma by inhibiting autophagy.

Objective:Hepatocellular carcinoma (HCC), the main type of liver cancer, has a high morbidity and mortality, and a poor prognosis. RNA helicase DDX5, which acts as a transcriptional co-regulator, is overexpressed in most malignant tumors and promotes cancer cell growth. Heat shock protein 90 (HSP90) is an important molecular chaperone in the conformational maturation and stabilization of numerous proteins involved in cell growth or survival.Methods:DDX5 mRNA and protein expression in surgically resected HCC tissues from 24 Asian patients were detected by quantitative real-time PCR and Western blot, respectively. The interaction of DDX5-HSP90 was determined by molecular docking, immunoprecipitation, and laser scanning confocal microscopy. The autophagy signal was detected by Western blot. The cell functions and signaling pathways of DDX5 were determined in 2 HCC cell lines. Two different murine HCC xenograft models were used to determine the function of DDX5 and the therapeutic effect of an HSP90 inhibitor.Results:HSP90 interacted directly with DDX5 and inhibited DDX5 protein degradation in the AMPK/ULK1-regulated autophagy pathway. The subsequent accumulation of DDX5 protein induced the malignant phenotype of HCC by activating the β-catenin signaling pathway. The silencing of DDX5 or treatment with HSP90 inhibitor both blocked in vivo tumor growth in a murine HCC xenograft model. High levels of HSP90 and DDX5 protein were associated with poor prognoses.Conclusions:HSP90 interacted with DDX5 protein and subsequently protected DDX5 protein from AMPK/ULK1-regulated autophagic degradation. DDX5 and HSP90 are therefore potential therapeutic targets for HCC.

Heat Shock Protein 70 (HSP70) Protein-Protein Interactions and a Putative Mechanism for the Potential Benefits of Heat Therapy for Type 2 Diabetes Mellitus

The use of heat therapy to treat diseases was very common in Africa. To date, the number of people using this form of therapy is very much on the decrease. Data on the molecular action mechanisms of how it might induce beneficial effects remain unknown. The aim of the present study was to make a contribution towards understanding the putative implication of Heat Shock Protein 70 (HSP70) in the pathophysiology of Type 2 Diabetes Mellitus (T2DM) and the hypothetical benefits of heat therapy through the establishment of a network of protein-protein interactions between Krüppel Like Factor 14 (KLF14), Transcription Factor 7 Like 2 (TCF7L2), Peroxisome Proliferator-Activated Receptor Gamma (PPARG) and HSP70 (HSPA4). Data were generated by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software version 10.0. This was used to identify the known and predicted protein-protein interactions (including direct or physical and indirect or functional associations) in the KLF14, TCF7L2, PPARG and HSP70 protein networks. With the active prediction methods (Gene Fusion, Neighborhood, Co-occurrence, Co-expression, Experiments, Databases and Text Mining) as interaction sources, Medium Confidence (0.400) and maximum number of interactions were used to show no more than 50 at the first shell and none at the second shell parameters. Fifty (50) proteins were identified to interact with these four proteins, namely KLF14, TCF7L2, PPARG and HSP70, resulting in a network diagram with 54 nodes (gene/proteins) and 485 edges, representing protein-protein associations. The network showed that HSP70 strongly interacts with other heat shock proteins like HSP90. The HSPBP, a cytoplasmic co-chaperone 1, inhibits HSPA1A chaperone activity by changing the conformation of its ATP-binding domain, thus interfering with this function. Based on the data generated by this <i>in silico</i> study, the potential beneficial effects of heat therapy on T2DM could probably be coordinated by the HSP70 protein-protein interactions involved in cell life and in the susceptibility to T2DM.

Does HSP90 play an important role in psoriasis?

