Heat Shock Protein A5 Research Articles

Identification and prognostic analysis of ferroptosis‑related gene HSPA5 to predict the progression of lung squamous cell carcinoma.

Ferroptosis, an iron-dependent form of regulated cell death driven by excessive lipid peroxidation, is implicated in the development and therapeutic responses of cancer. However, the role of ferroptosis-related gene profiles in lung squamous cell carcinoma (LSCC) remains largely unknown. The present study aimed to identify the prognostic roles of ferroptosis-related genes in LSCC. Sequencing data from the Cancer Genome Atlas were analyzed and ferroptosis-related gene expression between tumor and para-tumor tissue was identified. The prognostic role of these genes was also assessed using Kaplan-Meier analyses and univariate and multivariate Cox proportional hazards regression model analyses. Immunological correlation, tumor stemness, drug sensitivity and the transcriptional differences of heat shock protein (HSP)A5 in LSCC were also analyzed. Thereafter, the expression of HSPA5 in 100 patients with metastatic LSCC was evaluated using immunohistochemistry (IHC) and the clinical significance of these markers with different risk factors was assessed. Of the 22 ferroptosis-related genes, the expression of HSPA5, HSPB1, glutathione peroxidase 4, Fanconi anemia complementation group D2, CDGSH iron sulfur domain 1, farnesyl-diphosphate farnesyltransferase 1, nuclear factor erythroid 2 like 2, solute carrier (SLC)1A5, ribosomal protein L8, nuclear receptor coactivator 4, transferrin receptor and SLC7A11 was significantly increased in LSCC compared with adjacent tissues. However, only high expression of HSPA5 was able to predict progression-free survival (PFS) and disease-free survival in LSCC. Although HSPA5 was also significantly elevated in patients with lung adenocarcinoma, HSPA5 expression did not predict the prognosis of patients with lung adenocarcinoma. Of note, a higher expression of HSPA5 was related to higher responses to chemotherapy but not to immunotherapy. In addition, HSPA5 expression was positively correlated with 'ferroptosis', 'cellular responses to hypoxia', 'tumor proliferation signature', 'G2M checkpoint', 'MYC targets' and 'TGFB'. IHC analysis also demonstrated that a high expression of HSPA5 in patients with metastatic LSCC in the study cohort was associated with shorter PFS and overall survival. In conclusion, the present study demonstrated that the expression of the ferroptosis-related gene HSPA5 may be a negative prognostic marker for LSCC.

Identification of ferroptosis‑associated genes in chronic kidney disease.

Ferroptosis serves a pivotal role in developing chronic kidney disease (CKD). The present study aimed to detect and confirm the relevance of potential ferroptosis-related genes in CKD using bioinformatics and experimentation strategies. The original GSE15072 mRNA expression dataset was retrieved from the Gene Expression Omnibus database. Subsequently, the potential differentially expressed genes associated with ferroptosis of CKD were screened using R software. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analyses, correlation analysis and protein-protein interactions (PPI) were performed for differentially expressed ferroptosis-associated genes (DFGs). Lastly, the expression levels of the top nine DFGs were measured in the kidney tissue of Adriamycin-induced CKD rats and healthy controls via reverse transcription-quantitative (RT-q)PCR analysis. Overall, 49 DFGs among 21 patients with CKD and nine healthy controls were identified. GO and KEGG enrichment analyses demonstrated that these DFGs were primarily involved in 'ferroptosis' and 'mitophagy'. PPI findings indicated that these ferroptosis-associated genes interacted with one another. RT-qPCR of CKD tissue from the rat model revealed that STAT3, MAPK14, heat shock protein (HSP)A5, MTOR and solute carrier family 2 member 1 (SLC2A1) mRNA levels in CKD were upregulated. Overall, 49 potential ferroptosis-associated genes of CKD were identified via bioinformatics analyses. STAT3, MAPK14, HSPA5, MTOR and SLC2A1 may influence CKD onset by regulating ferroptosis. The present results add to the existing body of knowledge about CKD and may be useful in the treatment of CKD.

