Hspb1 and Lgals3 in spinal neurons are closely associated with autophagy following excitotoxicity based on machine learning algorithms.

Excitotoxicity represents the primary cause of neuronal death following spinal cord injury (SCI). While autophagy plays a critical and intricate role in SCI, the specific mechanism underlying the relationship between excitotoxicity and autophagy in SCI has been largely overlooked. In this study, we isolated primary spinal cord neurons from neonatal rats and induced excitotoxic neuronal injury by high concentrations of glutamic acid, mimicking an excitotoxic injury model. Subsequently, we performed transcriptome sequencing. Leveraging machine learning algorithms, including weighted correlation network analysis (WGCNA), random forest analysis (RF), and least absolute shrinkage and selection operator analysis (LASSO), we conducted a comprehensive investigation into key genes associated with spinal cord neuron injury. We also utilized protein-protein interaction network (PPI) analysis to identify pivotal proteins regulating key gene expression and analyzed key genes from public datasets (GSE2599, GSE20907, GSE45006, and GSE174549). Our findings revealed that six genes-Anxa2, S100a10, Ccng1, Timp1, Hspb1, and Lgals3-were significantly upregulated not only in vitro in neurons subjected to excitotoxic injury but also in rats with subacute SCI. Furthermore, Hspb1 and Lgals3 were closely linked to neuronal autophagy induced by excitotoxicity. Our findings contribute to a better understanding of excitotoxicity and autophagy, offering potential targets and a theoretical foundation for SCI diagnosis and treatment.

Under Stress: Searching for Genes Involved in the Response of Abies pinsapo Boiss to Climate Change.

Currently, Mediterranean forests are experiencing the deleterious effects of global warming, which mainly include increased temperatures and decreased precipitation in the region. Relict Abies pinsapo fir forests, endemic in the southern Iberian Peninsula, are especially sensitive to these recent environmental disturbances, and identifying the genes involved in the response of this endangered tree species to climate-driven stresses is of paramount importance for mitigating their effects. Genomic resources for A. pinsapo allow for the analysis of candidate genes reacting to warming and aridity in their natural habitats. Several members of the complex gene families encoding late embryogenesis abundant proteins (LEAs) and heat shock proteins (HSPs) have been found to exhibit differential expression patterns between wet and dry seasons when samples from distinct geographical locations and dissimilar exposures to the effects of climate change were analyzed. The observed changes were more perceptible in the roots of trees, particularly in declining forests distributed at lower altitudes in the more vulnerable mountains. These findings align with previous studies and lay the groundwork for further research on the molecular level. Molecular and genomic approaches offer valuable insights for mitigating climate stress and safeguarding this endangered conifer.

Induction of heat shock protein expression in SP2/0 transgenic cells and its effect on the production of monoclonal antibodies.

The objective of the current investigation was to evaluate the induction of heat shock proteins (HSPs) in SP2/0 transgenic cells and the effect of these proteins on the production of monoclonal antibodies (mAbs). The SP2/0 cell line expressing the PSG-026 antibody, a biosimilar candidate of golimumab, the culture parameters, and the target protein expression were not justified for industrial production and were used for the experiments. Paracetamol and heat shock were used as chemical and physical inducers of HSPs, respectively. The results showed that paracetamol and heat shock increased the expression of HSP70 and HSP27 at the mRNA and protein levels. The expression of HSPs was greater in paracetamol-treated cells than in heat shock-treated cells. Paracetamol treatment at concentrations above 0.5 mM significantly reduced cell viability and mAb expression. However, treatment with 0.25 mM paracetamol results in delayed cell death and increased mAb production. Heat shock treatment at 45°C for 30 minutes after enhanced mAb expression was applied after pre-treatment with paracetamol. In bioreactor cultures, pretreatment of cells with paracetamol improved cell viability and shortened the lag phase, resulting in increased cell density. The production of mAbs in paracetamol-treated cultures was markedly greater than that in the control. Analysis of protein quality and charge variants revealed no significant differences between paracetamol-treated and control cultures, indicating that the induction of HSPs did not affect protein aggregation or charge variants. These findings suggest that inducing and manipulating HSP expression can be a valuable strategy for improving recombinant protein production in biopharmaceutical processes.

The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

A study on expression of GRP78 and CHOP in neutrophil endoplasmic reticulum and their relationship with neutrophil apoptosis in the development of sepsis.