Psoriasis is a chronic, inflammatory skin disease. Environmental, genetic, autoinflammatory and autoimmune factors play a role in the pathogenesis of disease. It is believed that heat shock protein 90 (HSP90) is an interleukin-17 (IL-17) receptor, which plays an essential role in the psoriasis pathogenesis. To evaluate the expression of the gene encoding HSP90 protein in keratinocytes of patients with psoriasis depending on its duration, recurrences, exacerbating factors, therapy form, and the coexistence of metabolic disorders and cardiovascular diseases. Skin samples from 40 psoriatic patients were investigated in this study. Control skin biopsies were collected from 20 healthy volunteers. HSP90 expression level was measured by qRT-PCR reaction. This study has shown an increased mRNA expression of HSP90 in psoriatic patients as compared to healthy volunteers. A positive correlation of HSP90 expression and the frequency of exacerbations was found. A negative correlation between the HSP90 activity and the age of patients was demonstrated in the coexistence of psoriasis with hyperlipidaemia or diabetes. Among the factors exacerbating psoriasis, acute infections induced HSP90 expression most significantly. HSP90 plays a role in the pathogenesis of psoriasis. The expression of HSP90 increases with the frequency of exacerbations of psoriasis throughout the year. Hyperlipidaemia or diabetes associated with psoriasis in young adults, and acute infections and emotional stress increase the expression of HSP90. The expression of HSP90 in psoriatic patients is not dependent on the type of psoriasis, comorbidity of cardiovascular diseases, smoking and alcohol addiction.

Genomic Identification, Evolution and Sequence Analysis of the Heat-Shock Protein Gene Family in Buffalo.

Heat-shock proteins (HSP) are conserved chaperones crucial for protein degradation, maturation, and refolding. These adenosine triphosphate dependent chaperones were classified based on their molecular mass that ranges between 10–100 kDA, including; HSP10, HSP40, HSP70, HSP90, HSPB1, HSPD, and HSPH1 family. HSPs are essential for cellular responses and imperative for protein homeostasis and survival under stress conditions. This study performed a computational analysis of the HSP protein family to better understand these proteins at the molecular level. Physiochemical properties, multiple sequence alignment, and phylogenetic analysis were performed for 64 HSP genes in the Bubalus bubalis genome. Four genes were identified as belonging to the HSP90 family, 10 to HSP70, 39 to HSP40, 8 to HSPB, one for each HSPD, HSPH1, and HSP10, respectively. The aliphatic index was higher for HSP90 and HSP70 as compared to the HSP40 family, indicating their greater thermostability. Grand Average of hydropathicity Index values indicated the hydrophilic nature of HSP90, HSP70, and HSP40. Multiple sequence alignment indicated the presence of highly conserved consensus sequences that are plausibly significant for the preservation of structural integrity of proteins. In addition, this study has expanded our current knowledge concerning the genetic diversity and phylogenetic relatedness of HSPs of buffalo with other mammalian species. The phylogenetic tree revealed that buffalo is more closely related to Capra hircus and distantly associated with Danio rerio. Our findings provide an understanding of HSPs in buffalo at the molecular level for the first time. This study highlights functionally important HSPs and indicates the need for further investigations to better understand the role and mechanism of HSPs.

Xiaoyaosan (Tiaogan-Liqi therapy) protects peritoneal macrophages from corticosterone-induced stress by regulating the interaction between glucocorticoid receptor and ABCA1.

BackgroundPrevious studies have reported that Xiaoyaosan (XYS), Tiaogan-Liqi therapy, has a protective function in depressive disorder, and can regulate body weight and corticosterone (CORT) level. However, little is known about the effect of XYS in treating atherosclerosis. This study aimed to explore the influence XYS on macrophage foam cell formation and related mechanism.MethodsRat peritoneal macrophages (PMs) were separated and stimulated with CORT and oxidized low density lipoprotein (ox-LDL). The serum was obtained from rats treated with different doses of XYS and was added into the medium for macrophages. Then, the cell activity and lipid content of PMs were measured through Cell Counting Kit-8 (CCK-8) assay and oil red staining, respectively. The expressions of glucocorticoid receptor (GR), ATP binding cassette subfamily A member 1 (ABCA1), and heat shock protein 90 (HSP90) were detected. In addition, overexpression of GR and ABCA1 was performed and the effect on XYS treatment was subsequently assessed.ResultsThe CCK-8 assay showed the serum increased cell activity of CORT-induced stress PMs in a XYS dose-dependent manner. Oil red staining and enzyme-linked immunosorbent assay (ELISA) showed that the serum decreased lipids of PMs. In the XYS treated groups, HSP90 protein was decreased and protein levels of ABCA1 and GR were increased in cytoplasm, while GR protein in nucleus was decreased. Co-immunoprecipitation (Co-IP) assay indicated GR might interact with HSP90 and be involved with the function of XYS. Furthermore, overexpression of GR attenuated the protective function of XYS on CORT-induced stress in PMs, while overexpression of ABCA1 enhanced that.ConclusionsThis study denoted that XYS could protect PMs from CORT-induced stress by regulating the interaction of GR and ABCA1, which might contribute to the treatment of atherosclerosis.

