Human Heat Shock Protein Research Articles

Роль ендокринних факторів та маркерів клітинного стресу у прогнозуванні ефективності лікування клімактеричного синдрому в період перименопаузи

Women of menopausal age make up 10% of the world's population, and the problem of pathological menopause remains relevant today. Estrogen deficiency is considered to be the cause of menopause, so the use of replacement therapy is a pathogenetically sound method. It is possible that the immune system also plays a role in shaping the clinical picture. There is a certain relationship between estrogen levels and markers of intracellular stress. Studying the level of heat shock proteins as proven factors of cellular stress, along with endocrine factors, can become a reliable marker for the prognosis, course and compensation of disorders of the neurovegetative state of women in this period of life. Purpose - to study the role of levels of antimullerian, follicle-stimulating, luteinizing, thyroid-stimulating hormones, levels of antibodies (IgG) to human heat shock protein (Hsp60) and its bacterial homologue (Groel) in the prognosis, diagnosis and treatment of psychopathological manifestations in perimenopausal women. Materials and methods. The study examined 158 patients with perimenopausal menopausal syndrome between October 2019 and March 2022. The patients were divided into two groups. The 1st (main) group included 80 patients who agreed to receive treatment according to the standard regimen (combined treatment in a cyclic regimen (estrogens + gestagens), and the 2nd (comparative) group included 78 women who refused the proposed treatment Results. A decrease (p<0.001) in the risk of not achieving a high treatment effect was found for the main group, OR=0.12 (95% CI: 0.06-0.26) compared with the control group. It was found that a higher level of antibodies to GroEl, OR=0.989 (95% CI: 0.984-0.996), revealed a lower (p=0.001) risk of not achieving a high treatment effect. A higher body mass index (BMI) of a woman increases the risk of not achieving a high treatment effect (p<0.001), OR=1.19 (95% CI: 1.10-1.30) for every 1 kg/m2. When standardized by BMI, an integral indicator of psychopathological manifestations, taking into account the method of treatment and the level of antimullerian hormone, a decrease (p=0.039) in the risk of failure to achieve a high treatment effect was found for the main group, OR=0.16 (95% CI: 0.07-0.39) compared with the control group. Conclusions. It has been established that the most informative indicator for predicting the effectiveness of treatment of women with psychopathological manifestations is antimüllerian hormone. The data also show that the determination of the level of antibodies to GroEl correlates with the success of treatment of women with psychopathological manifestations of menopausal syndrome. The study was conducted in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Local Ethics Committee of the institutions mentioned in the paper. Informed consent of women was obtained for the study. No conflict of interests was declared by the authors.

Роль білків теплового шоку в прогнозуванні перебігу клімактеричного синдрому

The problem of menopause has been relevant for many decades. A pathogenetically justified method of treatment, taking into account the change in the hormonal background of a woman, is hormone replacement therapy. Determination of immune markers of cellular homeostasis is one of the promising directions, therefore, the study of changes in the levels of antibodies to HSP60 can become a reliable marker of both the course and compensation of disorders of the neurovegetative state in perimenopause. Purpose - to study the role of anti-Müllerian, follicle-stimulating, luteinizing, thyroid-stimulating hormones, antibodies (IgG) to human heat shock protein 60 (HSP60) and its bacterial homologue (GroEl), and obstetric history on the diagnosis and treatment of vasomotor symptoms in perimenopausal women. Materials and methods. 158 patients with climacteric syndrome in perimenopause were examined in the period October 2019 - March 2022. The Group 1 (main) included 80 patients who agreed to treatment and received it according to the standard scheme: combined treatment in a cyclical regimen - estrogens (17β-estradiol) + progestogens (dydrogesterone); the Group 2 (comparison) included 78 women who refused treatment due to estrogen deficiency. Results. Revealed a decrease (p<0.01) in the risk of not achieving a high treatment effect with vasomotor symptoms for treated patients, OR=0.04 (95% CI: 0.01-0.18) compared to the group of untreated patients. With the number of deliveries, the risk of not achieving a high treatment effect in women with vasomotor symptoms increases (p=0.01), OR=2.27 (95% CI: 1.2-4.27) for each delivery. A lower (p=0.039) risk of not achieving a high treatment effect was found in women with vasomotor symptoms at a higher GroEl level, ОR=0.994 (95% CI: 0.987-1.000) for each ng. When standardizing by the thyrotropin hormone and the manifestation of vasomotor symptoms before or without treatment, a decrease (p<0.001) in the risk of not achieving a high effect of treatment in women with vasomotor symptoms for treated patients was found, OR=0.04 (95% CI: 0.01-0.20). Conclusions. This study found that there is no informative indicator for predicting the effectiveness of treatment in women with vasomotor symptoms. It was established that the HSP60 indicator does not affect, and the GroEl indicator affects the effectiveness of the treatment of vasomotor clinical manifestations. The research was carried out in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the Local Ethics Committee of the institutions. The informed consent of the women was obtained for conducting the studies. No conflict of interest was declared by the authors.

