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International audience; Cystic Fibrosis is a lethal monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The most frequent mutation is the deletion of phenylalanine at position 508 of the protein. This F508del-CFTR mutation leads to misfolded protein that is detected by the quality control machinery within the endoplasmic reticulum and targeted for destruction by the proteasome. Modulating quality control proteins as molecular chaperones is a promising strategy for attenuating the degradation and stabilizing the mutant CFTR at the plasma membrane. Among the molecular chaperones, the small heat shock protein HspB1 and HspB4 were shown to promote degradation of F508del-CFTR. Here, we investigated the impact of HspB5 expression and phosphorylation on transport to the plasma membrane, function and stability of F508del-CFTR. We show that a phosphomimetic form of HspB5 increases the transport to the plasma membrane, function and stability of F508del-CFTR. These activities are further enhanced in presence of therapeutic drugs currently used for the treatment of cystic fibrosis (VX-770/Ivacaftor, VX-770+VX-809/Orkambi). Overall, this study highlights the beneficial effects of a phosphorylated form of HspB5 on F508del-CFTR rescue and its therapeutic potential in cystic fibrosis.

Background αB‐crystallin is a promiscuous protein involved in numerous cell functions. Mutations in CRYAB have been found in patients with different pathological phenotypes that are not properly understood. Patients can present different diseases like cataracts, muscle weakness, myopathy, cardiomyopathy, respiratory insufficiency or dysphagia, but also a variable combination of these pathologies has been found. These mutations can show either autosomal dominant or recessive mode of inheritance and variable penetrance and expressivity. This is the first report of congenital cataracts and myopathy described in childhood due to a CRYAB mutation with autosomal dominant mode of inheritance. Methods The whole exome sequence was subjected to phenotype‐driven analysis and a novel variant in CRYAB was detected: c.514delG, p.(Ala172ProfsTer14). The mutation was located in the C‐terminal domain of the protein, which is essential for chaperone activity. The deduced protein was analyzed searching for alterations of the relevant physico‐chemical properties described for this domain. A muscle biopsy was also tested for CRYAB with immunohistochemical and histoenzymatic techniques. Results CRYAB displayed a mild immunoreactivity in the subsarcolemmal compartment with no pathological sarcoplasmic accumulation. It agrees with an alteration of the physico‐chemical properties predicted for the C‐terminal domain: hydrophobicity, stiffness, and isomerization. Conclusions The described mutation leads to elongation of the protein at the carboxi‐terminal domain (CTD) with altered properties, which are essential for solubility and activity. It suggests that can be the cause of the severe conditions observed in this patient., The isolation of DNA and whole exome sequencing led us to discover a novel mutation in a CRYAB which causes cataracts, myopathies and other conditions observed in the proband. The predicted altered physico‐chemical properties of the C‐terminal are related with low activity and low solubility. This new condition agrees with the muscle biopsy where the cell is depleted and the mutated protein is found attached to the membrane cell.

AIMS: Multiple sclerosis (MS) is a chronic neurodegenerative disease characterised by demyelination, inflammation and neurodegeneration throughout the central nervous system. Although spinal cord pathology is an important factor contributing to disease progression, few studies have examined MS lesions in the spinal cord and how they differ from brain lesions. Here we have compared brain and spinal cord white and grey matter from MS and control tissues focussing on small heat shock proteins (HSPB) and HSP16.2.METHODS: Western blotting was used to examine protein levels of HSPB1, HSPB5, HSPB6, HSPB8 and HSP16.2 in brain and spinal cord from MS and age-matched non-neurological controls. Immunohistochemistry was used to examine expression of the HSPs in MS spinal cord lesions and controls. Expression levels were quantified using ImageJ.RESULTS: Western blotting revealed significantly higher levels of HSPB1, HSPB6 and HSPB8 in MS and control spinal cord compared to brain tissues. No differences in HSPB5 and HSP16.2 protein levels were observed although HSPB5 protein levels were higher in brain WM versus GM. In MS spinal cord lesions, increased HSPB1 and HSPB5 expression was observed in astrocytes, and increased neuronal expression of HSP16.2 was observed in normal appearing grey matter and type 1 grey matter lesions.CONCLUSIONS: The high constitutive expression of several HSPBs in spinal cord and increased expression of HSPBs and HSP16.2 in MS illustrate differences between brain and spinal cord in health and upon demyelination. Regional differences in HSP expression may reflect differences in astrocyte cytoskeleton composition and influence inflammation, possibly affecting the effectiveness of pharmacological agents. This article is protected by copyright. All rights reserved.