We investigated the relationship between neutrophil apoptosis and endoplasmic reticulum stress (ERS) in sepsis and its mechanism. A prospective cohort study was conducted by recruiting a total of 58 patients with sepsis. Peripheral blood samples were collected on 1, 3, 5 and 7 days after admission to the ICU. The expressions of endoplasmic reticulum specific glucose regulatory protein 78 (GRP78), C/EBP homologous protein (CHOP), apoptosis signal-regulating kinase 1 (ASK1), Bcl-2-like 11 (BIM), death receptor 5 (DR5), c-Jun N-terminal kinases (JNK) and p38 were detected by Western blot and PCR. The subcellular location of CHOP and GRP78 was observed by immunofluorescence analysis. Spearman correlation was used to analyze the correlation between the expression of chop protein and the apoptosis rate of peripheral blood neutrophils. Healthy volunteers in the same period were selected as the healthy control group. The expression of GRP78 protein was significantly elevated on the first day of ICU admission and showed a decreasing trend on the third, fifth and seventh day, but was significantly higher than the corresponding healthy control group. The expression of CHOP protein reached the highest level on the third day. The expression of chop protein in each group was significantly higher than that in the corresponding healthy control group. Immunofluorescence staining clearly showed that the CHOP protein accumulated in the nucleus, with an elevation in the intensity of GRP78. The neutrophil apoptosis rate of sepsis patients on the 1st, 3rd, 5th and 7th day of ICU stay was significantly higher than that of the healthy control group, with the highest apoptosis rate on the 3rd day, and then decreased gradually. CHOP protein expression level was significantly positively correlated with neutrophil apoptosis rate in sepsis patients. Endoplasmic reticulum stress occurs in neutrophils during the development of sepsis. GRP78 protein and CHOP protein may be involved in the pathological process of neutrophil apoptosis in sepsis.

eIF4G as a switch for heat shock mRNA translation.

The heat shock response is crucial for cell survival. In this issue of Molecular Cell, Desroches Altamirano et al.1 demonstrate that a temperature-induced conformational change in the translation initiation factor eIF4G is a key mechanism regulating translation during the heat shock response.

Temperature Effects on Expression Levels of hsp Genes in Eggs and Second-Stage Juveniles of Meloidogyne hapla Chitwood, 1949.

Meloidogyne hapla is one of the most important nematode pathogens. It is a sedentary, biotrophic parasite of plants that overwinters in the soil or in diseased roots. The development of M. hapla is temperature dependent. Numerous studies have been performed on the effect of temperature on the development of M. hapla, but only a few of them analyzed the heat shock protein (hsp) genes. The aim of the study was to perform expression profiling of eight hsp genes (Mh-hsp90, Mh-hsp1, Mh-hsp4, Mh-hsp6, Mh-hsp60, Mh-dnj19, Mh-hsp43, and Mh-hsp12.2) at two development stages of M. hapla, i.e., in eggs and second-stage juveniles (J2). The eggs and J2 were incubated under cold stress (5 °C), heat stress (35 °C, 40 °C), and non-stress (10 °C, 20 °C, and 30 °C) conditions. Expression profiling was performed by qPCR. It was demonstrated that only two genes, Mh-hsp60 and Mh-dnj19, have been upregulated by heat and cold stress at both development stages. Heat stress upregulated the expression of more hsp genes than cold stress did. The level of upregulation of most hsp genes was more marked in J2 than in eggs. The obtained results suggest that the Mh-hsp90 and Mh-hsp1 genes can be used as bioindicators of environmental impacts on nematodes of the Meloidogyne genus.

Skeletal Muscle Heat Shock Protein Content and the Repeated Bout Effect.

The "Repeated Bout Effect" (RBE) occurs when a skeletal muscle is preconditioned with a few lengthening contractions (LC) prior to exposing the muscle to a greater number of LC. The preconditioning (PC) results in significantly less damage and preservation of force. Since it takes only a few LC to increase muscle heat shock protein (HSP) content, it was of interest to examine the relationship between HSPs and the RBE. To do this, one tibialis anterior (TA) muscle from Sprague-Dawley rats (n = 5/group) was preconditioned with either 0, 5, or 15 lengthening contractions (LC) and exposed to a treatment of 60 LC 48 h later. Preconditioning TA muscles with 15 LC, but not 5 LC, significantly elevated muscle αB-crystallin (p < 0.05), HSP25 (p < 0.05), and HSP72 content (p < 0.001). These preconditioned TA muscles also showed a significantly (p < 0.05) reduced loss of active torque throughout the subsequent 60 LC. While there was a trend for all preconditioned muscles to maintain higher peak torque levels throughout the 60 LC, no significant differences were detected between the groups. Morphologically, preconditioned muscles appeared to show less discernible muscle fiber damage. In conclusion, an elevated skeletal muscle HSP content from preconditioning may contribute to the RBE.

Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

An experimental framework to assess biomolecular condensates in bacteria.

High-resolution imaging of biomolecular condensates in living cells is essential for correlating their properties to those observed through in vitro assays. However, such experiments are limited in bacteria due to resolution limitations. Here we present an experimental framework that probes the formation, reversibility, and dynamics of condensate-forming proteins in Escherichia coli as a means to determine the nature of biomolecular condensates in bacteria. We demonstrate that condensates form after passing a threshold concentration, maintain a soluble fraction, dissolve upon shifts in temperature and concentration, and exhibit dynamics consistent with internal rearrangement and exchange between condensed and soluble fractions. We also discover that an established marker for insoluble protein aggregates, IbpA, has different colocalization patterns with bacterial condensates and aggregates, demonstrating its potential applicability as a reporter to differentiate the two in vivo. Overall, this framework provides a generalizable, accessible, and rigorous set of experiments to probe the nature of biomolecular condensates on the sub-micron scale in bacterial cells.

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

Gender associations between phthalate exposure and biomarkers of oxidative stress: A prospective cohort study.

Previous epidemiologic research has shown that phthalate exposure in pregnant women is related to adverse birth outcomes in a sex-specific manner. However, the biological mechanism of phthalate exposure that causes these birth outcomes remains poorly defined. In this research, we investigated the association between phthalate exposure and placental oxidative stress in a large population-based cohort study, aiming to initially explore the relationship between phthalate exposure and gene expression in placental oxidative stress in a sex-specific manner. Quantitative PCR was performed to measure the expression of placental inflammatory mRNAs (HO-1, HIF1α, and GRP78) in 2469 placentae. The multiple linear regression models were used to investigate the associations between mRNA and urinary phthalate monoesters. Phthalate metabolites monomethyl phthalate (MMP) and mono-n-butyl phthalate (MBP) were positively correlated with higher HIF1α expression in placentae of male fetuses (p < .05). Mono-benzyl phthalate (MBzP) increased the expression of HO-1, HIF1α, and GRP78 in placentae of male fetuses, and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) up-regulated the expression of HIF1α and GRP78. Additionally, mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP) was negatively correlated with HO-1, HIF1α, and GRP78 in placentae of female fetuses. Maternal phthalate exposure was associated with oxidative stress variations in placental tissues. The associations were closer in the placentas of male fetuses than in that of female ones. The placenta oxidative stress is worth further investigation as a potential mediator of maternal exposure-induced disease risk in children.

Heat Shock Response and Heat Shock Proteins: Current Understanding and Future Opportunities in Human Diseases.

The heat shock response is an evolutionarily conserved mechanism that protects cells or organisms from the harmful effects of various stressors such as heat, chemicals toxins, UV radiation, and oxidizing agents. The heat shock response triggers the expression of a specific set of genes and proteins known as heat shock genes/proteins or molecular chaperones, including HSP100, HSP90, HSP70, HSP60, and small HSPs. Heat shock proteins (HSPs) play a crucial role in thermotolerance and aiding in protecting cells from harmful insults of stressors. HSPs are involved in essential cellular functions such as protein folding, eliminating misfolded proteins, apoptosis, and modulating cell signaling. The stress response to various environmental insults has been extensively studied in organisms from prokaryotes to higher organisms. The responses of organisms to various environmental stressors rely on the intensity and threshold of the stress stimuli, which vary among organisms and cellular contexts. Studies on heat shock proteins have primarily focused on HSP70, HSP90, HSP60, small HSPs, and ubiquitin, along with their applications in human biology. The current review highlighted a comprehensive mechanism of heat shock response and explores the function of heat shock proteins in stress management, as well as their potential as therapeutic agents and diagnostic markers for various diseases.

Heat shock protein-related diagnostic signature and molecular subtypes in ankylosing spondylitis: new pathogenesis insights.