Structural Characterization of Human Heat Shock Protein 90 N-Terminal Domain and Its Variants K112R and K112A in Complex with a Potent 1,2,3-Triazole-Based Inhibitor.

Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that stabilizes client proteins in a folded and functional state. It is composed of two identical and symmetrical subunits and each monomer consists of three domains, the N-terminal (NTD), the middle (MD), and the C-terminal domain (CTD). Since the chaperone activity requires ATP hydrolysis, molecules able to occupy the ATP-binding pocket in the NTD act as Hsp90 inhibitors, leading to client protein degradation and cell death. Therefore, human Hsp90 represents a validated target for developing new anticancer drugs. Since protozoan parasites use their Hsp90 to trigger important transitions between different stages of their life cycle, this protein also represents a profitable target in anti-parasite drug discovery. Nevertheless, the development of molecules able to selectively target the ATP-binding site of protozoan Hsp90 is challenging due to the high homology with the human Hsp90 NTD (hHsp90-NTD). In a previous work, a series of potent Hsp90 inhibitors based on a 1,4,5-trisubstituted 1,2,3-triazole scaffold was developed. The most promising inhibitor of the series, JMC31, showed potent Hsp90 binding and antiproliferative activity in NCI-H460 cells in the low-nanomolar range. In this work, we present the structural characterization of hHsp90-NTD in complex with JMC31 through X-ray crystallography. In addition, to elucidate the role of residue 112 on the ligand binding and its exploitability for the development of selective inhibitors, we investigated the crystal structures of hHsp90-NTD variants (K112R and K112A) in complex with JMC31.

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.

Is the small heat shock protein HSPB7 (cvHsp) a genuine actin-binding protein?

It is postulated that the small heat shock proteins directly interact with actin, affect formation and stabilize actin filaments. To verify this suggestion, we have analyzed interaction of recombinant human small heat shock protein HspB7 with skeletal muscle actin. In blot overlay HspB7 binds both G- and F-actin. The sites of interaction are located in the C-terminal large core domain of actin. In the course of ultracentrifugation F-actin and F-actin/tropomyosin complexes were pelleted and trapped HspB7. However, HspB7 pelleting was nonspecific and saturation was not achieved even at very high HspB7 concentration. HspB7 was unable to retard or prevent heat-induced F-actin aggregation. Native gel electrophoresis and chemical crosslinking failed to detect interaction of G-actin with HspB7, although both these methods clearly demonstrated formation of complexes formed by G-actin with DNAse I and cofilin-2. It is concluded that HspB7 is not a genuine actin-binding protein and its effect on actin filaments seems to be determined by interaction of HspB7 with minor regulatory proteins of actin filaments.

Roles of heat-shock protein 90 and its four domains (N, LR, M and C) in calcium oxalate stone-forming processes.

Human heat-shock protein 90 (HSP90) has four functional domains, including NH2-terminal (N), charged linker region (LR), middle (M) and COOH-terminal (C) domains. In kidney stone disease (or nephrolithiasis/urolithiasis), HSP90 serves as a receptor for calcium oxalate monohydrate (COM), which is the most common crystal to form kidney stones. Nevertheless, roles of HSP90 and its four domains in kidney stone formation remained unclear and under-investigated. We thus examined and compared their effects on COM crystals during physical (crystallization, growth and aggregation) and biological (crystal–cell adhesion and crystal invasion through extracellular matrix (ECM)) pathogenic processes of kidney stone formation. The analyses revealed that full-length (FL) HSP90 obviously increased COM crystal size and abundance during crystallization and markedly promoted crystal growth, aggregation, adhesion onto renal cells and ECM invasion. Comparing among four individual domains, N and C domains exhibited the strongest promoting effects, whereas LR domain had the weakest promoting effects on COM crystals. In summary, our findings indicate that FL-HSP90 and its four domains (N, LR, M and C) promote COM crystallization, crystal growth, aggregation, adhesion onto renal cells and invasion through the ECM, all of which are the important physical and biological pathogenic processes of kidney stone formation.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-022-04483-z.