Myofibrillar myopathy (MFM) is a group of inherited muscular disorders characterized by myofibrils dissolution and abnormal accumulation of degradation products. So far causative mutations have been identified in nine genes encoding Z-disk proteins, including αB-crystallin (CRYAB), a small heat shock protein (also called HSPB5).Here, we report a case study of a 63-year-old Polish female with a progressive lower limb weakness and muscle biopsy suggesting a myofibrillar myopathy, and extra-muscular multisystemic involvement, including cataract and cardiomiopathy. Five members of the proband's family presented similar symptoms. Whole exome sequencing followed by bioinformatic analysis revealed a novel D109A mutation in CRYAB associated with the disease.Molecular modeling in accordance with muscle biopsy microscopic analyses predicted that D109A mutation influence both structure and function of CRYAB due to decreased stability of oligomers leading to aggregate formation. In consequence disrupted sarcomere cytoskeleton organization might lead to muscle pathology. We also suggest that mutated RQDE sequence of CRYAB could impair CRYAB chaperone-like activity and promote aggregation of lens crystallins.

The αB-crystallin (HSPB5) protein is modulated in response to a wide variety of stressors generated by multiple physio-pathological conditions, sustained by reactive oxygen species (ROS) production. In cardiac muscle tissue, this protein regulates various cellular processes, such as protein degradation, apoptosis and the stabilization of cytoskeletal elements. In this work, we studied the role of HSPB5 expression, activation and localization in HL-1 murine cardiomyocytes exposed to pro-oxidant and non-cytotoxic H2O2 concentration, as well as in cardiac tissue isolated from mice following an acute, non-damaging endurance exercise. Our results demonstrated that HSPB5 is the most abundant HSP in both cardiac muscle tissue and HL-1 cells when compared to HSPB1 or HSPA1A (≈3–8 fold higher protein concentrations, p < 0.01). The acute exposure of cardiac muscle cells to sustainable level of H2O2 “in vitro” or to aerobic non-damaging exercise “in vivo” determined a fast and specific increase of HSPB5 phosphorylation (from 3 up to 25 fold increase, p < 0.01) correlated to an increase in lipid peroxidation (p < 0.05). In both experimental models, p-HSPB5 likely facilitated both the interaction with β-actin, desmin, and α-Filamin 1, the last one identified as new HSPB5 substrate in cardiac cells, as well as the sub-localization of HSPB5 within the same cellular compartment or the re-localization between compartments (i.e., nucleus and cytosol). Taken together, these data point out the role of “oxidative eustress” induced by physiological conditions in activating the molecular machinery devoted to cardiomyocytes’ protection and candidate HSPB5 as a putative molecular mediator for the health benefits induced in cardiac tissue by exercise training.

Sensorineural hearing loss is the most common long-term deficit after pneumococcal meningitis (PM), occurring in up to 30% of surviving patients. The infection and the following overshooting inflammatory host response damage the vulnerable sensory cells of the inner ear, resulting in loss of hair cells and spiral ganglion neurons, ultimately leading to elevated hearing thresholds. Here, we tested the oto-protective properties of the small heat shock protein alpha B-crystallin (HspB5) with previously reported anti-inflammatory, anti-apoptotic and neuroprotective functions, in an experimental model of PM-induced hearing loss. We analyzed the effect of local and systemic delivery of HspB5 in an infant rat model of PM, as well as ex vivo, using whole mount cultures. Cytokine secretion profile, hearing thresholds and inner ear damage were assessed at predefined stages of the disease up to 1 month after infection. PM was accompanied by elevated pro-inflammatory cytokine concentrations in the cerebrospinal fluid (CSF), leukocyte and neutrophil infiltration in the perilymphatic spaces of the cochlea with neutrophils extracellular trap formation during the acute phase of the disease. Elevated hearing thresholds were measured after recovery from meningitis. Intracisternal but not intraperitoneal administration of HspB5 significantly reduced the levels of TNF-α, IL-6 IFN-γ and IL-10 in the acute phase of the disease. This resulted in a greater outer hair cell survival, as well as improved hearing thresholds at later stages. These results suggest that high local concentrations of HspB5 are needed to prevent inner ear damage in acute PM. HspB5 represents a promising therapeutic option to improve the auditory outcome and counteract hearing loss after PM.

