ABSTRACT
Heat shock protein 20 (Hsp20) is a small molecule heat shock protein that plays an important role in plant growth, development, and stress resistance. Little is known about the function of Hsp20 family genes in apple (Malus domestica). Here, we performed a genome-wide analysis of the apple Hsp20 gene family, and a total of 49 Hsp20s genes were identified from the apple genome. Phylogenetic analysis revealed that the 49 genes were divided into 11 subfamilies, and MdHsp18.2b, a member located in the CI branch, was selected as a representative member for functional characterization. Treatment with NaCl and Botryosphaeria dothidea (B. dothidea), the causal agent of apple ring rot disease, significantly induced MdHsp18.2b transcription level. Further analysis revealed that overexpressing MdHsp18.2b reduced the resistance to salt stress but enhanced the resistance to B. dothidea infection in apple calli. Moreover, MdHsp18.2b positively regulated anthocyanin accumulation in apple calli. Physiology assays revealed that MdHsp18.2b promoted H2O2 production, even in the absence of stress factors, which might contribute to its functions in response to NaCl and B. dothidea infection. Hsps usually function as homo- or heterooligomers, and we found that MdHsp18.2b could form a heterodimer with MdHsp17.9a and MdHsp17.5, two members from the same branch with MdHsp18.2b in the phylogenetic tree. Therefore, we identified 49 Hsp20s genes from the apple genome and found that MdHsp18.2b was involved in regulating plant resistance to salt stress and B. dothidea infection, as well as in regulating anthocyanin accumulation in apple calli.
Subject(s)
Gene Expression Regulation, Plant , HSP20 Heat-Shock Proteins , Malus , Phylogeny , Plant Diseases , Plant Proteins , Malus/genetics , Malus/microbiology , Malus/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Diseases/microbiology , Plant Diseases/genetics , HSP20 Heat-Shock Proteins/genetics , HSP20 Heat-Shock Proteins/metabolism , Ascomycota/physiology , Ascomycota/genetics , Ascomycota/pathogenicity , Multigene Family , Disease Resistance/genetics , Anthocyanins/metabolismABSTRACT
HSP20 emerges as a novel regulator of autophagy in the heart. Nonetheless, the detailed function of HSP20 in the liver and its effect on autophagy remain unknown. Here, we observed that HSP20 expression is increased in liver tissues from mice and patients with metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as nonalcoholic fatty liver disease. Liver-specific downregulation of HSP20 mitigates hepatic steatosis and insulin resistance in obese mice, while upregulating HSP20 promotes lipid deposition and hepatocyte cell death. Mechanistically, liquid chromatography-tandem mass spectrometry revealed that HSP20 interacts with phosphorylated extracellular regulated protein kinase 2 (ERK2) and prevents its dephosphorylation by dual specificity phosphatase 6, leading to ERK2-mediated repression of autophagy and resulting in aggravated saturated fatty acid (SFA)-triggered hepatocyte death. Importantly, such adverse effects could be ameliorated by ERK inhibitor. Our data reveal a framework of how HSP20 increases susceptibility of SFA-induced liver injury through enhancing ERK2 phosphorylation, which represents a plausible therapeutic intervention to combat MASLD.
Subject(s)
Autophagy , HSP20 Heat-Shock Proteins , Mitogen-Activated Protein Kinase 1 , Animals , Humans , Male , Mice , Autophagy/drug effects , Autophagy/physiology , Hepatocytes/metabolism , Hepatocytes/drug effects , HSP20 Heat-Shock Proteins/metabolism , HSP20 Heat-Shock Proteins/genetics , Insulin Resistance/physiology , Liver/metabolism , Liver/pathology , Liver/drug effects , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Non-alcoholic Fatty Liver Disease/metabolism , PhosphorylationABSTRACT
The tardigrade Ramazzottius varieornatus has remarkable resilience to a range of environmental stresses. In this study, we have characterised two members of the small heat shock protein (sHSP) family in R. varieornatus, HSP20-3 and HSP20-6. These are the most highly upregulated sHSPs in response to a 24 h heat shock at 35 0C of adult tardigrades with HSP20-3 being one of the most highly upregulated gene in the whole transcriptome. Both R. varieornatus sHSPs and the human sHSP, CRYAB (HSPB5), were produced recombinantly for comparative structure-function studies. HSP20-3 exhibited a superior chaperone activity than human CRYAB in a heat-induced protein aggregation assay. Both tardigrade sHSPs also formed larger oligomers than CRYAB as assessed by size exclusion chromatography and transmission electron microscopy of negatively stained samples. Whilst both HSP20-3 and HSP20-6 formed particles that were variable in size and larger than the particles formed by CRYAB, only HSP20-3 formed filament-like structures. The particles and filament-like structures formed by HSP20-3 appear inter-related as the filament-like structures often had particles located at their ends. Sequence analyses identified two unique features; an insertion in the middle region of the N-terminal domain (NTD) and preceding the critical-sequence identified in CRYAB, as well as a repeated QNTN-motif located in the C-terminal domain of HSP20-3. The NTD insertion is expected to affect protein-protein interactions and subunit oligomerisation. Removal of the repeated QNTN-motif abolished HSP20-3 chaperone activity and also affected the assembly of the filament-like structures. We discuss the potential contribution of HSP20-3 to protein condensate formation.