Mebendazole's Conformational Space and Its Predicted Binding to Human Heat-Shock Protein 90.

Recent experimental evidence suggests that mebendazole, a popular antiparasitic drug, binds to heat shock protein 90 (Hsp90) and inhibits acute myeloid leukemia cell growth. In this study we use quantum mechanics (QM), molecular similarity, and molecular dynamics (MD) calculations to predict possible binding poses of mebendazole to the adenosine triphosphate (ATP) binding site of Hsp90. Extensive conformational searches and minimization of the five mebendazole tautomers using the MP2/aug-cc-pVTZ theory level resulted in 152 minima. Mebendazole-Hsp90 complex models were subsequently created using the QM optimized conformations and protein coordinates obtained from experimental crystal structures that were chosen through similarity calculations. Nine different poses were identified from a total of 600 ns of explicit solvent, all-atom MD simulations using two different force fields. All simulations support the hypothesis that mebendazole is able to bind to the ATP binding site of Hsp90.

The effects of type III antifreeze protein and human heat shock protein 70 added to the vitrification medium of mouse embryos on in vitro embryonic development rates

The effect of antifreeze protein type III (AFPIII) and human heat shock protein 70 (HHSP70), added to the vitrification medium of mouse embryos, on post-freeze/thaw in vitro embryonic growth rates and cell numbers were investigated. In total 20 female mice were synchronized. After synchronization, 2 females and 1 male were mated in the same cage. Twenty-four h after mating, the embryos were collected at the pronuclear stage. In total 8 groups were established, including a positive control group (C+), a negative control group (C-), and treatment groups that were vitrified in a medium added with 200, 400 and 800 ng/ml of AFPIII (AFPIII200, AFPIII400, AFPIII800), and 1, 2 and 4 µg/ml of HHSP70 (HHSP70-1, HHSP70-2, HHSP70-4). Solid surface vitrification (SSV) medium was prepared for the vitrification of the embryos. Once thawed, in vitro development rates of embryos were followed at 24, 48, 72 and 96 h. Four embryos, which progressed to the full blastocyst stage, were taken from each group and stained with the Hoeschst 33258 and propidium iodine (PI) dyes to determine the inner cell mass (ICM), trophectoderm (TE) and total cell numbers. The groups showed statistically significant difference for in vitro embryonic development rates at 48, 72 and 96 h (p

Insights on Human Small Heat Shock Proteins and Their Alterations in Diseases.

The family of the human small Heat Shock Proteins (HSPBs) consists of ten members of chaperones (HSPB1-HSPB10), characterized by a low molecular weight and capable of dimerization and oligomerization forming large homo- or hetero-complexes. All HSPBs possess a highly conserved centrally located α-crystallin domain and poorly conserved N- and C-terminal domains. The main feature of HSPBs is to exert cytoprotective functions by preserving proteostasis, assuring the structural maintenance of the cytoskeleton and acting in response to cellular stresses and apoptosis. HSPBs take part in cell homeostasis by acting as holdases, which is the ability to interact with a substrate preventing its aggregation. In addition, HSPBs cooperate in substrates refolding driven by other chaperones or, alternatively, promote substrate routing to degradation. Notably, while some HSPBs are ubiquitously expressed, others show peculiar tissue-specific expression. Cardiac muscle, skeletal muscle and neurons show high expression levels for a wide variety of HSPBs. Indeed, most of the mutations identified in HSPBs are associated to cardiomyopathies, myopathies, and motor neuropathies. Instead, mutations in HSPB4 and HSPB5, which are also expressed in lens, have been associated with cataract. Mutations of HSPBs family members encompass base substitutions, insertions, and deletions, resulting in single amino acid substitutions or in the generation of truncated or elongated proteins. This review will provide an updated overview of disease-related mutations in HSPBs focusing on the structural and biochemical effects of mutations and their functional consequences.