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by &beta, cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer&rsquo, s disease, alone or co-deposited with &beta, amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal &ldquo, cross-talk&rdquo, We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17&ndash, 29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17&ndash, 29 (3 &micro, M) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17&ndash, 29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17&ndash, 29 (20 &micro, M). We found a complete inhibition of hA17&ndash, 29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Activated microglia and macrophages play a key role in driving demyelination during multiple sclerosis (MS), but the factors responsible for their activation remain poorly understood. Here, we present evidence for a dual-trigger role of IFN-gamma and alpha B-crystallin (HSPB5) in this context. In MS-affected brain tissue, accumulation of the molecular chaperone HSPB5 by stressed oligodendrocytes is a frequent event. We have shown before that this triggers a TLR2-mediated protective response in surrounding microglia, the molecular signature of which is widespread in normal-appearing brain tissue during MS. Here, we show that IFN-gamma, which can be released by infiltrated T cells, changes the protective response of microglia and macrophages to HSPB5 into a robust pro-inflammatory classical response. Exposure of cultured microglia and macrophages to IFN-gamma abrogated subsequent IL-10 induction by HSPB5, and strongly promoted HSPB5-triggered release of TNF-alpha, IL-6, IL-12, IL-1 beta and reactive oxygen and nitrogen species. In addition, high levels of CXCL9, CXCL10, CXL11, several guanylate-binding proteins and the ubiquitin-like protein FAT10 were induced by combined activation with IFN-gamma and HSPB5. As immunohistochemical markers for microglia and macrophages exposed to both IFN-gamma and HSPB5, these latter factors were found to be selectively expressed in inflammatory infiltrates in areas of demyelination during MS. In contrast, they were absent from activated microglia in normal-appearing brain tissue. Together, our data suggest that inflammatory demyelination during MS is selectively associated with IFN-gamma-induced re-programming of an otherwise protective response of microglia and macrophages to the endogenous TLR2 agonist HSPB5.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal unstressed cells as well as in many cancer cells where they are over-expressed. These proteins are characterized by cell physiology dependent changes in their oligomerization and phosphorylation status. These structural changes allow them to interact with many different client proteins that subsequently display modified activity and/or half-life. Nowdays, the protein interactomes of small Hsps are under intense investigations and will represent, when completed, key parameters to elaborate therapeutic strategies aimed at modulating the functions of these chaperones. Here, we have analyzed the potential pro-cancerous roles of several client proteins that have been described so far to interact with HspB1 (Hsp27) and its close members HspB5 (αB-crystallin) and HspB4 (αA-crystallin).

Aims: Molecular chaperones constitute protectors of intracellular protein integrity and seem to confer short-term defence against various cell insults. Myocardial damage is associated to a loss of protective chaperones. Ischemic post-conditioning (IPost-Co) is a procedure that seems to protect against reperfusion injury. However, little is known on alpha-crystallin-B-chain (cryab/HspB5) evolution in IPost-Co. Here we have investigated cryab in myocardial ischemia and IPost-Co. Methods and results: Pigs underwent closed-chest 1.5 h mid-left anterior descending (LAD) balloon occlusion and were either sacrificed without reperfusion (I; N = 10), subjected to 2.5 h of reperfusion and sacrificed (I/R; N = 5); or subjected to IPost-Co before reperfusion and sacrificed 2.5 h afterwards (IPost-Co; N = 5). A sham-operated group was included (N = 6). Proteomic analysis (2-D-electrophoresis/MALDI-TOF/TOF) revealed cryab as a single spot (20 kDa; pI7.6). Myocardial cryab-20-protein and cryab-gene expression levels were decreased after ischemia and I/R(P < 0.05). After IPost-Co, cryab-20-protein and cryab-gene expression levels were similar to those found in the heart of sham-operated animals (P < 0.05). There was a direct correlation between LVEF-improvement after IPost-Co and myocardial cryab-20-protein levels. In a mice proof-of-principle study, cryab-20-peptide was synthesized and administered 1 h before LAD-ligation and ECG-proven MI. A 59% reduction in infarct size was achieved in cryab-20-treated animals (P < 0.05). Conclusions: Ischemia and reperfusion induce a decrease in myocardial cryab-20-protein levels together with a clinical impairment of cardiac function. IPost-Co induces a clinical improvement of cardiac function and a preservation of cryab-20 levels. Intervention studies on amice-MI model showed that cryab-20-peptide administration reduces infarct size. All together our results show a significant cardioprotective effect of cryab. (C) 2016 Elsevier Ireland Ltd. All rights reserved.

