O-GlcNAc modification of HSP27 alters its protein interactions and promotes refolding of proteins through the BAG3/HSP70 co-chaperone.

Almost all types of cellular stress induce post-translational O-GlcNAc modifications of proteins, and this increase promotes cell survival. We previously demonstrated that O-GlcNAc on certain small heat shock proteins (sHSPs), including HSP27, directly increases their chaperone activity as one potential protective mechanism. Here, we furthered our use of synthetic proteins to prepare biotinylated sHSPs and show that O-GlcNAc modification of HSP27 also changes how it interacts within the sHSP system and the broader HSP network. Specifically, we show that O-GlcNAc modified HSP27 binds more strongly to the co-chaperone protein BAG3, which then promotes refolding of a model substrate by HSP70. We use proteomics to identify other potential HSP27 interactions that are changed by O-GlcNAc, including one that we confirm with another sHSP, αB-crystallin. These findings add additional evidence for O-GlcNAc as a switch for regulating protein-protein interactions and for modifications of chaperones as one mechanism by which O-GlcNAc protects against protein aggregation.

Generation of human induced pluripotent stem cell (hiPSC) lines derived from three patients carrying the pathogenic CRYAB (A527G) mutation and one non-carrier family member.

A newly identified pathogenic variant (A527G) in alpha B-crystallin (αB-crystallin) has been linked to congenital cataract and young-onset dilated cardiomyopathy (DCM) within a Dutch family, although the disease mechanism remains unclear. Four human induced pluripotent stem cell (hiPSC) clones were generated from three symptomatic patients carrying the A527G variant, and one healthy proband. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai viral pluripotency vectors. The established hiPSCs clones exhibited regular ESC-like morphology, expression of pluripotency markers, and normal karyotyping. These hiPSC lines can facilitate future studies to understand the chaperone function and its role in DCM disease progression.

Modulation of Alzheimer's Disease Aß40 Fibril Polymorphism by the Small Heat Shock Protein αB-Crystallin.

Deposition of amyloid plaques in the brains of Alzheimer's disease (AD) patients is a hallmark of the disease. AD plaques consist primarily of the beta-amyloid (Aß) peptide but can contain other factors such as lipids, proteoglycans, and chaperones. So far, it is unclear how the cellular environment modulates fibril polymorphism and how differences in fibril structure affect cell viability. The small heat-shock protein (sHSP) alpha-B-Crystallin (αBC) is abundant in brains of AD patients, and colocalizes with Aß amyloid plaques. Using solid-state NMR spectroscopy, we show that the Aß40 fibril seed structure is not replicated in the presence of the sHSP. αBC prevents the generation of a compact fibril structure and leads to the formation of a new polymorph with a dynamic N-terminus. We find that the N-terminal fuzzy coat and the stability of the C-terminal residues in the Aß40 fibril core affect the chemical and thermodynamic stability of the fibrils and influence their seeding capacity. We believe that our results yield a better understanding of how sHSP, such as αBC, that are part of the cellular environment, can affect fibril structures related to cell degeneration in amyloid diseases.

CRYAB suppresses ferroptosis and promotes osteogenic differentiation of human bone marrow stem cells via binding and stabilizing FTH1.