A Novel Bead-Based Immunoassay for the Measurement of Heat Shock Proteins 27 and 70.

Heat shock proteins (HSPs) play an essential role in protecting proteins from denaturation and are implicated in diverse pathophysiological conditions like cardiovascular diseases, cancer, infections, and neurodegenerative diseases. Scientific evidence indicates that if HSP expression falls below a certain level, cells become sensitive to oxidative damage that accelerates protein aggregation diseases. On the other hand, persistently enhanced levels of HSP can lead to inflammatory and oncogenic changes. To date, although techniques for measuring HSPs exist, these assays are limited for use in specific sample types or are time consuming. Therefore, in the present study, we developed a single-molecule assay digital ELISA technology (Single Molecule Array—SIMOA) for the measurement of HSPs, which is time effective and can be adapted to measure multiple analytes simultaneously from a single sample. This technique combines two distinct HSP-specific antibodies that recognize different epitopes on the HSP molecule. A recombinant human HSP protein was used as the standard material. The assay performance characteristics were evaluated by repeated testing of samples spiked with HSP peptide at different levels. The limit of detection was 0.16 and 2 ng/mL for HSP27 and HSP70, respectively. The inter- and intra-assay coefficients of variation were less than 20% in all tested conditions for both HSPs. The HSP levels assayed after serial dilution of samples portrayed dilutional linearity (on average 109%, R2 = 0.998, p < 0.001, for HSP27 and 93%, R2 = 0.994, p < 0.001, for HSP70). A high linear response was also demonstrated with admixtures of plasma exhibiting relatively very low and high levels of HSP70 (R2 = 0.982, p < 0.001). Analyte spike recovery varied between 57% and 95%. Moreover, the relative HSP values obtained using Western blotting correlated significantly with HSP values obtained with the newly developed SIMOA assay (r = 0.815, p < 0.001 and r = 0.895, p < 0.001 for HSP70 and HSP27, respectively), indicating that our method is reliable. In conclusion, the assay demonstrates analytical performance for the accurate assessment of HSPs in various sample types and offers the advantage of a huge range of dilution linearity, indicating that samples with HSP concentration highly above the calibration range can be diluted into range without affecting the precision of the assay.

Photobiomodulation therapy on expression of HSP70 protein and tissue repair in experimental acute Achilles tendinitis.

The aim of the present study was to investigate the effects of photobiomodulation (PBM) therapy on the expression of heat shock protein 70 (HSP70) and tissue repair in an experimental model of collagenase-induced Achilles tendinitis. Thirty Wistar rats (aged 12weeks) were randomly distributed among control group (n = 8), tendinitis group (n = 11), and LED group (n = 11). Tendinitis was induced in the tendinitis and LED groups through a peritendinous injection of collagenase (100μl). The LED group animals received the first irradiation 1h after injury. A 630 ± 20nm, 300-mW continuous wave light-emitting diode (LED), spot size 1cm2, was placed in contact with the skin. One point over the tendon was irradiated for 30s, delivering 9J (9J/cm2). LED irradiation was performed once daily for 7days, with the total energy delivered being 63J. The tendons were surgically removed and expression of the HSP70 protein was calculated using semi-quantitative analyses of immunohistochemistry (HSCORE). Number of fibroblasts and amount of collagen were measured using histological and histochemical analyses. An increase in the mean HSCORE for HSP70, in the number of fibroblasts, and in the amount of collagen were found in the LED group compared with those in the tendinitis and control group (P≤ 0.05). PBM therapy increased the expression of the HSP70, number of fibroblasts, and amount of collagen in the acute Achilles tendinitis in rats.

The HSP90 Inhibitor, 17-AAG, Influences the Activation and Proliferation of T Lymphocytes via AKT/GSK3β Signaling in MRL/lpr Mice.