Subject(s)
Heat-Shock Proteins, Small , Humans , Heat-Shock Proteins, Small/metabolism , Amino Acid Sequence , HSP20 Heat-Shock Proteins/genetics , HSP20 Heat-Shock Proteins/metabolism , Molecular Chaperones/metabolism , Heat-Shock ResponseABSTRACT
Heat shock protein B6 (HSPB6) plays a certain role in the formation of several cancers, whereas its effect on osteosarcoma remains unclear. In this study, the effect of HSPB6 on osteosarcoma was validated through numerous experiments. HSPB6 was down-regulated in osteosarcoma. As indicated by the result of CCK-8 and colony formation assays, HSPB6 overexpression was likely to inhibit the osteosarcoma cells proliferation, whereas the flow cytometry analysis suggested that apoptosis of osteosarcoma cells was increased after HSPB6 overexpression. Furthermore, transwell and wound healing assays suggested that when HSPB6 was overexpressed, osteosarcoma cells migration and invasion were declined. Moreover, the western blotting assay suggested that the protein level of p-ERK1/2 was down-regulated in osteosarcoma when HSPB6 was overexpressed. Besides, the effect of HSPB6 on osteosarcoma in vivo was examined. As indicated by the result, HSPB6 overexpression was likely to prevent osteosarcoma growth and lung metastasis in vivo. As revealed by the findings of this study, HSPB6 overexpression exerted anticancer effects in osteosarcoma through the ERK signaling pathway and HSPB6 may be suitable target for osteosarcoma molecular therapies.
Subject(s)
Bone Neoplasms , Osteosarcoma , Humans , Apoptosis , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , HSP20 Heat-Shock Proteins/genetics , HSP20 Heat-Shock Proteins/metabolism , MAP Kinase Signaling System , Osteosarcoma/pathology , Signal TransductionABSTRACT
Background and Objectives: Colon cancer (CC) is the second most common cancer in Saudi Arabia, and the number of new cases is expected to increase by 40% by 2040. Sixty percent of patients with CC are diagnosed in the late stage, causing a reduced survival rate. Thus, identifying a new biomarker could contribute to diagnosing CC in the early stages, leading to delivering better therapy and increasing the survival rate. Materials and Methods: HSPB6 expression was investigated in extracted RNA taken from 10 patients with CC and their adjacent normal tissues, as well as in DMH-induced CC and a colon treated with saline taken from a male Wistar rat. Additionally, the DNA of the LoVo and Caco-2 cell lines was collected, and bisulfite was converted to measure the DNA methylation level. This was followed by applying 5-aza-2'-deoxycytidine (AZA) to the LoVo and Caco-2 cell lines for 72 h to see the effect of DNA methylation on HSPB6 expression. Finally, the GeneMANIA database was used to find the interacted genes at transcriptional and translational levels with HSPB6. Results: We found that the expression of HSPB6 was downregulated in 10 CC tissues compared to their adjacent normal colon tissues, as well as in the in vivo study, where its expression was lower in the colon treated with the DMH agent compared to the colon treated with saline. This suggests the possible role of HSPB6 in tumor progression. Moreover, HSPB6 was methylated in two CC cell lines (LoVo and Caco-2), and demethylation with AZA elevated its expression, implying a mechanistic association between DNA methylation and HSPB6 expression. Conclusions: Our findings indicate that HSPB6 is adversely expressed with tumor progression, and its expression may be controlled by DNA methylation. Thus, HSPB6 could be a good biomarker employed in the CC diagnostic process.