BACKGROUND: Bone formation and homeostasis are greatly dependent on the osteogenic differentiation of human bone marrow stem cells (BMSCs). Therefore, revealing the mechanisms underlying osteogenic differentiation of BMSCs will provide new candidate therapeutic targets for osteoporosis. METHODS: The osteogenic differentiation of BMSCs was measured by analyzing ALP activity and expression levels of osteogenic markers. Cellular Fe and ROS levels and cell viability were applied to evaluate the ferroptosis of BMSCs. qRT-PCR, Western blotting, and co-immunoprecipitation assays were harnessed to study the molecular mechanism. RESULTS: The mRNA level of CRYAB was decreased in the plasma of osteoporosis patients. Overexpression of CRYAB increased the expression of osteogenic markers including OCN, OPN, RUNX2, and COLI, and also augmented the ALP activity in BMSCs, on the contrary, knockdown of CRYAB had opposite effects. IP-MS technology identified CRYAB-interacted proteins and further found that CRYAB interacted with ferritin heavy chain 1 (FTH1) and maintained the stability of FTH1 via the proteasome mechanism. Mechanically, we unraveled that CRYAB regulated FTH1 protein stability in a lactylation-dependent manner. Knockdown of FTH1 suppressed the osteogenic differentiation of BMSCs, and increased the cellular Fe and ROS levels, and eventually promoted ferroptosis. Rescue experiments revealed that CRYAB suppressed ferroptosis and promoted osteogenic differentiation of BMSCs via regulating FTH1. The mRNA level of FTH1 was decreased in the plasma of osteoporosis patients. CONCLUSIONS: Downregulation of CRYAB boosted FTH1 degradation and increased cellular Fe and ROS levels, and finally improved the ferroptosis and lessened the osteogenic differentiation of BMSCs.

Biological effects and mechanism of ß-amyloid aggregation inhibition by penetrable recombinant human HspB5-ACD structural domain protein.

Alzheimer's disease (AD) is a global medical challenge. Studies have shown that neurotoxicity caused by pathological aggregation of ß-amyloid (Aß) is an important factor leading to AD. Therefore, inhibiting the pathological aggregation of Aß is the key to treating AD. The recombinant human HspB5-ACD structural domain protein (AHspB5) prepared by our group in the previous period has been shown to have anti-amyloid aggregation effects, but its inability to penetrate biological membranes has limited its development. In this study, we prepared a recombinant fusion protein (T-AHspB5) of TAT and AHspB5. In vitro experiments showed that T-AHspB5 inhibited the formation of Aß1-42 protofibrils and had the ability to penetrate the blood-brain barrier; in cellular experiments, T-AHspB5 prevented Aß1-42-induced oxidative stress damage, apoptosis, and inflammatory responses in neuronal cells, and its mechanism of action was related to microglia activation and mitochondria-dependent apoptotic pathway. In animal experiments, T-AHspB5 improved memory and cognitive dysfunction and inhibited pathological changes of AD in APP/PS1 mice. In conclusion, this paper is expected to reveal the intervention mechanism and biological effect of T-AHspB5 on pathological aggregation of Aß1-42, provide a new pathway for the treatment of AD, and lay the foundation for the future development and application of T-AHspB5.

Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation.

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Understanding the structural and functional changes and biochemical pathomechanism of the cardiomyopathy-associated p.R123W mutation in human αB-crystallin.

αB-crystallin, a member of the small heat shock protein (sHSP) family, is expressed in diverse tissues, including the eyes, brain, muscles, and heart. This protein plays a crucial role in maintaining eye lens transparency and exhibits holdase chaperone and anti-apoptotic activities. Therefore, structural and functional changes caused by genetic mutations in this protein may contribute to the development of disorders like cataract and cardiomyopathy. Recently, the substitution of arginine 123 with tryptophan (p.R123W mutation) in human αB-crystallin has been reported to trigger cardiomyopathy. In this study, human αB-crystallin was expressed in Escherichia coli (E. coli), and the missense mutation p.R123W was created using site-directed mutagenesis. Following purification via anion exchange chromatography, the structural and functional properties of both proteins were investigated and compared using a wide range of spectroscopic and microscopic methods. The p.R123W mutation induced significant alterations in the secondary, tertiary, and quaternary structures of human αB-crystallin. This pathogenic mutation resulted in an increased ß-sheet structure and formation of protein oligomers with larger sizes compared to the wild-type protein. The mutant protein also exhibited reduced chaperone activity and lower thermal stability. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) demonstrated that the p.R123W mutant protein is more prone to forming amyloid aggregates. The structural and functional changes observed in the p.R123W mutant protein, along with its increased propensity for aggregation, could impact its proper functional interaction with the target proteins in the cardiac muscle, such as calcineurin. Our results provide an explanation for the pathogenic intervention of p.R123W mutant protein in the occurrence of hypertrophic cardiomyopathy (HCM).