ObjectiveTo explore the molecular mechanism of 17-AAG in the treatment of systemic lupus erythematosus (SLE), and the effects of the heat shock protein 90 (HSP90) inhibitor 17-AAG on the activation and proliferation of lymphocytes and the AKT/GSK3β signaling pathway in MRL/lpr mice were detected.MethodsMRL/lpr mice were randomly divided into the control group and the experimental group. The experimental group was injected intraperitoneally with 17-AAG, and T lymphocytes were separated by magnetic beads. Lymphocyte proliferation was detected by MTT and flow cytometry (FCM), and the expression of the HSP90 protein and PI3K/AKT signaling pathway-related proteins was detected by Western blotting. Renal histopathology and immune complex deposition were also observed in both groups.ResultsImmune complex deposition and inflammation decreased in kidneys from MRL/lpr mice in the experimental group. HSP90 protein expression, T lymphocyte proliferation and phosphorylated AKT and GSK3β levels also decreased in the experimental group.Conclusion17-AAG can inhibit the activation and proliferation of T lymphocytes and downregulate the AKT/GSK3β signaling pathway, which may be relevant for the treatment of SLE.

Effects of transport stress on pathological injury and expression of main heat shock proteins in the caprine stomach

BackgroundTransportation is necessary to introduce new breeds of goats to the farm and move the adult meat goat from the farm to the slaughterhouse. However, these actions may give rise to transport stress. Heat shock proteins (HSPs) are playing some important regulate roles during transport stress. The aim of this study was to evaluate the effects of transport stress on the pathological injury and HSPs expression in the stomach of goats. A total of three batches of Ganxi goats from western Jiangxi province were enrolled in this study. For each batch, twelve healthy adult male goats were randomly divided into three groups (four goats per batch and per group): Control group, stress group transported during 2 h and stress group transported during 6 h.ResultsOur results showed that the different degrees of stomach walls damage, with the change of expression levels of heat shock protein 27 (HSP27), heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90), occurred after goats transportation. In rumen, the mRNA and protein expressions of HSP27 and HSP70 were increased after transport stress, but not HSP90. In reticulum, all three HSPs mRNA and protein levels were upregulated after 2 h transport, but decreased after 6 h transport. In omasum, HSP27 and HSP70 mRNA and protein were increased after transport stress, however, HSP90 mRNA level only had a slightly enhancement after transport stress. In abomasum, HSP70 and HSP90 mRNA and protein levels were increased after transport stress, but HSP27 was decreased after transport stress.ConclusionsTaken together, these results revealed that the pathological changes in the gastric tissues and the stomach HSPs expression in goats are related to transport stress and duration. Moreover, this study also provides some new data to advocate reducing transport stress of goats and improving animal welfare.

Characterization, expression profiling, and thermal tolerance analysis of heat shock protein 70 in pine sawyer beetle, Monochamus alternatus hope (Coleoptera: Cerambycidae).

Monochamus alternatus Hope (Coleoptera: Cerambycidae) warrants attention as a dominant transmission vector of the pinewood nematode, and it exhibits tolerance to high temperature. Heat shock protein 70 (HSP70) family members, including inducible HSP70 and heat shock cognate protein 70 (HSC70), are major contributors to the molecular chaperone networks of insects under heat stress. In this regard, we specifically cloned and characterized three MaltHSP70s and three MaltHSC70s. Bioinformatics analysis on the deduced amino acid sequences showed these genes, having close genetic relationships with HSP70s of Coleopteran species, collectively shared conserved signature structures and ATPase domains. Subcellular localization prediction revealed the HSP70s of M. alternatus were located not only in the cytoplasm and endoplasmic reticulum but also in the nucleus and mitochondria. The transcript levels of MaltHSP70s and MaltHSC70s in each state were significantly upregulated by exposure to 35-50°C for early 3 h, while MaltHSP70s reached a peak after exposure to 45°C for 2-3 h in contrast to less-upregulated MaltHSC70s. In terms of MaltHSP70s, the expression threshold in females was lower than that in males. Also, both fat bodies and Malpighian tubules were the tissues most sensitive to heat stress in M. alternatus larvae. Lastly, the ATPase activity of recombinant MaltHSP70-2 in vitro remained stable at 25-40°C, and this recombinant availably enhanced the thermotolerance of Escherichia coli. Overall, our findings unraveled HSP70s might be the intrinsic mediators of the strong heat tolerance of M. alternatus due to their stabilized structure and bioactivity.

HSP90 Inhibitor, 17-DMAG, Alone and in Combination with Lapatinib Attenuates Acquired Lapatinib-Resistance in ER-positive, HER2-Overexpressing Breast Cancer Cell Line.