Alpha B-Crystallin in Muscle Disease Prevention: The Role of Physical Activity.

HSPB5 or alpha B-crystallin (CRYAB), originally identified as lens protein, is one of the most widespread and represented of the human small heat shock proteins (sHSPs). It is greatly expressed in tissue with high rates of oxidative metabolism, such as skeletal and cardiac muscles, where HSPB5 dysfunction is associated with a plethora of human diseases. Since HSPB5 has a major role in protecting muscle tissues from the alterations of protein stability (i.e., microfilaments, microtubules, and intermediate filament components), it is not surprising that this sHSP is specifically modulated by exercise. Considering the robust content and the protective function of HSPB5 in striated muscle tissues, as well as its specific response to muscle contraction, it is then realistic to predict a specific role for exercise-induced modulation of HSPB5 in the prevention of muscle diseases caused by protein misfolding. After offering an overview of the current knowledge on HSPB5 structure and function in muscle, this review aims to introduce the reader to the capacity that different exercise modalities have to induce and/or activate HSPB5 to levels sufficient to confer protection, with the potential to prevent or delay skeletal and cardiac muscle disorders.

Plasmodium falciparum HSP40 protein eCiJp traffics to the erythrocyte cytoskeleton and interacts with the human HSP70 chaperone HSPA1.

Renovation of host erythrocytes is vital for pathogenesis by Plasmodium falciparum. These changes are mediated by parasite proteins that translocate beyond the parasitophorous vacuolar membrane in an unfolded state, suggesting protein folding by chaperones is imperative for the functionality of exported proteins. We report a type IV P. falciparum heat-shock protein 40, PF11_0034, that localizes to the cytoplasmic side of J-dots and interacts with the erythrocyte cytoskeleton, and therefore named eCiJp (erythrocyte cytoskeleton-interacting J protein). Recombinant eCiJp binds to the human heat-shock protein 70 HsHSPA1 and promotes its ATPase activity. In addition, eCiJp could suppress protein aggregation. Our data suggest that eCiJp recruits HsHSPA1 to the host erythrocyte cytoskeleton, where it may become involved in remodeling of the erythrocyte cytoskeleton and/or folding of exported parasite proteins.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87–118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Quaternary Structure and Hetero-Oligomerization of Recombinant Human Small Heat Shock Protein HspB7 (cvHsp).

In this study, a reliable and simple method of untagged recombinant human HspB7 preparation was developed. Recombinant HspB7 is presented in two oligomeric forms with an apparent molecular weight of 36 kDa (probably dimers) and oligomers with an apparent molecular weight of more than 600 kDa. By using hydrophobic and size-exclusion chromatography, we succeeded in preparation of HspB7 dimers. Mild oxidation promoted the formation of large oligomers, whereas the modification of Cys 126 by iodoacetamide prevented it. The deletion of the first 13 residues or deletion of the polySer motif (residues 17–29) also prevented the formation of large oligomers of HspB7. Cys-mutants of HspB6 and HspB8 containing a single-Cys residue in the central part of the β7 strand in a position homologous to that of Cys137 in HspB1 can be crosslinked to the wild-type HspB7 through a disulfide bond. Immobilized on monoclonal antibodies, the wild-type HspB6 interacted with the wild-type HspB7. We suppose that formation of heterodimers of HspB7 with HspB6 and HspB8 may be important for the functional activity of these small heat shock proteins.

Trends in HSPB5 research: a 36-year bibliometric analysis.

HSPB5 (heat shock protein B5), also known as αB-crystallin, is one of the most widespread and populous of the ten human small heat shock proteins (sHsps). Over the past decades, extensive research has been conducted on HSPB5. However, few studies have statistically analyzed these publications. Herein, we conducted a bibliometric analysis to track the global research trend and current development status of HSPB5 research from the Web of Science Core Collection (WoSCC) database between 1985 and 2020. Our results demonstrate that 1220 original articles cited 54,778 times in 391 scholarly journals were published. Visualization analyses reveal that the Journal of Biological Chemistry was the most influential journal with 85 articles. The USA dominated this field with 520 publications (42.62%), followed by Japan with 149 publications (12.21%), and Kato contributed the largest number of publications. Most related publications were published in journals focusing on biochemistry molecular biology, cell biology, neurosciences neurology, and ophthalmology. In addition, keyword co-occurrence analyses identify three predominant research topics: expression of HSPB5, chaperone studies for HSPB5, and pathological studies of HSPB5. This study provides valuable guidance for researchers and leads to collaborative opportunities between diverse research interests to be integrated for HSPB5 research.