Phosphorylation of αB-Crystallin Involves Interleukin-1ß-Mediated Intracellular Retention in Retinal Müller Cells: A New Mechanism Underlying Fibrovascular Membrane Formation.

Purpose: Chronic inflammation plays a pivotal role in the pathology of proliferative diabetic retinopathy (PDR), in which biological alterations of retinal glial cells are one of the key elements. The phosphorylation of αB-crystallin/CRYAB modulates its molecular dynamics and chaperone activity, and attenuates αB-crystallin secretion via exosomes. In this study, we investigated the effect of phosphorylated αB-crystallin in retinal Müller cells on diabetic mimicking conditions, including interleukin (IL)-1ß stimuli. Methods: Human retinal Müller cells (MIO-M1) were used to examine gene and protein expressions with real-time quantitative PCR, enzyme linked immunosorbent assay (ELISA), and immunoblot analyses. Cell apoptosis was assessed by Caspase-3/7 assay and TdT-mediated dUTP nick-end labeling staining. Retinal tissues isolated from the Spontaneously Diabetic Torii (SDT) fatty rat, a type 2 diabetic animal model with obesity, and fibrovascular membranes from patients with PDR were examined by double-staining immunofluorescence. Results: CRYAB mRNA was downregulated in MIO-M1 cells with the addition of 10 ng/mL IL-1ß; however, intracellular αB-crystallin protein levels were maintained. The αB-crystallin serine 59 (Ser59) residue was phosphorylated with IL-1ß application in MIO-M1 cells. Cell apoptosis in MIO-M1 cells was induced by CRYAB knockdown. Immunoreactivity for Ser59-phosphorylated αB-crystallin and glial fibrillary acidic protein was colocalized in glial cells of SDT fatty rats and fibrovascular membranes. Conclusions: The Ser59 phosphorylation of αB-crystallin was modulated by IL-1ß in Müller cells under diabetic mimicking inflammatory conditions, suggesting that αB-crystallin contributes to the pathogenesis of PDR through an anti-apoptotic effect.

Human Mutated MYOT and CRYAB Genes Cause a Myopathic Phenotype in Zebrafish.

Myofibrillar myopathies (MFMs) are a group of hereditary neuromuscular disorders sharing common histological features, such as myofibrillar derangement, Z-disk disintegration, and the accumulation of degradation products into protein aggregates. They are caused by mutations in several genes that encode either structural proteins or molecular chaperones. Nevertheless, the mechanisms by which mutated genes result in protein aggregation are still unknown. To unveil the role of myotilin and αB-crystallin in the pathogenesis of MFM, we injected zebrafish fertilized eggs at the one-cell stage with expression plasmids harboring cDNA sequences of human wildtype or mutated MYOT (p.Ser95Ile) and human wildtype or mutated CRYAB (p.Gly154Ser). We evaluated the effects on fish survival, motor behavior, muscle structure and development. We found that transgenic zebrafish showed morphological defects that were more severe in those overexpressing mutant genes. which developed a myopathic phenotype consistent with that of human myofibrillar myopathy, including the formation of protein aggregates. Results indicate that pathogenic mutations in myotilin and αB-crystallin genes associated with MFM cause a structural and functional impairment of the skeletal muscle in zebrafish, thereby making this non-mammalian organism a powerful model to dissect disease pathogenesis and find possible druggable targets.

Disordered region encodes α-crystallin chaperone activity toward lens client γD-crystallin.

Small heat-shock proteins (sHSPs) are a widely expressed family of ATP-independent molecular chaperones that are among the first responders to cellular stress. Mechanisms by which sHSPs delay aggregation of client proteins remain undefined. sHSPs have high intrinsic disorder content of up to ~60% and assemble into large, polydisperse homo- and hetero-oligomers, making them challenging structural and biochemical targets. Two sHSPs, HSPB4 and HSPB5, are present at millimolar concentrations in eye lens, where they are responsible for maintaining lens transparency over the lifetime of an organism. Together, HSPB4 and HSPB5 compose the hetero-oligomeric chaperone known as α-crystallin. To identify the determinants of sHSP function, we compared the effectiveness of HSPB4 and HSPB5 homo-oligomers and HSPB4/HSPB5 hetero-oligomers in delaying the aggregation of the lens protein γD-crystallin. In chimeric versions of HSPB4 and HSPB5, chaperone activity tracked with the identity of the 60-residue disordered N-terminal regions (NTR). A short 10-residue stretch in the middle of the NTR ("Critical sequence") contains three residues that are responsible for high HSPB5 chaperone activity toward γD-crystallin. These residues affect structure and dynamics throughout the NTR. Abundant interactions involving the NTR Critical sequence reveal it to be a hub for a network of interactions within oligomers. We propose a model whereby the NTR critical sequence influences local structure and NTR dynamics that modulate accessibility of the NTR, which in turn modulates chaperone activity.