Simple SummaryLapatinib is a tyrosine kinase inhibitor widely used as a treatment for a Human Epidermal growth factor Receptor 2 (HER2) (+) breast cancer patients. However, when resistance is acquired through continued exposure, and it is associated with a poor prognosis for patients. In this study, we identified HSP90 as a common node for acquired resistance to lapatinib in two lapatinib resistant cell lines using proteomic analysis. Notably, in vitro and in vivo studies demonstrated synergy effect between lapatinib and an HSP90 inhibitor were observed in the estrogen receptor (+) HER2 (+) breast cancer cell only. These results could be a potential strategy for future clinical trials for HSP90 inhibitors in treatment—refractory HER2 (+) metastatic cancer patientsLapatinib, a Human Epidermal growth factor Receptor 2 (HER2)-targeting therapy in HER2-overexpressing breast cancer, has been widely used clinically, but the prognosis is still poor because most patients acquire resistance. Therefore, we investigated mechanisms related to lapatinib resistance to evaluate new therapeutic targets that may overcome resistance. Lapatinib-resistant cell lines were established using SKBR3 and BT474 cells. We evaluated cell viability and cell signal changes, gene expression and protein changes. In the xenograft mouse model, anti-tumor effects were evaluated using drugs. Analysis of the protein interaction network in two resistant cell lines with different lapatinib resistance mechanisms showed that HSP90 protein was commonly increased. When Heat Shock Protein 90 (HSP90) inhibitors were administered alone to both resistant cell lines, cell proliferation and protein expression were effectively inhibited. However, inhibition of cell proliferation and protein expression with a combination of lapatinib and HSP90 inhibitors showed a more synergistic effect in the LR-BT474 cell line than the LR-SKBR3 cell line, and the same result was exhibited with the xenograft model. These results suggest that HSP90 inhibitors in patients with lapatinib-resistant Estrogen Receptor (ER) (+) HER2 (+) breast cancer are promising therapeutics for future clinical trials.

Effects of Combine Exercise on HSP70 and SOD1 Expression of Aorta, Skeletal Muscle and Myocardium in High Fat Diet induced Obese Aging Rats

PURPOSE: Exercise improve myocardial cell protection and vascular function through cell repair and suppression of oxidative stress in cardiovascular diseases caused by aging. This study aimed to investigate the effect of combine exercise on HSP70 and SOD1 protein expression of aorta, skeletal muscle and myocardium in high fat diet induced obese aging rats.METHODS: Male 50-week-old Sprague Dawley rats (n=40) were divided into normal diet (ND, n=10), normal diet+exercise (NDEx, n=10), high fat diet (HFD, n=10), and high fat diet+exercise (HFDEx, n=10) groups. After six weeks on a high fat diet to induce obesity, a 12-week combine exercise program was implemented, which combine exercise (treadmill running+ladder climbing) three times a week for 45 minutes per session.RESULTS: Body weight was significantly decreased after 12 weeks combine exercise program compared to the ND group (p&lt;.05) and HFDEx group compared to the HFD group (p&lt;.05), respectively. After completing the 12-week exercise program, heat shock protein 70 (HSP70) and superoxide dismutase 1 (SOD1) expressions were significantly (p&lt;.05) higher in the NDEx group compared to the ND group in the myocardium. Also, SOD1 protein expression was significantly (p&lt;.05) higher in the NDEx group compared to the ND group and HFDEx group compared to the HFD group in the skeletal muscle.CONCLUSIONS: In conclusion, combine exercise intervention of high fat diet-induced obesity resulted in decreased cell repair protein and antioxidant enzyme protein in the myocardium. Therefore, it is thought that combine exercise intervention for obese induced rats improved the cell repair protein and antioxidant enzyme activity of the myocardium.

Effects of vitamin D3 on the level of heat shock protein 70 and oxidative stress in human sperm: a pilot study

BackgroundVitamin D3 (Vit.D3) is an antioxidant that can improve sperm motility. However, the specific mechanisms of Vit.D3’s effect on sperm are not yet elucidated. Since heat shock protein 70 (HSP70) and oxidative stress (OS) are effective in sperm motility and it has been shown that vitamin D3 is associated with these factors, thus this pilot study evaluated the effects of Vit.D3 on HSP70 and OS levels in normal and asthenozoospermic semen samples.ResultsIn this study, the level of HSP70 proteins was evaluated by western blotting and immunocytochemistry in eight normal and nine asthenozoospermic samples. Additionally, malondialdehyde (MDA) assay and nitroblue tetrazolium (NBT) test were used for measuring OS. The results showed that the amount of HSP70 protein was higher in the groups treated with Vit.D3 compared to the control groups. Likewise, in normal and asthenozoospermic samples, OS was increased in treatment groups, but the increase was insignificant in asthenozoospermic samples.ConclusionAccording to the data, HSP70 and OS levels were increased in the sperm cells after incubation with Vit.D3; thus, vitamin D3 might improve sperm motility via these factors in vitro.