Switching Kogenate FS® to Kovaltry® for the Treatment of Severe Haemophilia A Patients: A Successful Single Center Experience

Background: Haemophilia A is an X-linked hereditary disorder caused by deficient levels of the coagulation protein factor VIII (FVIII). Standard therapy for haemophilia A comprises replacement of FVIII with plasma-derived or recombinant FVIII concentrate, which can be administered as on-demand or prophylaxis regimen. The most challenging complication of replacement therapy in hemophilia A is the occurrence of alloantibodies against infused factor FVIII, thus predisposing the patients to an increased morbidity and disability. Historically, first FVIII was isolated from cryoprecipitated plasma concentrates. Thirty years ago, after the cloning of F8 gene, therapy for hemophilia A patients underwent a new exciting era that developed technological strategies aimed to improve haemostatic efficacy and safety of the replacement factor. In this line, a new recombinant FVIII, BAY 81-8973 (octocog alfa; Kovaltry®, Bayer) has recently been introduced onto pharmaceutical market for treatment of these patients. Kovaltry® is an unmodified, full-length, recombinant FVIII concentrate with the same amino acid sequence as Kogenate FS® (Bayer), but produced with innovative manufacturing technologies that include production without addition of human- or animal-derived raw materials from purification or formulation processes, inclusion of a higher degree of sialic acid capping of N-terminal glycans on the molecular surface, coexpression of the human chaperone protein heat shock protein 70, and a 20-nm nanofiltration step to remove small non-enveloped viruses and potential protein aggregates. On this basis we considered beneficial for our patients changing Kogenate FS® therapy for Kovaltry®.Objective: We aimed to demonstrate that switching from Kogenate FS® to Kovaltry® could be easily performed without increasing the risk of bleeding or FVIII consumption in severe haemophilia A patients.Methods: Sixty two adult and pediatric patients with severe haemophilia A, without inhibitors, treated with Kogenate FS® either on on-demand treatment (n=13) or on prophylaxis regimen (n=49), were recruited. On November 2016, we began to switch Kogenate FS® treatment to Kovaltry® in these patients.The trend of prophylaxis with Kovaltry® was evaluated by determining mean injection frequency (per week), mean dose of each injection (IU/kg), mean total dose injected/week (IU), baseline and post-infusion levels of FVIII and annual bleeding rate (ABR). Same data for Kogenate FS® were collected retrospectively from clinical histories of patients.For determining baseline and post-infusion levels of Kovaltry®, whole blood was collected in 3.2% sodium citrate tubes (BD Vacutainer, Becton Dickinson, San José, CA, USA) at pre-dosing and 30 minutes after Kovaltry® infusion. Plasma FVIII:C was measured by chromogenic assay.Results: Switching from Kogenate FS® to Kovaltry® caused no changes, except in two patients, either in mean injection frequency or mean dose of each injection or mean total dose injected/week or baseline and post-infusion levels of FVIII or annual bleeding rate. Increment observed in mean total dose injected/week in two of the pediatric patients was due to an increased physical activity (patient 2) and to weight gain (patient 8). For sake of example, Table 1 shows these variables for 10 pediatric patients under prophylactic treatment.None of the patients treated with Kovaltry® developed inhibitors.Conclusion: Changing severe haemophilia A treatment from Kogenate FS® to Kovaltry® can be performed easily and did not pose any risk to the patient since Kovaltry® proved to be equally effective than Kogenate FS® to prevent bleeding episodes and did not induce the occurrence of inhibitors. [Display omitted] DisclosuresÁlvarez-Roman:Novo Nordisk: Consultancy; Shire: Consultancy. Jimenez-Yuste:Novo Nordisk: Consultancy, Honoraria, Research Funding; Roche: Consultancy.

Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22).

The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28–32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

Small Hsps as Therapeutic Targets of Cystic Fibrosis Transmembrane Conductance Regulator Protein.