The small heat shock protein αB-Crystallin protects versus withaferin A-induced apoptosis and confers a more metastatic phenotype in cisplatin-resistant ovarian cancer cells.

Since a majority of ovarian tumors recur in a drug-resistant form leaving patients few treatment options, the goal of this study was to explore phenotypic and molecular characteristics of a cisplatin-resistant ovarian cancer cell line (OVCAR8R) as compared to its cisplatin-sensitive syngeneic counterpart (OVCAR8) and to explore the effectiveness of a novel chemotherapeutic, Withaferin A (WA). In addition to unique morphological characteristics, the small heat shock proteins (Hsps) αB-Crystallin (HspB5) and Hsp27 are constitutively expressed along with increased expression of vimentin in OVCAR8R cells, while OVCAR8 cells do not endogenously express these Hsps, supporting that Hsp overexpression may confer resistance to chemotherapy and promote more aggressive tumor types. WA increases apoptosis in a dose-dependent manner in OVCAR8 cells, while OVCAR8R cells remain more viable at comparable doses of WA coincident with the upregulation of αB-Crystallin. To determine the significance of αB-Crystallin in conferring a more aggressive phenotype, αB-Crystallin was silenced by CRISPR-Cas9 in OVCAR8R cells. The morphology of the OVCAR8R clones in which αB-Crystallin was silenced reverted to the morphology of the original cisplatin-sensitive OVCAR8 cells. Further, cisplatin-resistant OVCAR8R cells constitutively express higher levels of vimentin and migrate more readily than cisplatin-sensitive OVCAR8 and OVCAR8R cells in which αB-Crystallin was silenced. Transient overexpression of wildtype αB-Crystallin, but not a chaperone-defective-mutant, alters the morphology of these cells to closely resemble the cisplatin-resistant OVCAR8R cells and protects versus WA-induced apoptosis. Together, this research supports the potential effectiveness of WA as a therapy for ovarian cancer cells that have not yet acquired resistance to platinum-based therapies, and importantly, underscores that αB-Crystallin contributes to a more aggressive cellular phenotype and as such, may be a promising molecular target for a better clinical outcome.

HSPB5 suppresses renal inflammation and protects lupus-prone NZB/W F1 mice from severe renal damage.

BACKGROUND: Lupus nephritis (LN) is an inflammatory disease of the kidneys affecting patients with systemic lupus erythematosus. Current immunosuppressive and cytotoxic therapies are associated with serious side effects and fail to protect 20-40% of LN patients from end-stage renal disease. In this study, we investigated whether a small heat shock protein, HSPB5, can reduce kidney inflammation and the clinical manifestations of the disease in NZB/W F1 mice. Furthermore, we investigated whether HSPB5 can enhance the effects of methylprednisolone, a standard-of-care drug in LN, in an endotoxemia mouse model. METHODS: NZB/W F1 mice were treated with HSPB5, methylprednisolone, or vehicle from 23 to 38 weeks of age. Disease progression was evaluated by weekly proteinuria scores. At the end of the study, the blood, urine, spleens, and kidneys were collected for the assessment of proteinuria, blood urea nitrogen, kidney histology, serum IL-6 and anti-dsDNA levels, immune cell populations, and their phenotypes, as well as the transcript levels of proinflammatory chemokine/cytokines in the kidneys. HSPB5 was also evaluated in combination with methylprednisolone in a lipopolysaccharide-induced endotoxemia mouse model; serum IL-6 levels were measured at 24 h post-endotoxemia induction. RESULTS: HSPB5 significantly reduced terminal proteinuria and BUN and substantially improved kidney pathology. Similar trends, although to a lower extent, were observed with methylprednisolone treatment. Serum IL-6 levels and kidney expression of BAFF, IL-6, IFNγ, MCP-1 (CCL2), and KIM-1 were reduced, whereas nephrin expression was significantly preserved compared to vehicle-treated mice. Lastly, splenic Tregs and Bregs were significantly induced with HSPB5 treatment. HSPB5 in combination with methylprednisolone also significantly reduced serum IL-6 levels in endotoxemia mice. CONCLUSIONS: HSPB5 treatment reduces kidney inflammation and injury, providing therapeutic benefits in NZB/W F1 mice. Given that HSPB5 enhances the anti-inflammatory effects of methylprednisolone, there is a strong interest to develop HSBP5 as a therapeutic for the treatment of LN.