Autoantibody against Tumor-Associated Antigens as Diagnostic Biomarkers in Hispanic Patients with Hepatocellular Carcinoma.

Background: Tumor-associated antigens (TAAs) have been investigated for many years as potential early diagnosis tools, especially for hepatocellular carcinoma (HCC). Nonetheless, very few studies have focused on the Hispanic HCC group that may be associated with distinct etiological risk factors. In the present study, we investigated novel anti-TAA autoantibodies as diagnostic biomarkers for Hispanic HCC patients. Methods: Novel TAA targets were identified by the serological proteome analysis (SERPA) and from differentially expressed HCC driver genes via bioinformatics. The autoantibody levels were validated by enzyme-linked immunosorbent assay (ELISA). Results: Among 19 potential TAA targets, 4 anti-TAA autoantibodies were investigated as potential diagnostic biomarkers with significantly high levels in Hispanic HCC sera, including DNA methyltransferase 3A (DNMT3A), p16, Hear shock protein 60 (Hsp60), and Heat shock protein A5 (HSPA5). The area under the ROC curve (AUC) value of the single autoantibodies varies from 0.7505 to 0.8885. After combining all 4 autoantibodies, the sensitivity of the autoantibody panel increased to 75% compared to the single one with the highest value of 45.8%. In a separate analysis of the Asian cohort, autoantibodies against HSPA5 and p16 showed significantly elevated levels in HCC compared to normal healthy controls, but not for DNMT3A or HSP60. Conclusion: Anti-DNMT3A, p16, HSPA5, and HSP60 autoantibodies have the potential to be diagnostic biomarkers for Hispanic HCC patients, of which DNMT3A and HSP60 might be exclusive for Hispanic HCC diagnosis.

Early Aerobic Exercise Promotes Neurological Function Recovery of Rats after Cerebral Ischemia/Reperfusion by Upregulating the Expression of Heat Shock Protein A5.

The neuroprotective function of heat shock protein A5 (HSPA5) in ischemic stroke has been confirmed. This study aimed to investigate the effects of early aerobic exercise on neurological function recovery from cerebral ischemia/reperfusion and to determine whether these effects are associated with the expression level of HSPA5 in the ischemic penumbra. A total of 72 male Sprague-Dawley rats were randomly assigned to the ischemia and exercise group [middle cerebral artery occlusion (MCAO)-Ex, n=18], ischemia and sedentary group (MCAO-St, n=18), sham-surgery and exercise group (Sham-Ex, n=18), or sham-surgery and sedentary group (Sham-St, n=18). The MCAO-Ex and MCAO-St groups were subjected to MCAO for 60 min, whereas the Sham-Ex and Sham-St groups were subjected to an identical operation without MCAO. Rats in the MCAO-Ex and Sham-Ex groups then ran on a treadmill for 30 min once a day for 5 consecutive days. After reperfusion, the motor function of the rats was scored by the Bederson neurological function test, balance beam test, and screen test. Nissl staining was conducted to assess morphological and structural change of nerve cells in the ischemic penumbra. The reverse transcription-quantitative polymerase chain reaction was applied to detect the mRNA expression of HSPA5. Western blot analysis was conducted to determine the protein expression of HSPA5. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out in the ischemic penumbra after MCAO. Rats receiving early treadmill exercise had lower Bederson neurological function, balance beam, and screen test scores on the 3rd, 7th, and 14th days after MCAO; in addition, more neurons survived in the ischemic penumbra after MCAO, and higher mRNA and protein expression of HSPA5 and fewer TUNEL-positive stained cells were observed. Our study demonstrated that early aerobic exercise can improve neurological function recovery after ischemia/reperfusion. Furthermore, the increased level of HSPA5 in the ischemic penumbra might be one of the mechanisms of enhanced neurological function recovery.