Distinct expression and localization patterns of HSP70 in developmental reproductive organs of rams.

Heat shock protein 70 (HSP70) has been widely reported to play a vital role in maintaining intracellular homeostasis, mainly through cellular protection and immune regulation. The expression and function of HSP70 can vary depending upon species and age. To explore the expression signatures and regulatory functions of HSP70 in the reproductive organs of male sheep, we evaluated the expression and distribution patterns of HSP70 in the testes and epididymides (caput, corpus, and cauda) of Tibetan sheep at three developmental stages (i.e., 3months, 1year and 3years after birth) by qRT-PCR, Western blot and immunofluorescence. HSP70 was found to be expressed in testes, caput, corpus, and cauda epididymides throughout the developmental stages but is mainly expressed postpuberty (1year and 3years old). Immunofluorescence results revealed that in the testes, a positive reaction for HSP70 protein was mainly seen in round spermatids and luminal sperms from the groups aged 1year and 3years. In caput epididymides, the positive signals for HSP70 protein was notably observed in sperm and principal cells of the epididymal epithelium from the groups aged 1year and 3years, and positive signals in the epididymal interstitium were found in all three age groups. In corpus and cauda epididymides, HSP70 protein was present in the epididymal epithelium and interstitium, and the positive signals gradually increased with age. In conclusion, these findings suggest that Tibetan sheep HSP70 may play a crucial role in further development and maturation of postmeiotic germ cells and participate in regulation of intraepididymal homeostasis maintenance in Tibetan sheep.

Effect of ethanol on the changes in testicular protein expression in adult male rats.

Ethanol consumption is a major cause of male infertility, but the exact mechanism is still largely unknown. This study attempted to investigate the effect of ethanol on sperm morphology, acrosome reaction status and the alteration of the testicular protein expressions. Fourteen male rats were divided into control and ethanol groups (n=7/each group). Ethanol-treated rats received ethanol (5g/kg, 40% v/v) via oral gavage for consecutive 14days. Testosterone hormone, sperm parameters, and testicular and epididymal histopathologies were evaluated. In addition, the expressions of testicular proteins including androgen receptor (AR), heat shock protein 70 (HSP70), steroidogenic acute regulatory protein (StAR) and tyrosine-phosphorylated (TyrPho) proteins were investigated. The results showed that ethanol significantly increased percentage of abnormal sperm morphology and acrosome-reacted spermatozoa. Some seminiferous and ductus epididymal histopathologies were observed in ethanol-treated rats. Significantly, ethanol reduced serum testosterone and expressions of testicular AR and TyrPho proteins. However, the overexpression of StAR and HSP70 proteins in ethanol testis was found. It was concluded that the changes in testicular protein expressions may be involved in mechanism of male infertility caused from ethanol consumption.

Repressed Gene Expression of Photosynthetic Antenna Proteins Associated with Yellow Leaf Variation as Revealed by Bulked Segregant RNA-seq in Tea Plant Camellia sinensis.

The young leaves and shoots of albino tea cultivars are usually characterized as having a yellow or pale color, high amino acid, and low catechin. Increasing attention has been paid to albino tea cultivars in recent years because their tea generally shows high umami and reduced astringency. However, the genetic mechanism of yellow-leaf variation in albino tea cultivar has not been elucidated clearly. In this study, bulked segregant RNA-seq (BSR-seq) was performed on bulked yellow- and green-leaf hybrid progenies from a leaf color variation population. A total of 359 and 1134 differentially expressed genes (DEGs) were identified in the yellow and green hybrid bulked groups (Yf vs Gf) and parent plants (Yp vs Gp), respectively. The significantly smaller number of DEGs in Yf versus Gf than in Yp versus Gp indicated that individual differences could be reduced within the same hybrid progeny. Analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes revealed that the photosynthetic antenna protein was most significantly enriched in either the bulked groups or their parents. Interaction was found among light-harvesting chlorophyll a/b -binding proteins (LHC), heat shock proteins (HSPs), and enzymes involved in cuticle formation. Combined with the transcriptomic expression profile, results showed that the repressed genes encoding LHC were closely linked to aberrant chloroplast development in yellow-leaf tea plants. Furthermore, the photoprotection and light stress response possessed by genes involved in HSP protein interaction and cuticle formation were discussed. The expression profile of DEGs was verified via quantitative real-time PCR analysis of the bulked samples and other F1 individuals. In summary, using BSR-seq on a hybrid population eliminated certain disturbing effects of genetic background and individual discrepancy, thereby helping this study to intensively focus on the key genes controlling leaf color variation in yellow-leaf tea plants.