Effects of the CRYAB gene on stem cell-like properties of colorectal cancer and its mechanism.

Aims: Alpha B-crystallin (CRYAB), a known molecular chaperone, is involved in the occurrence and development of various tumor types. However, the function of CRYAB in colorectal cancer stem cells (CSCs) remains unknown. This study aimed to elucidate the role and possible regulatory mechanisms of CRYAB in the cancer stem cell-like phenotype of colorectal cancer (CRC). Subjects and Methods: The expression of CRYAB in patients with CRC and lymph node metastasis at various stages and its relationship with overall survival were detected using the TCGA database. In this study, CRC-CSCs were enriched from HCT116 and Caco2 cells with serum-free suspension culture. The CRYAB gene, stemness-related genes, and mesenchymal markers were detected via quantitative real-time PCR (qRT-PCR) in CRC cells. Then, CRYAB-HCT116S and CRYAB-Caco2S cell lines were established by lentivirus-mediated overexpression of CRYAB. Self-renewal ability and stemness features were measured by the sphere formation assay and flow cytometry. The tumorigenesis capacity in vivo was inspected in nude mice. The functions of CRYAB on CSC proliferation, migration, and invasion were examined using colony formation and the transwell assay. Finally, the Wnt/ß-catenin pathway-related mRNAs and proteins were detected via qRT-PCR and western blotting. Results: The expression of CRYAB in CRC is related to the clinical phase and prognosis, except with lymphoid metastasis. CRYAB expression was elevated in CSCs. Upregulation of CRYAB enhanced the expression of CSC-related genes and mesenchymal markers. The capacity to form colonospheres, tumorigenesis, cell proliferation, and metastasis were significantly advanced in CRYAB-overexpressed cells. Moreover, CRYAB dramatically suppressed ß-catenin degradation and downregulated the expression of p-GSK-3ß. Conclusions: CRYAB maintains CSC formation via the Wnt/ß-catenin pathway in CRCs, which may, therefore, function as vital molecular targets for CRC therapeutic strategies.

CRYAB reduces cigarette smoke-induced inflammation, apoptosis, and oxidative stress by retarding PI3K/Akt and NF-κB signaling pathways in human bronchial epithelial cells.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a familiar airway disease characterized by chronic immune response in the lungs. More and more evidences have assured that cigarette smoking is the primary reason for the progression of COPD, but its related regulatory mechanism requires further clarification. The α-B-crystallin (CRYAB) has been identified to exhibit vital functions in different diseases, and is down-regulated in the alveoli of mice mediated by cigarette smoke extract (CSE). METHODS: The messenger RNA expression of CRYAB was assessed by reverse transcription--quantitative polymerase chain reaction. The proteins' expressions were tested using Western blot method. The cytotoxicity was measured by lactate dehydrogenase assay. The levels of malondialdehyde, superoxide dismutase, catalase, myeloperoxidase, tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 were assessed through enzyme-linked-immunosorbent serologic assay (ELISA). RESULTS: In this study, it was discovered that the expression of CRYAB was markedly decreased with the increased time of cigarette smoking. Moreover, CRYAB overexpression increased cell viability and decreased cell apoptosis induced by cigarette smoke. In addition, the strengthened oxidative stress and inflammation mediated by CSE treatment was relieved after overexpression of CRYAB. Eventually, results OF Western blot method confirmed that CRYAB retarded the activation of phosphatidylinositol 3-kinase-Ak strain transforming (PI3K-Akt) and nuclear factor kappa B (NF-κB) signaling pathways. CONCLUSION: Our results manifested that CRYAB reduced cigarette smoke-induced inflammation, apoptosis, and oxidative stress in normal and diseased bronchial epithelial (NHBE) and human bronchial epithelial (BEAS-2B) cells by suppressing PI3K/Akt and NF-κB signaling pathways, which highlighted the functioning of CRYAB in preventing or treating COPD.