Comparative study of SARS-CoV-2 infection in different cell types: Biophysical-computational approach to the role of potential receptors

Cellular susceptibility to SARS-CoV-2 infection in the respiratory tract has been associated with the ability of the virus to interact with potential receptors on the host membrane. We have modeled viral dynamics by simulating various cellular systems and artificial conditions, including macromolecular crowding, based on experimental and transcriptomic data to infer parameters associated with viral growth and predict cell susceptibility. We have accomplished this based on the type, number and level of expression of the angiotensin-converting enzyme 2 (ACE2), transmembrane serine 2 (TMPRSS2), basigin2 (CD147), FURIN protease, neuropilin 1 (NRP1) or other less studied candidate receptors such as heat shock protein A5 (HSPA5) and angiotensin II receptor type 2 (AGTR2). In parallel, we studied the effect of simulated artificial environments on the accessibility to said proposed receptors. In addition, viral kinetic behavior dependent on the degree of cellular susceptibility was predicted. The latter was observed to be more influenced by the type of proteins and expression level, than by the number of potential proteins associated with the SARS CoV-2 infection. We predict a greater theoretical propensity to susceptibility in cell lines such as NTERA-2, SCLC-21H, HepG2 and Vero6, and a lower theoretical propensity in lines such as CaLu3, RT4, HEK293, A549 and U-251MG. An important relationship was observed between expression levels, protein diffusivity, and thermodynamically favorable interactions between host proteins and the viral spike, suggesting potential sites of early infection other than the lungs. This research is expected to stimulate future quantitative experiments and promote systematic investigation of the effect of crowding presented here.

Abstract 14305: Diastolic Dysfunction and Activation of Ventricular and Atrial Unfolded Protein Response in a Novel Gottingen Miniswine Model of Heart Failure With Preserved Ejection Fraction

Introduction: During cardiac hypertrophy, the unfolded protein response (UPR) is activated which includes upregulation of chaperones to assist with protein folding and protein ubiquination for proteolytic degradation. We investigated the expression level of a key chaperone Hspa5 (heat shock protein A5) and ubiquinated protein levels in left ventricular (LV) and atrial (LA) tissue obtained from a preclinical porcine model heart failure with preserved ejection fraction (HFpEF) involving obesity and hypertension. Methods: Female Gottingen minipigs were fed standard or western diet (WD) for 20 weeks to induce obesity and metabolic syndrome and subsequent HFpEF. Those fed WD were also administered 11-deoxycorticosterone acetate (DOCA) (50 mg/kg, SQ) to induce hypertension. LV functional assessment was performed via transthoracic echocardiography and invasive hemodynamics at 20 weeks. In addition, UPR and proteasomal function were assayed following 20 weeks of HFpEF in ventricular and left atrial extracts via RT-qPCR and immunoblot. Results: Minipigs fed WD+DOCA demonstrated hypercholesterolemia, hypertension, LV hypertrophy and increased LVEDP. LVEF was >50% in HFpEF and control groups. We observed significant LV diastolic dysfunction as measured by E/e’ and LA dysfunction as decreased LA fractional area of change (LAFAC) at 20 weeks. LV extracts from HFpEF swine demonstrated a marked increase in Hspa5 without measurable increases in standard fetal genes (e.g. Myh7 ). LA extracts from HFpEF swine exhibited robust accumulation of ubiquitylated proteins compared to control swine. There was a positive correlation between Hspa5 and E/e’ ratio, and the inverse for LA ubiquitylated proteins and LA function. Conclusions: Coordinate with myocardial stiffening, hypertrophy and functional decline, HFpEF robustly augments proteostasis in both LV myocardium and LA recapitulating the clinical presentation of HFpEF patients.