Subject(s)
Colonic Neoplasms , Humans , Rats , Animals , Male , Decitabine/pharmacology , Caco-2 Cells , Cell Line, Tumor , Promoter Regions, Genetic , Rats, Wistar , Colonic Neoplasms/genetics , DNA Methylation , Gene Expression Regulation, Neoplastic , HSP20 Heat-Shock Proteins/geneticsABSTRACT
Inflammation and oxidative stress contribute to the progression of acute lung injury (ALI). MicroRNA-23a-5p (miR-23a-5p) has been reported to regulate inflammation and oxidative stress; however, its role in ALI is still poorly elucidated. Mice were intravenously treated with the miR-23a-5p antagomir, agomir, or the negative controls for 3 consecutive days and then received a single intratracheal injection of lipopolysaccharide (LPS, 5 mg/kg) to induce ALI. Pulmonary function, bronchoalveolar lavage fluids (BALFs), arterial blood gas, and molecular biomarkers associated with inflammation and oxidative stress were analyzed. In addition, murine peritoneal macrophages were isolated and treated with LPS to verify the role of miR-23a-5p in vitro. We detected an elevation of miR-23a-5p expression in the lungs from ALI mice. The miR-23a-5p antagomir was prevented, whereas the miR-23a-5p agomir aggravated inflammation, oxidative stress, lung tissue injury, and pulmonary dysfunction in LPS-treated mice. Besides, the miR-23a-5p antagomir also reduced the productions of proinflammatory cytokines and free radicals in LPS-treated primary macrophages, which were further augmented in cells following the miR-23a-5p agomir treatment. Additional findings demonstrated that the miR-23a-5p agomir exacerbated LPS-induced ALI via activating apoptosis signal-regulating kinase 1 (ASK1), and that pharmacological or genetic inhibition of ASK1 significantly repressed the deleterious effects of the miR-23a-5p agomir. Moreover, we proved that the miR-23a-5p agomir activated ASK1 via directly reducing heat shock protein 20 (HSP20) expression. miR-23a-5p is involved in the regulation of LPS-induced inflammation, oxidative stress, lung tissue injury, and pulmonary dysfunction by targeting HSP20/ASK1, and it is a valuable therapeutic candidate for the treatment of ALI.
Subject(s)
Acute Lung Injury/pathology , Gene Expression Regulation , HSP20 Heat-Shock Proteins/metabolism , Lipopolysaccharides/toxicity , MAP Kinase Kinase Kinase 5/metabolism , MicroRNAs/genetics , Oxidative Stress , Acute Lung Injury/chemically induced , Acute Lung Injury/genetics , Acute Lung Injury/metabolism , Animals , Apoptosis , Cytokines , HSP20 Heat-Shock Proteins/genetics , Inflammation/etiology , Inflammation/metabolism , Inflammation/pathology , MAP Kinase Kinase Kinase 5/genetics , Male , Mice , Mice, Inbred C57BL , Signal TransductionABSTRACT
The cytoskeleton has a primary role in cardiomyocyte function, including the response to mechanical stimuli and injury. The small heat shock protein 20 (Hsp20) conveys protective effects in cardiac muscle that are linked to serine-16 (Ser16) Hsp20 phosphorylation by stress-induced PKA, but the link between Hsp20 and the cytoskeleton remains poorly understood. Herein, we demonstrate a physical and functional interaction of Hsp20 with the cytoskeletal protein 14-3-3. We show that, upon phosphorylation at Ser16, Hsp20 translocates from the cytosol to the cytoskeleton where it binds to 14-3-3. This leads to dissociation of 14-3-3 from the F-actin depolymerization regulator cofilin-2 (CFL2) and enhanced F-actin depolymerization. Importantly, we demonstrate that the P20L Hsp20 mutation associated with dilated cardiomyopathy exhibits reduced physical interaction with 14-3-3 due to diminished Ser16 phosphorylation, with subsequent failure to translocate to the cytoskeleton and inability to disassemble the 14-3-3/CFL2 complex. The topological sequestration of Hsp20 P20L ultimately results in impaired regulation of F-actin dynamics, an effect implicated in loss of cytoskeletal integrity and amelioration of the cardioprotective functions of Hsp20. These findings underscore the significance of Hsp20 phosphorylation in the regulation of actin cytoskeleton dynamics, with important implications in cardiac muscle physiology and pathophysiology.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Cytoskeleton/metabolism , HSP20 Heat-Shock Proteins/metabolism , Myocardium/metabolism , 14-3-3 Proteins/metabolism , Actins/metabolism , Animals , Cofilin 2/metabolism , HEK293 Cells , HSP20 Heat-Shock Proteins/genetics , Humans , Mice , Mutation , Phosphorylation , Protein Binding , Protein Processing, Post-TranslationalABSTRACT