Exploitation of High Tumour GSH Levels for Targeted siRNA Delivery in Rhabdomyosarcoma Cells.

Metastatic alveolar rhabdomyosarcoma (aRMS) is an aggressive paediatric cancer with a poor prognosis. Downregulation of critical tumour genes using targeted siRNA remains an obstacle, but association with nanoparticles could help to deliver, protect, target, and enhance penetration. siRNA towards two genes was investigated: (i) Human αB-crystallin (CRYAB) and Heat Shock Protein Family B (Small) Member 2 (HSPB2), and (ii) Keratin 17 (KRT17). A mesoporous silica based nanosystem was linked to siRNA via disulfide bonds and loaded with IR820 dye. Transfection efficiency and signalling was evaluated, and the metabolic effects and cell proliferation were monitored in 2D culture and 3D spheroid models. The bound siRNA was protected from degradation with RNase I for at least 24 h. The delivered siRNA showed significant suppression of viability; 53.21 ± 23.40% for CRYAB and HSPB2 siRNA, and 88.06 ± 17.28% for KRT17 siRNA. After 72 h this increased to >50% cell apoptosis and necrosis. Intracellular total glutathione (GSH) levels were also compared with fibroblasts, and the RMS cell lines showed a several-fold increase. IR820 cellular uptake rate and penetration depth was significantly improved by nanoparticle delivery. Targetted siRNA delivery may pave the way for less invasive and more effective treatments of aRMS.

Effects of methanol and formic acid on CRYB, ALDH2, and ATP5A1 of RGCs.

OBJECTIVE: To explore the toxicity of methanol and its metabolite, formic acid on αB-crystallin(CRYB), aldehyde dehydrogenase (ALDH2), and ATPsynthase (ATP5A1) of rat retinal ganglion cells (RGCs). METHODS: RGCs are cultured in vitro in a toxic environment with 15/30/60 mM methanol or formic acid, respectively. Then, the morphological changes of RGCs and protein and mRNA levels of ALDH2, ATP5A1, and CRYB in rat RGCs were evaluated. RESULTS: 1) Compared to the toxicity of 15 mM formic acid on RGCs, 30 mM of formic acid environment significantly promoted apoptosis, and cell death occurred in the 60-mM formic acid group 24 h later. The toxicity of methanol for inducing apoptosis was not as obvious as formic acid. 2) In the 15-mM group, the level of CRYB protein was down-regulated after stimulating with both methanol and formic acid for 48 h, and ATP5A1 protein level decreased significantly with formic but not methanol. No change in ALDH2 was observed in methanol or formic acid. With a prolonged duration (>7 d) or high concentration (>30 mM) stimulation, cells treated with both methanol and formic acid showed severe apoptosis, rendering it challenging to collect a sufficient number of cells for protein detection. 3) In the 48-h group, no significant effect was detected on the mRNA of CRYB, ATP5A1, and ALDH2 by both 15/30 mM formic acid and 15 mM methanol. Conversely, 30 mM methanol had a significant up-regulation effect on the expression of the three genes, while no significant effect was observed in the 7-d groups. CONCLUSIONS: Formic acid exerted stronger toxicity on CRYB, ATP5A1, and ALDH2 than methanol and played a regulatory role at the translation level, while the effect of methanol is still uncertain, needing additional investigation.