Effect Of Aerobic Exercise On Cardiac Diastolic Dysfunction: Role Of Heat Shock Protein 27

PURPOSE: To evaluate the role of heat shock protein 27 (HSP27) in the effect of aerobic exercise on cardiac diastolic dysfunction in type 2 diabetic rats. METHODS: Forty male Sprague-Dawley rats were randomly divided into four groups: control (C), aerobic exercise (A), diabetes (D), and diabetes plus aerobic exercise (DA). Type 2 diabetes was induced by feeding with a high-fat high-sugar diet for 7 weeks followed by a single intraperitoneal injection of streptozotocin (30mg/kg) in the rats. Aerobic exercise was performed on a rodent treadmill at 21m/min for 60 min, 5 days per week for 8 weeks. Metabolic factors, such as fasting blood glucose (FBG), triglycerides (TG), cholesterol (CHOL) and lipoproteins, were determined by a standard procedure. Cardiac structure and function, such as left ventricular diastolic upper diameter (LVIDd), left ventricular diastolic terminal volume (EDV) and ejection fraction (EF), were measured using echocardiography. Myocardial HSP27 protein expression and phosphorylation were determined using western blot, and myocardial HSP27-titin colocalization were determined using double immunofluorescence. Two-way ANOVAs with post-hoc tests were used to assess differences between groups. RESULTS: No pathological changes were observed in the myocardial structure in all four groups. Compared to the C group, the D group had significantly higher levels of FBG, TG, and CHOL (all p<0.01), and significantly lower LVIDd and EDV (both p<0.01). Compared to the D group, the DA group had significantly lower levels of FBG, TG, and CHOL (all p<0.01) and significantly higher LVIDd and EDV (both p<0.05). Interestingly, compared to the C group, the D group showed significantly lower myocardial HSP27 phosphorylation and HSP27-titin colocalization (both p<0.05), while the DA group showed significantly higher myocardial HSP27 protein expression and phosphorylation, and HSP27-titin colocalization than the D group (all p<0.01). CONCLUSION: Decreases in myocardial HSP27 phosphorylation and HSP27-titin colocalization are likely involved in early diastolic dysfunction in diabetic rats. Increased HSP27 phosphorylation and titin colocalization may be an important mechanism by which aerobic exercise restores diastolic function in diabetic rats.

Pen-866, a miniature drug conjugate of a heat shock protein 90 (HSP90) ligand linked to SN38 for patients with advanced solid malignancies: Phase I and expansion cohort results.