Smoking increases the risk of cardiovascular diseases. The present study was designed to determine the effects of 2-month exposure to cigarette smoke (CS) on proteins in the left ventricles of spontaneously hypertensive rats (SHR) and to identify the molecular targets associated with the pathogenesis/progression of CS-induced cardiac hypertrophy. SHR and Wistar Kyoto rats (WKY) were exposed to CS at low (2 puffs/min for 40 min) or high dose (2 puffs/min for 120 min), 5 days a week for 2 months. Using the two-dimensional fluorescence difference gel electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry, we compared differences in the expression levels of proteins in the whole left ventricles induced by long-term smoking. High-dose CS mainly caused cardiac hypertrophy in SHR, but not WKY, but no change in blood pressure. Proteomic analysis identified 30 protein spots with significant alterations, with 14 up-regulated and 16 down-regulated proteins in the left ventricles of CS-exposed SHR, compared with control SHR. Among these proteins, two members of the heat shock proteins (HSP70 and HSP20) showed significant up-regulation in the left ventricles of CS high-dose SHR, and the results were confirmed by western blot analysis. Our findings suggested that HSPs play an important role in regulation of CS-induced cardiac hypertrophy.
Subject(s)
Cardiomegaly/etiology , Cardiomegaly/genetics , Cigarette Smoking/adverse effects , Cigarette Smoking/genetics , HSP20 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Proteomics/methods , Animals , Cardiomegaly/metabolism , Gene Expression , HSP20 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/genetics , Heart Ventricles/metabolism , Rats, Inbred SHR , Rats, Inbred WKY , Risk , Up-RegulationABSTRACT
Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.
Subject(s)
Aedes/genetics , Aedes/virology , Dengue Virus/physiology , Dengue/virology , HSP20 Heat-Shock Proteins/genetics , Insect Proteins/genetics , Mosquito Vectors/genetics , Mosquito Vectors/virology , Animals , Computational Biology/methods , Gene Expression Profiling , Host-Pathogen Interactions , Transcriptome , Virus ReplicationABSTRACT
The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.
Subject(s)
Autophagy , HSP20 Heat-Shock Proteins/metabolism , Heart Failure/metabolism , Metabolic Syndrome/metabolism , Animals , Beclin-1/genetics , Beclin-1/metabolism , Coronary Vessels/metabolism , Female , HSP20 Heat-Shock Proteins/genetics , Heart Failure/etiology , Metabolic Syndrome/complications , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Muscle, Skeletal/metabolism , Myocardium/metabolism , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , SwineABSTRACT
Small heat shock proteins (sHSPs) increase stress tolerance in a wide variety of organisms and enable them to endure changes in their environment. However, the molecular mechanism by which sHSPs protect plants against cold stress is unknown. Here, the sHSP of tomato named SlHSP17.7 (Solyc06g076540.1.1) has the characteristic of low temperature induced expression in BL21(DE3) E. coli and a molecular chaperone function in vitro. Overexpression of SlHSP17.7 showed a tolerant response to cold stress treatment due to an induce intracellular sucrose and less accumulation of ROS. Yeast two-hybrid assays showed that SlHSP17.7 is a binding partner of the cation/Ca2+ exchanger (SlCCX1-like; Solyc07g006370.1.1). This interaction was confirmed by pull down and bimolecular fluorescence complementation (BiFC) assays. High SlHSP17.7 and low SlCCX1-like levels alleviated programed cell death (PCD) under cold stress. Thus, SlHSP17.7 might be a cofactor of SlCCX1-like targeting endoplasmic reticulum (ER) membrane proteins, retaining intracellular Ca2+ homeostasis, and decreasing cold stress sensitivity. These findings provide a sound basis for genetic engineering of cold stress tolerance in tomato.