Oxidation of peroxiredoxin-4 induces oligomerization and promotes interaction with proteins governing protein folding and endoplasmic reticulum stress

Peroxiredoxins (PRDXs) catalyze the reduction of hydrogen peroxide (H2O2). PRDX4 is the only peroxiredoxin located within the endoplasmic reticulum (ER) and is the most highly expressed H2O2 scavenger in the ER. PRDX4 has emerged as an important player in numerous diseases, such as fibrosis and metabolic syndromes, and its overoxidation is a potential indicator of ER redox stress. It is unclear how overoxidation of PRDX4 governs its oligomerization state and interacting partners. Herein, we addressed these questions via nonreducing Western blots, mass spectrometry, and site-directed mutagenesis. We report that the oxidation of PRDX4 in lung epithelial cells treated with tertbutyl hydroperoxide caused a shift of PRDX4 from monomer/dimer to high molecular weight (HMW) species, which contain PRDX4 modified with sulfonic acid residues (PRDX4-SO3), as well as of a complement of ER-associated proteins, including protein disulfide isomerases important in protein folding, thioredoxin domain–containing protein 5, and heat shock protein A5, a key regulator of the ER stress response. Mutation of any of the four cysteines in PRDX4 altered the HMW species in response to tertbutyl hydroperoxide as well as the secretion of PRDX4. We also demonstrate that the expression of ER oxidoreductase 1 alpha, which generates H2O2 in the ER, increased PRDX4 HMW formation and secretion. These results suggest a link between SO3 modification in the formation of HMW PRDX4 complexes in cells, whereas the association of key regulators of ER homeostasis with HMW oxidized PRDX4 point to a putative role of PRDX4 in regulating ER stress responses.

Zika virus envelope – heat shock protein A5 (GRP78) binding site prediction

Recent studies reported the association of the Zika virus (ZIKV) with a stress response receptor on the host cell membrane that facilitates viral entry. This host receptor was the heat shock protein A5 (HSPA5), also termed glucose-regulating protein 78 (GRP78). In this study, structural bioinformatics and molecular dynamics simulations were utilized to suggest the binding site of ZIKV envelope protein during the interaction with cell-surface GRP78. The Pep42 cyclic peptide was used as a profiler, as it was reported earlier, to target GRP78 on the cancer cell membrane selectively. Sequence and structural alignments show that part of the ZIKV envelope protein (C308-C339 region), in addition to its cyclic nature, has somehow sequence and structural similarities to the cyclic Pep42. Three amino acids in the ZIKV envelope were identical to those in the Pep42 peptide. Cyclic peptides dynamics are studied, and its binding to GRP78 is predicted. Protein-protein docking is further performed to explore the binding characteristics of the ZIKV envelope to GRP78. Results revealed that the binding was favorable between ZIKV envelope protein and GRP78. The docking pose revealed the involvement of the substrate-binding domain ß of GRP78 and the domain III of the ZIKV envelope protein in viral recognition for the host-cell.

Natural products may interfere with SARS-CoV-2 attachment to the host cell

SARS-CoV-2 has been emerged in December 2019 in China, causing deadly (5% mortality) pandemic pneumonia, termed COVID-19. More than one host-cell receptor is reported to be recognized by the viral spike protein, among them is the cell-surface Heat Shock Protein A5 (HSPA5), also termed GRP78 or BiP. Upon viral infection, HSPA5 is upregulated, then translocating to the cell membrane where it is subjected to be recognized by the SARS-CoV-2 spike. In this study, some natural product compounds are tested against the HSPA5 substrate-binding domain β (SBDβ), which reported to be the recognition site for the SARS-CoV-2 spike. Molecular docking and molecular dynamics simulations are used to test some natural compounds binding to HSPA5 SBDβ. The results show high to a moderate binding affinity for the phytoestrogens (Diadiazin, Genistein, Formontein, and Biochanin A), chlorogenic acid, linolenic acid, palmitic acid, caffeic acid, caffeic acid phenethyl ester, hydroxytyrosol, cis-p-Coumaric acid, cinnamaldehyde, thymoquinone, and some physiological hormones such as estrogens, progesterone, testosterone, and cholesterol to the HSPA5 SBDβ. Based on its binding affinities, the phytoestrogens and estrogens are the best in binding HSPA5, hence may interfere with SARS-CoV-2 attachment to the stressed cells. These compounds can be successful as anti-COVID-19 agents for people with a high risk of cell stress like elders, cancer patients, and front-line medical staff.Communicated by Ramaswamy H. Sarma

Ebola virus glycoprotein GP1-host cell-surface HSPA5 binding site prediction.