3515 Background: PEN-866 is a miniature drug conjugate which links a HSP90 binding small molecule to a SN-38 cytotoxic payload. HSP90 is highly expressed in advanced malignancies. PEN-866 targets and binds to activated tumor HSP90 protein, releases its cytotoxic payload, and results in complete tumor regressions in multiple xenograft models. This first-in-human study assessed safety, tolerability, pharmacokinetics (PK), and preliminary efficacy of PEN-866. Methods: Patients (pts) with progressive, advanced solid malignancies were enrolled in escalating cohorts of 2-9 pts. The primary objective was to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of PEN-866 given weekly (3 out of 4 weeks in a 28-day cycle). Results: 30 pts were treated in 8 cohorts (range 30-360 mg flat dosing or 150–200 mg/m2 BSA-based dosing). As of 9Jan20, the total median/mean exposure was 7.05/12.4 weeks. No dose limiting toxicities (DLTs) occurred in the first 4 cohorts (30-240 mg; 14 pts). In cohort 5 (360 mg), 1 of 3 pts had a DLT of grade (G) 3 transient diarrhea, and 2 other pts had G3 uncomplicated transient neutropenia. A change to BSA-based dosing was instituted for cohort 6 (175 mg/m2), on which no DLTs were observed, although 1 pt experienced G3 uncomplicated transient neutropenia. At 200 mg/m2, 2 of 5 pts experienced DLTs (G5 dehydration, G3 fatigue). The MTD and RP2D were determined to be 175 mg/m2. The most frequent (≥20% pts) related adverse events were nausea (50%), fatigue (43%), diarrhea (40%), vomiting (27%), and anemia (23%). PK was nonlinear. Plasma exposures increased greater than dose proportionally. Median t1/2 ~7 h. Cleaved SN38 never exceeded 3% of PEN-866 plasma AUC at all dose levels. Tissue PK confirmed tumor accumulation and retention of both the conjugate and released payload. As of 9Jan20, 26 pts were evaluable for response. 11 pts had stable disease at 8 weeks, of which 7 lasted 12–58 weeks. One pt with anal squamous cell carcinoma achieved a confirmed partial response. Decreased target lesion size was observed in 6 additional pts. Conclusions: PEN-866 was well tolerated and demonstrated preliminary evidence of antitumor activity. PEN-866 will be evaluated in Phase 2a expansion cohorts enrolling multiple solid tumors (NCT03221400). Clinical trial information: NCT03221400 .

Genetic Modification of the HSP90 Gene Using CRISPR-Cas9 to Enhance Thermotolerance in T. Suecica

Phytoplankton are marine microorganisms that play a key role in the production of oxygen and serve as the foundation of the marine food chain. Over the past century, the population of phytoplankton has declined significantly with the onset of climate change. Although phytoplankton have the capacity to adapt to rising ocean temperatures, rapid environmental changes, including increased top-down control and thermal stratification, reduce populations before adaptations are incorporated into the genome. To enhance survival rates, thermotolerance in common algal strains can be enhanced through increased expression of the conserved Heat Shock Protein 90 (HSP90). Trials will be conducted on the common algal species, Tetraselmis suecica (T. suecica), for its considerable size, photosynthetic rate, and nutrient-rich properties. Thermotolerance will be augmented by splicing the HSP90 gene into the T. suecica metallothionein (Mt) promoter using CRISPR-Cas9. A period of incubation in a copper sulphate solution ensures Mt promoter stimulation, thereby increasing HSP90 expression. The efficacy of the proposed methods will be measured by comparing HSP90 protein production between transgenic and wild-type T. suecica cultures. The genomic incorporation of the modified HSP90 gene enables future populations to exhibit thermotolerance in the presence of heavy metals in the ocean beyond its basal level of expression. By accelerating the adaptation of thermotolerance, the overall fitness of T. suecica can be increased to re-establish its population under warmer oceanic conditions. By applying similar methods to other phytoplankton, the repopulation of various species can increase biodiversity and global net primary productivity.

The Heat Shock Protein HSP70 Promotes Th17 Genes’ Expression via Specific Regulation of microRNA

T helper cells type 17 (Th17) are orchestrators of autoimmune conditions, including multiple sclerosis (MS), but mechanisms of Th17 pathogenicity remain unknown. MicroRNAs (miRNA) are known to control T cells. To understand the function of miRNA in Th17, we have established a T cell line, EL4-TCR+, that resembles the expression pattern of the Th17 cells. Subsequently, we have evaluated the crosstalk between miRNA and Th17 genes’ expression using a combination of gene expression profiling, gene expression manipulation, RNA and protein immunoprecipitation, as well as confocal microscopy. We have found that Th17-related miRNA were strongly expressed in EL4-TCR+ cells following the binding of the cluster of differentiation 3 (CD3) component of the T cell receptor (TCR). Furthermore, a specific inhibition of these miRNA resulted in downregulation of the critical Th17 genes’ expression. Surprisingly, this mechanism relied on the function of the stress signal regulator heat shock protein 70 (HSP70). Upon activation, HSP70 co-localized intracellularly with miRNA processing proteins. Precipitation of HSP70 resulted in enrichment of the Th17-associated miRNA. Finally, HSP70 inhibition led to downregulation of the Th17 genes’ expression and ameliorated development of autoimmune demyelination. Our study demonstrated that HSP70 facilitates specific miRNA function leading to Th17 genes’ expression, a mechanism linking stress and autoimmunity.