Subject(s)
Cold-Shock Response/genetics , HSP20 Heat-Shock Proteins/genetics , Plant Proteins/genetics , Solanum lycopersicum/physiology , HSP20 Heat-Shock Proteins/metabolism , Solanum lycopersicum/genetics , Phylogeny , Plant Proteins/metabolism , ThermotoleranceABSTRACT
Physico-chemical properties of HspB6 S10F and P20L mutants with abrogated cardioprotective activity and associated with different forms of cardiomyopathy were analyzed. Under normal conditions both the wild-type HspB6 and its mutants formed small size oligomers (dimers) with apparent molecular weight of 50-60 kDa. Under crowding conditions (0.5 M trimethylamine N-oxide, TMAO) the wild-type HspB6 remained predominantly dimeric or formed small molecular weight complexes, whereas both mutants tended to form high molecular weight complexes. Catalytic subunit of cAMP-dependent protein kinase phosphorylated the wild-type HspB6 and its S10F mutant with comparable rate. The rate of P20L mutant phosphorylation was higher than that of the wild-type HspB6. S10F and P20L mutations did not affect interaction of phosphorylated HspB6 with universal adapter proteins 14-3-3. The wild-type HspB6 was resistant to heat-induced denaturation and aggregation, whereas both its mutants were denatured and started to aggregate at temperature much lower than its wild-type counterpart. Titration with fluorescent probe bis-ANS was accompanied by larger increase of fluorescence in the case of both mutants than in the case of the wild-type HspB6. Both mutants possessed higher chaperone-like activity than the wild-type protein. It is concluded that both S10F and P20L mutations are accompanied by increase of hydrophobicity of the very N-terminal region of HspB6 leading to increased aggregation at elevated temperature, formation of large complexes under crowding conditions and increased chaperone-like activity measured in vitro. Increased hydrophobicity and self-association can affect substrate specificity and interaction with certain target proteins thus leading to decrease or complete abrogation of cardioprotective activity.
Subject(s)
HSP20 Heat-Shock Proteins/chemistry , HSP20 Heat-Shock Proteins/genetics , Humans , Mutation , Phosphorylation , Protein Structure, QuaternaryABSTRACT
Probenecid has been used for decades in the treatment of gout but recently has also been found to improve outcomes in patients with heart failure via stimulation of the transient receptor potential vanilloid 2 (TRPV2) channel in cardiomyocytes. This study tested the use of probenecid on a novel mouse model of peripartum cardiomyopathy (PPCM) as a potential treatment option. A human mutation of the human heat shock protein 20 (Hsp20-S10F) in mice has been recently shown to result in cardiomyopathy, when exposed to pregnancies. Treatment with either probenecid or control sucrose water was initiated after the first pregnancy in both wild type and Hsp20-S10F mice. Serial echocardiography was performed during subsequent pregnancies and hearts were collected after the third pregnancies for staining and molecular analysis. Hsp20-S10F mice treated with probenecid had decreased mortality, hypertrophy, TRPV2 expression and molecular parameters of heart failure. Probenecid treatment also decreased apoptosis as evidenced by an increase in the level of Bcl-2/Bax. Probenecid improved survival in a novel mouse model of PPCM and may be an appropriate therapy for humans with PPCM as it has a proven safety and tolerability in patients with heart failure.