Ebola virus (EBOV) infection is a widespread infection that has created a bad memory in Africa. In the 2014 and 2015 outbreak, more than 28,000 infections were reported by the World Health Organization, with about 11,300 deaths in Guinea, Liberia, and Sierra Leone. Heat shock protein A5 (HSPA5), termed also GRP78, is a host cell chaperone protein responsible for the unfolded protein response in the endoplasmic reticulum. Under stress, HSPA5 is upregulated and becomes cell-surface exposed. Recent studies report the association of cell-surface HSPA5 with EBOV glycoproteins GP1 and GP2. In this study, structural and sequence analysis and molecular docking are used to predict the possible binding site between the cell-surface HSPA5 and EBOV GP1. The results show a promising binding site that supports the hypothesis of HSPA5 selectivity for binding to a specific peptide sequence (pep42). This study paves the way to suggest possible inhibitors to stop viral association with cell-surface receptors and subsequently reduce viral infection.Electronic supplementary materialThe online version of this article (10.1007/s12192-020-01106-z) contains supplementary material, which is available to authorized users.

Profiling the Expression of Endoplasmic Reticulum Stress Associated Heat Shock Proteins in Animal Epilepsy Models

Unfolded protein response is a signaling cascade triggered by misfolded proteins in the endoplasmic reticulum. Heat shock protein H4 (HSPH4) and A5 (HSPA5) are two chaperoning proteins present within the organelle, which target misfolded peptides during prolonged stress conditions. Epileptogenic insults and epileptic seizures are a notable source of stress on cells. To investigate whether they influence expression of these chaperones, we performed immunohistochemical stainings in brains from rats that experienced a status epilepticus (SE) as a trigger of epileptogenesis and from canine epilepsy patients. Quantification of HSPA5 and HSPH4 revealed alterations in hippocampus and parahippocampal cortex. In rats, SE induced up-regulation of HSPA5 in the piriform cortex and down-regulation of HSPA5 and HSPH4 in the hippocampus. Regionally restricted increases in expression of the two proteins has been observed in the chronic phase with spontaneous recurrent seizures. Confocal microscopy revealed a predominant expression of both proteins in neurons, no expression in microglia and circumscribed expression in astroglia. In canine patients, only up-regulation of HSPH4 expression was observed in Cornu Ammonis 1 region in animals diagnosed with structural epilepsy. This characterization of HSPA5 and HSPH4 expression provided extensive information regarding spatial and temporal alterations of the two proteins during SE-induced epileptogenesis and following epilepsy manifestations. Up-regulation of both proteins implies stress exerted on ER during these disease phases. Taken together suggest a differential impact of epileptogenesis on HSPA5 and HSPH4 expression and indicate them as a possible target for pharmacological modulation of unfolded protein response.

Pifithrin-μ incorporated in gold nanoparticle amplifies pro-apoptotic unfolded protein response cascades to potentiate synergistic glioblastoma therapy

Conventional radiotherapy has a pivotal role in the treatment of glioblastoma; nevertheless, its clinical utility has been limited by radiation resistance. There is emerging evidence that upregulated heat shock protein A5 (HSPA5) in cancer cells maintains or restores the homeostasis of a cellular microenvironment and results in cancer resistance in various treatments. Therefore, we describe a bioresponsive nanoplatform that can deliver a HSPA5 inhibitor (pifithrin-μ, PES) and radiosensitizer (gold nanosphere, AuNS), to expand the synergistic photothermal therapy and radiotherapy, as well as to monitor the progression of cancer therapy using computer tomography/magnetic resonance imaging. The nanoplatform (PES-Au@PDA, 63.3 ± 3.1 nm) comprises AuNS coated with the photothermal conversion agent polydopamine (PDA) for enhanced radiotherapy and photothermal therapy, as well as PES (loading efficiency of PES approximately 40%), a small molecular inhibitor against HSPA5 to amplify the pro-apoptotic unfolded protein response (UPR). The reported nanoplatform enables hyperthermia-responsive release of PES. Results from in vitro and in vivo studies demonstrate that PES-Au@PDA can specially activate pro-apoptotic UPR cascades, leading to remarkably improved radiotherapy and photothermal therapy efficiencies. Considered together, a versatile theranostic nanosystem is reported for promoting the synergistic radiophotothermal therapy by selectively activating pro-apoptotic UPR cascade pathways.