Contains fulltext : 137883.pdf (Publisher’s version ) (Open Access) BACKGROUND: The presence of hypoxia in head and neck squamous cell carcinoma (HNSCC) is associated with therapeutic resistance and increased risk of metastasis formation. alphaB-crystallin (HspB5) is a small heat shock protein, which is also associated with metastasis formation in HNSCC. In this study, we investigated whether alphaB-crystallin protein expression is increased in hypoxic areas of HNSCC biopsies and analyzed whether hypoxia induces alphaB-crystallin expression in vitro and in this way may confer hypoxic cell survival. METHODS: In 38 HNSCC biopsies, the overlap between immunohistochemically stained alphaB-crystallin and pimonidazole-adducts (hypoxiamarker) was determined. Moreover, expression levels of alphaB-crystallin were analyzed in HNSCC cell lines under hypoxia and reoxygenation conditions and after exposure to reactive oxygen species (ROS) and the ROS scavenger N-acetylcysteine (NAC). siRNA-mediated knockdown was used to determine the influence of alphaB-crystallin on cell survival under hypoxic conditions. RESULTS: In all biopsies alphaB-crystallin was more abundantly present in hypoxic areas than in normoxic areas. Remarkably, hypoxia decreased alphaB-crystallin mRNA expression in the HNSCC cell lines. Only after reoxygenation, a condition that stimulates ROS formation, alphaB-crystallin expression was increased. alphaB-crystallin mRNA levels were also increased by extracellular ROS, and NAC abolished the reoxygenation-induced alphaB-crystallin upregulation. Moreover, it was found that decreased alphaB-crystallin levels reduced cell survival under hypoxic conditions. CONCLUSIONS: We provide the first evidence that hypoxia stimulates upregulation of alphaB-crystallin in HNSCC. This upregulation was not caused by the low oxygen pressure, but more likely by ROS formation. The higher expression of alphaB-crystallin may lead to prolonged survival of these cells under hypoxic conditions.

Radostina V Cherneva,1 Ognian B Georgiev,1 Daniela S Petrova,1 Nedka L Trifonova,2 Maria Stamenova,2 Vesela I Ivanova,3 Veselin I Vlasov31Clinic of Internal Medicine, Division of Pulmonology, University Hospital Alexandrovska, Sofia, Bulgaria; 2Department of Biology, 3Department of Pathology, Medical University of Sofia, Sofia, BulgariaBackground: αB-crystallin (HspB5) is a chaperone whose role as a marker of innate immunity activation as well as its therapeutic potential have recently been investigated in several inflammatory diseases: multiple sclerosis, myocardial ischemia, and Guillain–Barré syndrome.Aim: The aim of this study is to determine the role of αB-crystallin in chronic obstructive pulmonary disease (COPD) pathogenesis and inflammation.Materials: Plasma levels of αB-crystallin were studied in 163 patients: 52 healthy non-COPD smokers; 20 COPD smokers in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I–II; 43 COPD smokers in GOLD stages III-IV. Forty-eight patients were diagnosed with acute inflammatory respiratory disease. The plasma levels of αB-crystallin antibodies were determined by an enzyme-linked immunosorbent assay (Calbiochem), and were confirmed with Western blotting. Tissue expression of the protein was compared in three different groups of patients: COPD smokers, COPD nonsmokers, and in patients with age-related emphysema.Results: The mean level of anti-αB-crystallin antibodies in non-COPD smokers was 0.291nm. In COPD smokers it was 0.352 nm and, in patients with inflammatory lung diseases, 0.433 nm. in patients with inflammatory lung diseases. There was a statistically significant difference between COPD smokers and healthy non-COPD smokers (P = 0.010). The same could be observed comparing the group of patients with acute inflammation and non-COPD healthy smokers (P = 0.007). There was no statistically significant difference between patients with mild/moderate inflammation and those with severe COPD. Tissue detection of the protein showed that it was significantly overexpressed in COPD smokers in comparison to COPD nonsmokers and was only slightly expressed in patients with age-related emphysema.Conclusion: αB-crystallin is increased in patients with inflammatory lung diseases. Though unspecific, it could be used in a panel of markers discerning COPD smokers from healthy nonsmokers. As αB-crystallin is a regulator of innate immunity and a therapeutic anti-inflammatory agent, its exact role in COPD pathogenesis and therapy should be explored further.Keywords: COPD, HspB5, chaperonopathology, pathogenesis

Historically, small heat shock proteins (sHSPs) have been extensively studied in the context of being intracellular molecular chaperones. However, recent studies looking at the role of sHSPs in neurological diseases have demonstrated a near universal upregulation of certain sHSPs in damaged and diseased brains. Initially, it was thought that sHSPs are pathological in these disease states because they are found in the areas of damage. However, transgenic overexpression and exogenous administration of sHSPs in various experimental disease paradigms have shown just the contrary – that sHSPs are protective, not pathological. This review examines sHSPs in neurological diseases and highlights the potential for using these neuroprotective sHSPs as novel therapeutics. It first addresses the endogenous expression of sHSPs in a variety of neurological disorders. Although many studies have examined the expression of sHSPs in neurological diseases, there are no review articles summarizing these data. Furthermore, it focuses on recent studies that have investigated the therapeutic potential of sHSPs for neurological diseases. Finally, it will explain what we think is the function of endogenous sHSPs in neurological diseases.