Subject(s)
Calcium Channels/genetics , Cardiomyopathies/drug therapy , HSP20 Heat-Shock Proteins/genetics , Heart Failure/drug therapy , Probenecid/pharmacology , TRPV Cation Channels/genetics , Animals , Apoptosis/drug effects , Cardiomyopathies/diagnostic imaging , Cardiomyopathies/genetics , Cardiomyopathies/pathology , Disease Models, Animal , Echocardiography , Female , Gene Expression Regulation/drug effects , Heart Failure/diagnostic imaging , Heart Failure/genetics , Heart Failure/pathology , Humans , Mice , Mutation/genetics , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/pathology , Peripartum Period/drug effects , Pregnancy , Pregnancy Complications, Cardiovascular/drug therapy , Pregnancy Complications, Cardiovascular/geneticsABSTRACT
Small heat shock proteins (shsps) are conserved across invertebrate species. They are implicated in the modulation of various biological processes, such as immune responses, abiotic stress tolerance metamorphosis, and embryonic development. Herein, we identified a heat shock protein 20 from the red swamp crayfish, Procambarus clarkii (named as Pc-Hsp20), and performed in vivo studies to elucidate its physiological functions in the innate immunity. The open reading frame of Pc-Hsp20 was 609 base pair, encoding a protein of 202 amino acid residues with a hsp20/alpha crystallin family domain. Pc-Hsp20 was ubiquitously expressed in various tissues; however, it was highest in the hepatopancreas. The challenge with immune elicitors remarkably enhanced the transcript level of Pc-Hsp20 in the hepatopancreas when compared with the control. Administration of double-stranded RNA could significantly reduce expression of the Pc-Hsp20 mRNAs, and most of the immune-related genes expression enhanced with a variable concentration in the hepatopancreas. Altogether, these results suggest that Pc-Hsp20 may participate in innate immunity against microbial pathogens.
Subject(s)
Arthropod Proteins/genetics , Astacoidea/immunology , HSP20 Heat-Shock Proteins/genetics , Hepatopancreas/physiology , Infections/immunology , Animals , Arthropod Proteins/metabolism , Cloning, Molecular , HSP20 Heat-Shock Proteins/metabolism , Immunity, Innate , Phylogeny , Protein Domains/genetics , RNA, Double-Stranded/immunology , Sequence Alignment , Stress, Physiological , Transcriptome , alpha-Crystallins/geneticsABSTRACT
Small heat shock proteins (sHSPs) have been thought to function as chaperones, protecting their targets from denaturation and aggregation when organisms are subjected to various biotic and abiotic stresses. We previously reported an sHSP from Oryza sativa (OsHSP20) that homodimerizes and forms granules within the cytoplasm but its function was unclear. We now show that OsHSP20 transcripts were significantly up-regulated by heat shock and high salinity but not by drought. A recombinant protein was purified and shown to inhibit the thermal aggregation of the mitochondrial malate dehydrogenase (MDH) enzyme in vitro, and this molecular chaperone activity suggested that OsHSP20 might be involved in stress resistance. Heterologous expression of OsHSP20 in Escherichia coli or Pichia pastoris cells enhanced heat and salt stress tolerance when compared with the control cultures. Transgenic rice plants constitutively overexpressing OsHSP20 and exposed to heat and salt treatments had longer roots and higher germination rates than those of control plants. A series of assays using its truncated mutants showed that its N-terminal arm plus the ACD domain was crucial for its homodimerization, molecular chaperone activity in vitro, and stress tolerance in vivo. The results supported the viewpoint that OsHSP20 could confer heat and salt tolerance by its molecular chaperone activity in different organisms and also provided a more thorough characterization of HSP20-mediated stress tolerance in O. sativa.
Subject(s)
Escherichia coli/metabolism , HSP20 Heat-Shock Proteins , Microorganisms, Genetically-Modified/metabolism , Oryza/genetics , Pichia/metabolism , Plant Proteins , Protein Multimerization , Salt Tolerance , Escherichia coli/genetics , HSP20 Heat-Shock Proteins/biosynthesis , HSP20 Heat-Shock Proteins/genetics , Microorganisms, Genetically-Modified/genetics , Pichia/genetics , Plant Proteins/biosynthesis , Plant Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Salt Stress/geneticsABSTRACT
Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.