MiR-181b regulates ER stress induced neuron death through targeting Heat Shock Protein A5 following intracerebral haemorrhage

Endoplasmic reticulum (ER) stress acts as a protein folding and contributes to neuronal damage and neurological deterioration following intracerebral hemorrhage (ICH). Heat Shock Protein A5 (HSPA5) serves as an essential regulator of the endoplasmic reticulum (ER) stress response. However, the specific mechanism has not been will identified. Primary cortical neurons from C57BL/6 mice were subjected to erythrocyte lysates. Cell viability, microRNA and HSPA5 levels, and ER stress was detected. The interaction between microRNA and the target HSPA5 was identified by dual luciferase reporter gene assay. In addition, inflammatory cytokines, brain edema, and neurological functions in ICH mice were also assessed. Erythrocyte lysates induced ER stress and neuron damage, downregulated miR-181b and upregulated HSPA5 levels. MiR-181b suppressed HSPA5 expression by directly binding its 3′-untranslated region. Correspondingly, our data demonstrated that overexpression of miR-181b attenuated erythrocyte lysates induced neuronal necrosis and apoptosis. In vivo, downregulated miR-181b increased the HSPA5 level, along with significant elevations of pro-inflammatory cytokines, brain edema, and neurological injury following ICH. HSPA5 pathway plays an important role in ER stress induced brain damage following ICH. In addition, miR-181b has neuroprotective effects that alleviates neurological injury and represents a promising therapeutic strategy in ICH.

Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation.

BackgroundDerailment of proteostasis, the homeostasis of production, function, and breakdown of proteins, contributes importantly to the self‐perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in humans. Autophagy plays an important role in proteostasis by degrading aberrant proteins and organelles. Herein, we investigated the role of autophagy and its activation pathway in experimental and clinical AF.Methods and ResultsTachypacing of HL‐1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B‐II expression, autophagic flux and autophagosome formation, and degradation of p62, resulting in reduction of Ca2+ amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4‐phenyl butyrate, overexpression of the ER chaperone‐protein heat shock protein A5, or overexpression of a phosphorylation‐blocked mutant of eukaryotic initiation factor 2α (eIF2α) prevents autophagy activation and Ca2+‐transient loss in tachypaced HL‐1 cardiomyocytes. Moreover, pharmacological inhibition of ER stress in tachypaced Drosophila confirms its role in derailing cardiomyocyte function. In vivo treatment with sodium salt of phenyl butyrate protected atrial‐tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L‐type Ca2+‐current reduction), cellular Ca2+‐handling/contractile dysfunction, and ER stress and autophagy; it also attenuated AF progression. Finally, atrial tissue from patients with persistent AF reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage.ConclusionsThese results identify ER stress–associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER‐stress inhibitor 4‐phenyl butyrate.

Effects of heat shock protein A5 induced autophagy on cerebral ischemia/reperfusion injury in mice