Heat shock proteins (HSP) have been associated with a range of persistent inflammatory disorders; however, little research has been conducted on the involvement of HSP in the development of ankylosing spondylitis (AS). The research aims to identify a diagnostic signature based on HSP-related genes and determine the molecular subtypes of AS. We gathered the transcriptional data of patients with AS from the GSE73754 dataset and conducted a literature search for HSP-related genes (HRGs). The logistic regression model was utilized for the identification of hub HRGs associated with AS. Subsequently, these HRGs were employed in the construction of a nomogram prediction model. We employed a consensus clustering approach to identify novel molecular subgroups. Subsequently, we conducted functional analyses, encompassing GO, KEGG, and GSEA, to elucidate the underlying mechanisms between these subgroups. To assess the immunological landscape, we employed the xCell algorithm. Through logistic regression analysis, the four core HRGs (CCT2, HSPA6, DNAJB14, and DNAJC5) were confirmed as potential biomarkers for AS. Subsequent stratification revealed two distinct molecular phenotypes, designated as Cluster 1 and Cluster 2. Notably, Cluster 2 was characterized by the upregulation of pathways pertinent to immune response and inflammation. Our research suggests that the CCT2, HSPA6, DNAJB14, and DNAJC5 exhibit potential as effective blood-based diagnostic biomarkers for AS. These findings contribute to a deeper comprehension of the underlying mechanisms involved in the development of AS and offer potential targets for personalized therapeutic interventions.

Exosomal HSPB1, interacting with FUS protein, suppresses hypoxia-induced ferroptosis in pancreatic cancer by stabilizing Nrf2 mRNA and repressing P450.

Ferroptosis is a new type of programmed cell death, which has been involved in the progression of tumours. However, the regulatory network of ferroptosis in pancreatic cancer is still largely unknown. Here, using datasets from GEO and TCGA, we screened HSPB1, related to the P450 monooxygenase signalling, a fuel of ferroptosis, to be a candidate gene for regulating pancreatic cancer cell ferroptosis. We found that HSPB1 was enriched in the exosomes derived from human pancreatic cancer cell lines SW1990 and Panc-1. Then, hypoxic SW1990 cells were incubated with exosomes alone or together with HSPB1 siRNA (si-HSPB1), and we observed that exosomes promoted cell proliferation and invasion and suppressed ferroptosis, which was reversed by si-HSPB1. Moreover, we found a potential binding affinity between HSPB1 and FUS, verified their protein interaction by using dual-colour fluorescence colocalization and co-IP assays, and demonstrated the promoting effect of FUS on oxidative stress and ferroptosis in hypoxic SW1990 cells. Subsequently, FUS was demonstrated to bind with and stabilize the mRNA of Nrf2, a famous anti-ferroptosis gene that negatively regulates the level of P450. Furthermore, overexpressing FUS and activating the Nrf2/HO-1 pathway (using NK-252) both reversed the inhibitory effect of si-HSPB1 on exosome functions. Finally, our in vivo studies showed that exosome administration promote tumour growth in nude mice of xenotransplantation, which was able to be eliminated by knockdown of HSPB1. In conclusion, exosomal HSPB1 interacts with the RNA binding protein FUS and decreases FUS-mediated stability of Nrf2 mRNA, thus suppressing hypoxia-induced ferroptosis in pancreatic cancer.

Determination of anti-cancer effects of Nigella sativa seed oil on MCF7 breast and AGS gastric cancer cells.

BACKGROUND: This study aimed to investigate the cytotoxic, apoptotic, invasion, metastasis, and heat shock proteins (HSPs) effects of N. sativa oil on breast and gastric cancer cells. METHODS: We assessed the cytotoxic and apoptotic effects of various concentrations of N. sativa oil (10-50-100-200 µg/mL) on MCF7 breast cancer and AGS, an adenocarcinoma of the gastric cell line, at 24, 48 and 72 h using the MTT test. Additionally, the expression of the Caspase-3, BCL2/Bax, MMP2-9 and HSP60-70 gene was examined using RT-PCR in cell lines treating with N. sativa. RESULTS: The MTT experiments demonstrate that N. sativa has a time and dose-dependent inhibitory effect on the proliferation of MCF7 and AGS cancer cells. The vitality rates of MCF7 and AGS cells treated with N. sativa were 77.04-67.50% at 24 h, 65.28-39.14% at 48 h, and 48.95-32.31% at 72 h. The doses of 100 and 200 µg/mL were shown to be the most effective on both cancer cells. RT-PCR analysis revealed that N. sativa oil extract increased caspase-3 levels in both cell lines at higher concentrations and suppressed BCL2/Bax levels. Exposure of MCF7 and AGS cell lines to N. sativa caused a significant decrease in the expression of MMP2-9 and HSP60-70 genes over time, particularly at a dosage of 200 µg/mL compared to the control group (p < 0.05). CONCLUSIONS: Our findings indicate that N. sativa oil has a dose-dependent effect on cytotoxicity and the expression of apoptotic, heat shock proteins, and matrix metalloproteinases genes in breast and gastric cancer.

The modulation of autophagy and unfolded protein response by ent-kaurenoid derivative CPUK02 in human colorectal cancer cells.