Subject(s)
HSP20 Heat-Shock Proteins/genetics , Leishmania donovani/isolation & purification , Leishmaniasis/parasitology , Molecular Diagnostic Techniques/methods , Polymerase Chain Reaction/methods , Animals , DNA Primers/chemistry , DNA, Protozoan/genetics , Humans , Leishmania donovani/genetics , Protozoan Proteins/genetics , Sensitivity and Specificity , Skin/parasitologyABSTRACT
BACKGROUND: Studies have shown that HSP20 (heat-shock protein 20) genes play important roles in regulating plant growth, development, and stress response. However, the grape HSP20 gene family has not been well studied. RESULTS: A total of 48 VvHSP20 genes were identified from the grape genome, which were divided into 11 subfamilies (CI, CII, CIII, CV, CVI, CVII, MI, MII, ER, CP and PX/Po) based on a phylogenetic analysis and subcellular localization. Further structural analysis showed that most of the VvHSP20 genes (93.8%) had no intron or only one intron, while genes that clustered together based on a phylogenetic tree had similar motifs and evolutionarily conserved structures. The HSP20s share a conservedα-crystalline domain (ACD) and the different components of the ACD domain suggest the functional diversity of VvHSP20s. In addition, the 48 VvHSP20 genes were distributed on 12 grape chromosomes and the majority of VvHSP20 genes were located at the proximal or distal ends of chromosomes. Chromosome mapping indicated that four groups of VvHSP20 genes were identified as tandem duplication genes. Phytohormone responsive, abiotic and biotic stress-responsive, and plant development-related cis-elements were identified from the cis-regulatory elements analysis of VvHSP20s. The expression profiles of VvHSP20s genes (VvHSP20-1, 11, 14, 17, 18, 19, 20, 24, 25, 28, 31, 39, 42, and 43) were largely similar between RNA-Seq and qRT-PCR analysis after hydrogen peroxide (H2O2) treatment. The results showed that most VvHSP20s were down-regulated by H2O2 treatment during fruit development. VvHSP20s genes were indeed found to be involved in the grape berry development and differences in their transcriptional levels may be the result of functional differentiation during evolution. CONCLUSIONS: Our results provide valuable information on the evolutionary relationship of genes in the VvHSP20 family, which is useful for future studies on the functional characteristics of VvHSP20 genes in grape.
Subject(s)
Genome, Plant/genetics , HSP20 Heat-Shock Proteins/genetics , Hydrogen Peroxide/pharmacology , Multigene Family , Vitis/genetics , Fruit/drug effects , Fruit/genetics , Fruit/growth & development , Gene Expression Profiling , Genome-Wide Association Study , HSP20 Heat-Shock Proteins/drug effects , Phylogeny , Plant Growth Regulators/metabolism , Plant Proteins/drug effects , Plant Proteins/genetics , Stress, Physiological , Vitis/drug effects , Vitis/growth & developmentABSTRACT
BACKGROUND: The protective effects of heat shock proteins (Hsps) were studied in some infectious and non-infectious diseases, but their specificity was slightly known in various disorders. Among Hsps, small Hsps (e.g. Hsp27 and Hsp20) have important roles in protein folding and translocation, and also in immunity. METHODS: In this study, overexpression of Hsp20 and Hsp27 was performed by transfection of the plasmids encoding Hsp20 and Hsp27 (pEGFP-Hsp20 and pEGFP-Hsp27) into Huh7.5, Hela and Vero cells using Lipofectamine along with heat shock. Then, their anti-herpes simplex virus-1 (HSV-1), anti- human immunodeficiency virus-1 (HIV-1) and anti-hepatitis C virus (HCV) effects, as well as cytotoxicity, were evaluated in vitro, for the first time. RESULTS: Our data showed that simultaneous treatment with Lipofectamine and heat shock augmented the rate of transfection and subsequently the expression of Hsps in these cells. Moreover, overexpression of Hsp20 in HCV-infected Huh7.5 cells, HIV-infected Hela cells and HSV-infected Vero cells reduced the replication of HCV, HIV and HSV, respectively. In contrast, overexpression of Hsp27 significantly decreased HSV replication similar to Hsp20, but it did not affect the replication of HIV and HCV. CONCLUSION: Generally, Hsp20 was identified as a novel anti-HCV, anti-HSV and anti-HIV agent, but Hsp27 was efficient in the suppression of HSV infection. These Hsps may act through suppression of virus entry and/ or through interaction with viral proteins. Thus, it is necessary to determine their exact mechanisms in the near future.