To determine the role of heat shock protein A5 (HSPA5) induced autophagy on cerebral ischemia/reperfusion in-jury in mice. Thirty-six BALB/c mice were randomly divided into sham group, ischcmia/reperfusion (I/R) group, vehicle + I/R group, 3-Methyladenine(3-MA) + I/R group, scramble siRNA group and HSPA5 siRNA + I/R group(n=6). In sham group, the operation was only performed, did not insert line switch. Focal cerebral ischemia was performed using the method of middle cerebral artery occlusion (MCAO) for 60 min and 24 h reperfusion. In vehicle + I/R group and 3-MA + I/R group, 2μl 0.9% NaCl or 3-MA(30 mg/ml) was admin-istered by intracerebroventricular injection 30 min before MCAO; In scramble siRNA + I/R group and HSPA5 siRNA + I/R group, 5μl scram-ble siRNA or HSPA5 siRNA(2μg/μl) was administered by intracerebroventricular injection 24 h before MCAO. Autophagosome in neuron, the expression of microtubule-associated protein light chain 3 (LC3)-Ⅱ/LC3-I in ischemic cortex, the degree of cerebral ischemic injury and neu-rological function score were detected. Initial electron microscopy showed that neuronal morphology appeared to be normal in the sham group. At 24 h after I/R, cell shrinkage, loss of cellular organelles and formation of autophagosomes were observed in the ischemic cerebral cortex of I/R group. In addition, autophagosomes were less frequently observed than that in I/R group. The expressions of LC3-Ⅱ/LC3-I and Beclin-1 protein were increased significantly in I/R group compared with that in sham group(P < 0.05). Compare with I/R group, the LC3-Ⅱ/LC3-I protein levels induced by I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group was decreased effectively (P < 0.05). In addi-tion, the cerebral ischemic injury and neurological symptoms after I/R in 3-MA + I/R group or HSPA5 siRNA + I/R group were exacerbated significantly (P < 0.05). These results suggest that HSPA5 induced autophagy may play a protective role in focal I/R damage in mice.

Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1 μg of isolated AApoAII fibrils into R1.P1-Apoa2c mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.

Downregulation of miRNA-134 protects neural cells against ischemic injury in N2A cells and mouse brain with ischemic stroke by targeting HSPA12B

MicroRNAs (miRNAs) have emerged as a major regulator in neurological diseases, and understanding their molecular mechanism in modulating cerebral ischemic injury may provide potential therapeutic targets for ischemic stroke. However, as one of 19 differentially expressed miRNAs in mouse brain with middle cerebral artery occlusion (MCAO), the role of miR-134 in ischemic injury is not well understood. In this study, the miR-134 expression level was manipulated both in oxygen–glucose deprivation (OGD)-treated N2A neuroblastoma cells in vitro and mouse brain with MCAO-induced ischemic stroke in vivo, and its possible targets of heat shock protein A5 (HSPA5) and HSPA12B were determined by bioinformatics analysis and dual luciferase assay. The results showed that overexpression of miR-134 exacerbated cell death and apoptosis both in vitro and in vivo. Conversely, downregulating miR-134 levels reduced cell death and apoptosis. Furthermore, non-expression of miR-134 enhanced HSPA12B protein levels in OGD-treated N2A cells as well as in the ischemic region. It could attenuate brain infarction size and neural cell damage, and improve neurological outcomes in mice with ischemic stroke, whereas upregulation of miR-134 had the opposite effect. In addition, HSPA12B was validated to be a target of miR-134 and its short interfering RNAs (siRNAs) could block miR-134 inhibitor-induced neuroprotection in OGD-treated N2A cells. In conclusion, downregulation of miR-134 could induce neuroprotection against ischemic injury in vitro and in vivo by negatively upregulating HSPA12B protein expression.

Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis.

To compare proteomic profiles of spermatozoa from patients with varicocele and poor sperm quality before and after varicocelectomy. This work was designed as a prospective and observational study. The study was based on 20 men with varicocele grade 3 and poor sperm quality undergoing varicocelectomy at the Fertility Unit of Royan institute in 2009. Two semen samples were collected, one before varicocelectomy and the other after surgery. Protein separation was done by two-dimensional protein electrophoresis, and analyzed by gel densitometry and mass spectrometry. Differential sperm protein expression levels were measured by gel densitometry. Comparison of the sperm parameters showed that sperm motility and concentration were increased after varicocelectomy. At the level of protein, a total of 3 protein spots were identified whose expression was significantly lower in sperm samples before varicocelectomy compared with after surgery including heat shock protein A5 (HSPA5), superoxide dismutase 1 (SOD1) and δ-subunit of the catalytic core of mitochondrial adenosine triphosphate synthase (ATP5D). High grade varicocoele affects sperm protein expression presumably because of increasing testicular temperature. These proteins play essential roles in sperm production, DNA integrity protection, and sperm motility. This novel study demonstrates that varicocelectomy can improve both sperm quality and proteins expression.