Human small heat shock proteins are molecular chaperones that regulate fundamental cellular processes in normal and pathological cells. Here, we have reviewed the role played by HspB1, HspB4 and HspB5 in the context of Cystic Fibrosis (CF), a severe monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR) some of which trigger its misfolding and rapid degradation, particularly the most frequent one, F508del-CFTR. While HspB1 and HspB4 favor the degradation of CFTR mutants, HspB5 and particularly one of its phosphorylated forms positively enhance the transport at the plasma membrane, stability and function of the CFTR mutant. Moreover, HspB5 molecules stimulate the cellular efficiency of currently used CF therapeutic molecules. Different strategies are suggested to modulate the level of expression or the activity of these small heat shock proteins in view of potential in vivo therapeutic approaches. We then conclude with other small heat shock proteins that should be tested or further studied to improve our knowledge of CFTR processing.

Prognostic significance of heat shock protein 90AA1 (HSP90α) in invasive breast cancer

AimsThe mechanisms that drive breast cancer (BC) progression and poor outcome are not fully understood. The human heat shock protein 90 alpha family class A member 1 (HSP90α) encoded by...

Delivery of functional exogenous proteins by plant-derived vesicles to human cells in vitro

Plant-derived extracellular vesicles (EVs) gain more and more attention as promising carriers of exogenous bioactive molecules to the human cells. Derived from various edible sources, these EVs are remarkably biocompatible, biodegradable and highly abundant from plants. In this work, EVs from grapefruit juice were isolated by differential centrifugation followed by characterization of their size, quantity and morphology by nanoparticle tracking analysis, dynamic light scattering, atomic force microscopy and cryo-electron microscopy (Cryo-EM). In Cryo-EM experiments, we visualized grapefruit EVs with the average size of 41 ± 13 nm, confirmed their round-shaped morphology and estimated the thickness of their lipid bilayer as 5.3 ± 0.8 nm. Further, using cell culture models, we have successfully demonstrated that native grapefruit-derived extracellular vesicles (GF-EVs) are highly efficient carriers for the delivery of the exogenous Alexa Fluor 647 labeled bovine serum albumin (BSA) and heat shock protein 70 (HSP70) into both human peripheral blood mononuclear cells and colon cancer cells. Interestingly, loading to plant EVs significantly ameliorated the uptake of exogenous proteins by human cells compared to the same proteins without EVs. Most importantly, we have confirmed the functional activity of human recombinant HSP70 in the colon cancer cell culture upon delivery by GF-EVs. Analysis of the biodistribution of GF-EVs loaded with 125I-labeled BSA in mice demonstrated a significant uptake of the grapefruit-derived extracellular vesicles by the majority of organs. The results of our study indicate that native plant EVs might be safe and effective carriers of exogenous proteins into human cells.

Identification of unknown acid-resistant genes of oral microbiotas in patients with dental caries using metagenomics analysis

Acid resistance is critical for the survival of bacteria in the dental caries oral micro-environment. However, there are few acid-resistant genes of microbiomes obtained through traditional molecular biology experimental techniques. This study aims to try macrogenomics technologies to efficiently identify acid-resistant genes in oral microbes of patients with dental caries. Total DNA was extracted from oral microbiota obtained from thirty dental caries patients and subjected to high-throughput sequencing. This data was used to build a metagenomic library, which was compared to the sequences of two Streptococcus mutant known acid-resistant genes, danK and uvrA, using a BLAST search. A total of 19 and 35 unknown gene sequences showed similarities with S. mutans uvrA and dnaK in the metagenomic library, respectively. Two unknown genes, mo-dnaK and mo-uvrA, were selected for primer design and bioinformatic analysis based on their sequences. Bioinformatics analysis predicted them encoding of a human heat-shock protein (HSP) 70 and an ATP-dependent DNA repair enzyme, respectively, closely related with the acid resistance mechanism. After cloning, these genes were transferred into competent Escherichia coli for acid resistance experiments. E. coli transformed with both genes demonstrated acid resistance, while the survival rate of E. coli transformed with mo-uvrA was significantly higher in an acidic environment (pH = 3). Through this experiment we found that identify unknown acid-resistant genes in oral microbes of patients with caries by establishing a metagenomic library is very efficient. Our results provide an insight into the mechanisms and pathogenesis of dental caries for their treatment without affecting oral probiotics.