Effect of Trehalose on Oligomeric State and Anti-Aggregation Activity of αB-Crystallin.

αB-Crystallin (αB-Cr), one of the main crystalline lens proteins, along with other crystallins maintains lens transparency suppressing protein aggregation and thus preventing cataractogenesis. αB-Cr belongs to the class of molecular chaperones; being expressed in many tissues it has a dynamic quaternary structure, which is essential for its chaperone-like activity. Shift in the equilibrium between ensembles of oligomers of different size allows regulating the chaperone activity. Trehalose is known to inhibit protein aggregation in vivo and in vitro, and it is widely used in biotechnology. The results of studying the effect of trehalose on the chaperone-like activity of crystallins can serve as a basis for the design of drugs delaying cataractogenesis. We have studied the trehalose effect on the quaternary structure and anti-aggregation activity of αB-Cr using muscle glycogen phosphorylase b (Phb) as a target protein. According to the dynamic light scattering data, trehalose affects the nucleation stage of Phb thermal aggregation at 48°C, and an increase in the αB-Cr adsorption capacity (AC0) is the main effect of trehalose on the aggregation process in the presence of the protein chaperone (AC0 increases 1.5-fold in the presence of 66 mM trehalose). According to the sedimentation analysis data, trehalose stabilizes the dimeric form of Phb at the stages of denaturation and dissociation and enhances the interaction of αB-Cr with the target protein. Moreover, trehalose shifts the equilibrium between the αB-Cr oligomers towards the smaller forms. Thus, trehalose affects the quaternary structure of αB-Cr and increases its anti-aggregation activity at the nucleation stage.

Restoration of miR-23a expression by chidamide sensitizes CML cells to imatinib treatment with concomitant downregulation of CRYAB.

MicroRNAs (miRNAs) are involved in various processes from the initiation and development of cancers, including chronic myeloid leukemia (CML). In this report, we aimed to investigate the roles of miR-23a in the regulation of imatinib mesylate (IM) sensitivity in CML cells and the possible mechanisms involved in this process. We demonstrated that the expression of miR-23a was markedly low in bone marrow mononuclear cells from patients in whom IM treatment had failed and imatinib-resistant K562/G01 cells when compared to patients with optimal responses and imatinib-sensitive K562 cells, respectively. Overexpression of miR-23a was shown to induce apoptosis of K562/G01 cells and sensitize these cells to imatinib treatment. With the aid of bioinformatics analysis, we revealed that CRYAB could be a potential downstream effector of miR-23a, contributing to miR-23a-mediated IM resistance. We also observed that the expression of CRYAB was inversely correlated with miR-23a expression in CML cell lines and patient samples. Importantly, chidamide upregulated miR-23a expression and reversed the IM resistance of CML cells. Together, these findings strongly suggest that miR-23a acts as a tumor suppressor by downregulating CRYAB expression. Restoration of miR-23a by chidamide may therefore have a therapeutic effect in controlling the sensitivity of CML cells to imatinib.

The crystallin alpha B (HSPB5)-tripartite motif containing 33 (TRIM33) axis mediates myocardial fibrosis induced by angiotensinogen II through transforming growth factor-ß (TGF-ß1)-Smad3/4 signaling.

Myocardial fibrosis, a common pathological manifestation of cardiac remodeling (CR), often leads to heart failure (HF) and even death. The underlying molecular mechanism of the role of TRIM33 in Ang II-induced myocardial fibrosis is not fully understood. We found that TRIM33 was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. Adult mice induced by Ang II were used as in vivo models, and Ang II-induced neonatal mouse primary cardiac fibroblasts (CFs) were used as in vitro models. The level of CF fibrosis in vitro was assessed by CF proliferation, migration, activation and extracellular matrix (ECM) synthesis. In addition, Masson staining, the heart weight/body weight (HW/BW) ratio and echocardiography were used to evaluate the in vivo effect of TRIM33. TRIM33 expression was specifically upregulated in CFs and myocardial tissue after Ang II stimulation. In in vitro experiments, we found that TRIM33 knockdown promoted Ang II-induced CF proliferation, while TRIM33 overexpression weakened Ang II-induced CF proliferation, migration, activation and collagen synthesis. Mechanistically, we showed that TRIM33, negatively regulated by HSPB5, mediated its antifibrotic effect by inhibiting the activation of TGF-ß1 and its downstream genes, Smad3 and Smad4. Finally, TRIM33 overexpression suppressed fibrosis and promoted cardiac repair and functional recovery in Ang II-induced mice. Our results clearly establish that TRIM33 limits cardiac fibrosis by hindering CF proliferation, migration, activation and collagen synthesis. Enhancing these beneficial functions of TRIM33 by a targeting vector might be a novel therapeutic strategy for CR.