Downregulation of miR-181b in mouse brain following ischemic stroke induces neuroprotection against ischemic injury through targeting heat shock protein A5 and ubiquitin carboxyl-terminal hydrolase isozyme L1

Understanding the molecular mechanism of cerebral hypoxic preconditioning (HPC)-induced endogenous neuroprotection may provide potential therapeutic targets for ischemic stroke. By using bioinformatics analysis, we found that miR-181b, one of 19 differentially expressed miRNAs, may target aconitate hydratase (ACO2), heat shock protein A5 (HSPA5), and ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) among 26 changed protein kinase C isoform-specific interacting proteins in HPC mouse brain. In this study, the role of miR-181b in oxygen-glucose deprivation (OGD)-induced N2A cell ischemic injury in vitro and mouse middle cerebral artery occlusion (MCAO)-induced cerebral ischemic injury in vivo, and its regulation of ACO2, HSPA5, and UCHL1 were further determined. We found that miR-181b expression levels significantly decreased in mouse brain following MCAO and in OGD-treated N2A cells. Up- and downregulation of miR-181b by transfection of pre- or anti-miR-181b could negatively regulate HSPA5 and UCHL1 (but not ACO2) protein levels as well as N2A cell death and programmed cell death in OGD-treated N2A cells. By using a T7 promoter-driven control dual luciferase assay, we confirmed that miR-181b could bind to the 3'-untranslated rergions of HSPA5 and UCHL1 mRNAs and repress their translations. miR-181b antagomir reduced caspase-3 cleavage and neural cell loss in cerebral ischemic cortex and improved neurological deficit of mice after MCAO. In addition, HSPA5 and UCHL1 short interfering RNAs (siRNAs) blocked anti-miR-181b-mediated neuroprotection against OGD-induced N2A cell injury in vitro. These results suggest that the downregulated miR-181b induces neuroprotection against ischemic injury through negatively regulating HSPA5 and UCHL1 protein levels, providing a potential therapeutic target for ischemic stroke.

Low level methylmercury enhances CNTF-evoked STAT3 signaling and glial differentiation in cultured cortical progenitor cells

Although many previous investigations have studied how mercury compounds cause cell death, sub-cytotoxic levels may affect mechanisms essential for the proper development of the nervous system. The present study investigates whether low doses of methylmercury (MeHg) and mercury chloride (HgCl2) can modulate the activity of JAK/STAT signaling, a pathway that promotes gliogenesis. We report that sub-cytotoxic doses of MeHg enhance ciliary neurotrophic factor (CNTF) evoked STAT3 phosphorylation in human SH-SY5Y neuroblastoma and mouse cortical neural progenitor cells (NPCs). This effect is specific for MeHg, since HgCl2 fails to enhance JAK/STAT signaling. Exposing NPCs to these low doses of MeHg (30–300nM) enhances CNTF-induced expression of STAT3-target genes such as glial fibrillary acidic protein (GFAP) and suppressors of cytokine signaling 3 (SOCS3), and increases the proportion of cells expressing GFAP following 2 days of differentiation. Higher, near-cytotoxic concentrations of MeHg and HgCl2 inhibit STAT3 phosphorylation and lead to increased production of superoxide. Lower concentrations of MeHg effective in enhancing JAK/STAT signaling (30nM) do not result in a detectable increase in superoxide nor increased expression of the oxidant-responsive genes, heme oxygenase 1, heat shock protein A5 and sirtuin 1. These findings suggest that low concentrations of MeHg inappropriately enhance STAT3 phosphorylation and glial differentiation, and that the mechanism causing this enhancement is distinct from the reactive oxygen species-associated cell death observed at higher concentrations of MeHg and HgCl2.