BACKGROUND: CPUK02 (15-Oxosteviol benzyl ester) is a semi-synthetic derivative of stevioside known for its anticancer effects. It has been reported that the natural compound of stevioside and its associated derivatives enhances the sensitivity of cancer cells to conventional anti-cancer agents by inducing endoplasmic reticulum (ER) stress. In response to ER stress, autophagy and unfolded protein responses (UPR) are activated to restore cellular homeostasis. Consequently, the primary aim of this study is to investigate the impact of CPUK02 treatment on UPR and autophagy markers in two colorectal cancer cell lines. METHODS: HCT116 and SW480 cell lines were treated with various concentrations of CPUK02 for 72 h. The expression levels of several proteins and enzymes were evaluated to investigate the influence of CPUK02 on autophagy and UPR pathways. These include glucose-regulated protein 78 (GRP78), Inositol-requiring enzyme 1-α (IRE1-α), spliced X-box binding protein 1 (XBP-1 s), protein kinase R-like ER kinase (PERK), C/EBP homologous protein (CHOP), Beclin-1, P62 and Microtubule-associated protein 1 light chain 3 alpha (LC3ßII). The evaluation was conducted using western blotting and quantitative real-time PCR techniques. RESULTS: The results obtained indicate that the treatment with CPUK02 reduced the expression of UPR markers, including GRP78 and IRE1-α at protein levels and XBP-1 s, PERK, and CHOP at mRNA levels in both HCT116 and SW480 cell lines. Furthermore, CPUK02 also influenced autophagy by decreasing Beclin-1 and increasing P62 and LC3ßII at mRNA levels in both HCT116 and SW480 treated cells. CONCLUSIONS: The study findings suggest CPUK02 may exert its cytotoxic effects by inhibiting UPR and autophagy flux in colorectal cancer cells.

A self-amplifying loop of TP53INP1 and P53 drives oxidative stress-induced apoptosis of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cell (BMSC) transplantation is a promising regenerative therapy; however, the survival rate of BMSCs after transplantation is low. Oxidative stress is one of the main reasons for the high apoptosis rate of BMSCs after transplantation, so there is an urgent need to explore the mechanism of oxidative stress-induced apoptosis of BMSCs. Our previous transcriptome sequencing results suggested that the expression of P53-induced nuclear protein 1 (TP53INP1) and the tumor suppressor P53 (P53) was significantly upregulated during the process of oxidative stress-induced apoptosis of BMSCs. The present study further revealed the role and mechanism of TP53INP1 and P53 in oxidative stress-induced apoptosis in BMSCs. Overexpression of TP53INP1 induced apoptosis of BMSCs, knockdown of TP53INP1 alleviated oxidative stress apoptosis of BMSCs. Under oxidative stress conditions, P53 is regulated by TP53INP1, while P53 can positively regulate the expression of TP53INP1, so the two form a positive feedback loop. To clarify the mechanism of feedback loop formation. We found that TP53INP1 inhibited the ubiquitination and degradation of P53 by increasing the phosphorylation level of P53, leading to the accumulation of P53 protein. P53 can act on the promoter of the TP53INP1 gene and increase the expression of TP53INP1 through transcriptional activation. This is the first report on a positive feedback loop formed by TP53INP1 and P53 under oxidative stress. The present study clarified the formation mechanism of the positive feedback loop. The TP53INP1-P53 positive feedback loop may serve as a potential target for inhibiting oxidative stress-induced apoptosis in BMSCs.

Escherichia coli-based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70.

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70 genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756 cells are unable to support λ bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756 cells grow adequately at temperatures ranging from 30 °C to 37 °C, but they die at elevated temperatures (>40 °C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

Heat shock proteins as a key defense mechanism in poultry production under heat stress conditions.

Over the past years, the poultry industry has been assigned to greater production performance but has become highly sensitive to environmental changes. The average world temperature has recently risen and is predicted to continue rising. In open-sided houses, poultry species confront high outside temperatures, which cause heat stress (HS) problems. Cellular responses are vital in poultry, as they may lead to identifying confirmed HS biomarkers. Heat shock proteins (HSP) are highly preserved protein families that play a significant role in cell function and cytoprotection against various stressors, including HS. The optimal response in which the cell survives the HS elevates HSP levels that prevent cellular proteins from damage caused by HS. The HSP have chaperonic action to ensure that stress-denatured proteins are folded, unfolded, and refolded. The HSP70 and HSP90 are the primary HSP in poultry with a defensive function during HS. HSP70 was the optimal biological marker for assessing HS among the HSP studied. The current review attempts to ascertain the value of HSP as a heat stress defense mechanism in poultry.