Targeting DNA topoisomerases or checkpoint kinases results in an overload of chaperone systems, triggering aggregation of a metastable subproteome.

A loss of the checkpoint kinase ataxia telangiectasia mutated (ATM) leads to impairments in the DNA damage response, and in humans causes cerebellar neurodegeneration, and an increased risk of cancer. A loss of ATM is also associated with increased protein aggregation. The relevance and characteristics of this aggregation are still incompletely understood. Moreover, it is unclear to what extent other genotoxic conditions can trigger protein aggregation as well. Here, we show that targeting ATM, but also ATR or DNA topoisomerases, results in the widespread aggregation of a metastable, disease-associated subfraction of the proteome. Aggregation-prone model substrates, including Huntingtin exon 1 containing an expanded polyglutamine repeat, aggregate faster under these conditions. This increased aggregation results from an overload of chaperone systems, which lowers the cell-intrinsic threshold for proteins to aggregate. In line with this, we find that inhibition of the HSP70 chaperone system further exacerbates the increased protein aggregation. Moreover, we identify the molecular chaperone HSPB5 as a cell-specific suppressor of it. Our findings reveal that various genotoxic conditions trigger widespread protein aggregation in a manner that is highly reminiscent of the aggregation occurring in situations of proteotoxic stress and in proteinopathies.

Cells are constantly perceiving and responding to changes in their surroundings, and challenging conditions such as extreme heat or toxic chemicals can put cells under stress. When this happens, protein production can be affected. Proteins are long chains of chemical building blocks called amino acids, and they can only perform their roles if they fold into the right shape. Some proteins fold easily and remain folded, but others can be unstable and often become misfolded. Unfolded proteins can become a problem because they stick to each other, forming large clumps called aggregates that can interfere with the normal activity of cells, causing damage. The causes of stress that have a direct effect on protein folding are called proteotoxic stresses, and include, for example, high temperatures, which make proteins more flexible and unstable, increasing their chances of becoming unfolded. To prevent proteins becoming misfolded, cells can make 'protein chaperones', a type of proteins that help other proteins fold correctly and stay folded. The production of protein chaperones often increases in response to proteotoxic stress. However, there are other types of stress too, such as genotoxic stress, which damages DNA. It is unclear what effect genotoxic stress has on protein folding. Huiting et al. studied protein folding during genotoxic stress in human cells grown in the lab. Stress was induced by either blocking the proteins that repair DNA or by 'trapping' the proteins that release DNA tension, both of which result in DNA damage. The analysis showed that, similar to the effects of proteotoxic stress, genotoxic stress increased the number of proteins that aggregate, although certain proteins formed aggregates even without stress, particularly if they were common and relatively unstable proteins. Huiting et al.'s results suggest that aggregation increases in cells under genotoxic stress because the cells fail to produce enough chaperones to effectively fold all the proteins that need it. Indeed, Huiting et al. showed that aggregates contain many proteins that rely on chaperones, and that increasing the number of chaperones in stressed cells reduced protein aggregation. This work shows that genotoxic stress can affect protein folding by limiting the availability of chaperones, which increases protein aggregation. Remarkably, there is a substantial overlap between proteins that aggregate in diseases that affect the brain ­ such as Alzheimer's disease ­ and proteins that aggregate after genotoxic stress. Therefore, further research could focus on determining whether genotoxic stress is involved in the progression of these neurological diseases.