Subject(s)
Antiviral Agents , HIV-1/physiology , HSP20 Heat-Shock Proteins/genetics , HSP27 Heat-Shock Proteins/genetics , Hepacivirus/physiology , Simplexvirus/physiology , Virus Replication/drug effects , Animals , Antiviral Agents/toxicity , Cell Survival/drug effects , Chlorocebus aethiops , HSP20 Heat-Shock Proteins/toxicity , HSP27 Heat-Shock Proteins/toxicity , HeLa Cells , Humans , Lipids , Transfection , Vero CellsABSTRACT
OBJECTIVE: To explore the role of heat shock protein 20 (HSP20)-mediated cardiomyocyte exosomes in the cardiac function in mice with myocardial infarction via the activation of the protein kinase B (Akt) signaling pathway. MATERIALS AND METHODS: A total of 30 mice were enrolled to establish the model of myocardial infarction. Next, these mice were divided into three groups, namely Blank group (healthy mice), Model group (mouse models of myocardial infarction), and HSP20 group (mouse models of myocardial infarction transfected with lentivirus to overexpress HSP20). After that, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining assay was performed to detect myocardial apoptosis. Reactive oxygen species (ROS) accumulation in myocardial tissues was determined via dihydroethidium (DHE) staining assay. Western blotting was employed to analyze the expression level of Akt. The expression levels of inflammatory factors tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1ß) in HSP20-mediated cardiomyocyte exosomes were measured through quantitative real time polymerase chain reaction (qRT-PCR). RESULTS: Compared with that in Blank group, the number of cardiomyocyte exosomes was increased in Model group and HSP20 group under anoxic conditions (p<0.05). The results of quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) proved that the HSP20 messenger ribonucleic acid (mRNA) expression in mediated cardiomyocyte exosomes was significantly lower in Model group than that in Blank group (p<0.05), while in HSP20 group, it was overtly higher than that in Model group but clearly lowered compared with that in Blank group (p<0.05). The protein expression of Akt in cardiomyocyte exosomes was evidently decreased in Model group compared with that in Blank group (p<0.05), while it was notably increased in HSP20 group compared with that in Model group (p<0.05). In comparison with Blank group, Model group had significantly elevated mRNA expression levels of TNF-α and IL-1ß. The mRNA expression levels of TNF-α and IL-1ß in HSP20 group were remarkably lower than those in Model group (p<0.05). The results of TUNEL assay revealed that the overexpression of HSP20 affected myocardial apoptosis. The myocardial apoptosis index in Model group [(38.42±2.52) %] was higher than that in Blank group [(9.74±1.21) %], HSP20 group had a significantly decreased myocardial apoptosis index [(22.36±2.13) %] in comparison with Model group (p<0.05). In accordance with DHE staining comparison, the accumulation of ROS in myocardial tissues in Model group was significantly higher than that in Blank group (p<0.05) and HSP20 group (p<0.05). CONCLUSIONS: We demonstrated that HSP20-mediated cardiomyocyte exosomes activate the AKT signaling pathway, repress TNF-α and IL-1ß factors, and alleviate myocardial infarction.
Subject(s)
Exosomes/metabolism , HSP20 Heat-Shock Proteins/metabolism , Myocardial Infarction/pathology , Myocytes, Cardiac/ultrastructure , Proto-Oncogene Proteins c-akt/metabolism , Animals , Apoptosis , Cells, Cultured , Disease Models, Animal , Exosomes/ultrastructure , HSP20 Heat-Shock Proteins/genetics , Humans , Interleukin-1beta/metabolism , Male , Mice , Microscopy, Electron, Transmission , Myocardium/cytology , Myocardium/pathology , Myocytes, Cardiac/metabolism , Primary Cell Culture , Reactive Oxygen Species/metabolism , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Preeclampsia (PE) remains the leading cause of maternal and fetal morbidity and mortality. Excessive apoptosis of the placenta and poor remodeling of spiral arteries caused by insufficient invasion of trophoblast cells into uterus have been implicated in the pathogenesis of PE. Accumulating evidence showed that heat shock protein 20 (HSP20) is closely associated with the proliferation, apoptosis, and metastasis of tumor cells. However, little is known about whether HSP20 plays a role in the development of PE. In this study, we detected the apoptosis index and the expressions of HSP20 and apoptosis-associated proteins in the placentas from PE and normal pregnancies. We found that HSP20 was reversely related to the apoptosis rate and the levels of proapoptotic proteins. Moreover, we identified that HSP20 could suppress the proliferation and apoptosis of trophoblast cells, turning them into a more invasive phenotype. Additionally, H2O2-induced oxidative stress was significantly alleviated, and several key proteins on the Akt signaling pathway were upregulated in HSP20-overexpressing trophoblast cells. These findings strongly suggested that HSP20 might play a role in the remodeling of spiral arteries through affecting the invasiveness of extravillous trophoblast cells via Akt signaling pathway, and the dysregulation of it might contribute to the pathophysiology of PE.