Heterologous overexpression of heat shock protein 20 genes of different species of yellow Camellia in Arabidopsis thaliana reveals their roles in high calcium resistance.

Yellow Camellia (Camellia sect. chrysantha) is a rare ornamental plant and an important germplasm resource globally. Camellia nitidissima thrives in normal acidic soils, while Camellia limonia can adapt to the calcareous soils found in karst areas. Our previous study on the karst adaptation of yellow camellias revealed that the expression levels of heat shock protein 20(HSP20) were higher in Camellia limonia than in Camellia nitidissima. However, the functions of the HSP20 gene of Camellia limonia remain unclear to data. In this study, the HSP20 genes of Camellia limonia (ClHSP20-OE lines) and Camellia. nitidissima (CnHSP20-OE lines) were cloned and overexpressed heterologously in Arabidopsis thaliana. Additionally, we overexpressed the HSP20 gene of Arabidopsis (AtHSP20-OE lines) was also overexpressed, and the T-DNA inserted mutants (athspmutant lines) were also used to determine the functions of HSP20 genes. Under high calcium stress, the chlorophyll, nitrogen, water content and humidity of leaves were increased in ClHSP20-OE lines, while those of other lines were declined. The size of the stomatal apertures, stomatal conductance, and the photosynthetic efficiency of ClHSP20-OE lines were higher than those of the other lines. However, the accumulation of H2O2 and O2- in the leaves of ClHSP20-OE lines was the lowest among all the lines. Energy spectrum scanning revealed that the percentage of calcium on the surfaces of the leaves of ClHSP20-OE lines was relatively low, while that of athspmutant lines was the highest. The ClHSP20 gene can also affected soil humidity and the contents of soil nitrogen, phosphorus, and potassium. Transcriptome analysis revealed that the expressions of FBA5 and AT5G10770 in ClHSP20-OE lines was significantly up-regulated compared to that of CnHSP20-OE lines. Compared to that of athspmutant lines, the expressions of DREB1A and AT3G30460 was significantly upregulated in AtHSP20-OE lines, and the expression of POL was down-regulated. Our findings suggest that the HSP20 gene plays a crucial role in maintained photosynthetic rate and normal metabolism by regulating the expression of key genes under high-calcium stress. This study elucidates the mechanisms underlying the karst adaptation in Camellia. limonia and provides novel insights for future research on karst plants.

Cellular functions of heat shock protein 20 (HSPB6) in cancer: A review.

Heat shock proteins (HSP) are a large family of peptide proteins that are widely found in cells. Studies have shown that the expression and function of HSPs in cells are very complex, and they can participate in cellular physiological and pathological processes through multiple pathways. Multiple heat shock proteins are associated with cancer cell growth, proliferation, metastasis, and resistance to anticancer drugs, and they play a key role in cancer development by ensuring the correct folding or degradation of proteins in cancer cells. As research hotspots, HSP90, HSP70 and HSP27 have been extensively studied in cancer so far. However, HSP20, also referred to as HSPB6, as a member of the small heat shock protein family, has been shown to play an important role in the cardiovascular system, but little research has been conducted on HSP20 in cancer. This review summarizes the current cellular functions of HSP20 in different cancer types, as well as its effects on cancer proliferation, progression, prognosis, and its other functions in cancer, to illustrate the close association between HSP20 and cancer. We show that, unlike most HSPs, HSP20 mainly plays an active anticancer role in cancer development, which is expected to provide new ideas and help for cancer diagnosis and treatment and research.

Genome-wide analysis of the HSP20 gene family and its response to heat and drought stress in Coix (Coix lacryma-jobi L.).

BACKGROUND: Heat shock protein 20 (HSP20) is a member of the heat stress-related protein family, which plays critical roles in plant growth, development, and response to abiotic stresses. Although many HSP20 genes have been associated with heat stress in numerous types of plants, little is known about the details of the HSP20 gene family in Coix. To investigate the mechanisms of the ClHSP20 response to heat and drought stresses, the ClHSP20 gene family in Coix was identified and characterized based on genome-wide analysis. RESULTS: A total of 32 putative ClHSP20 genes were identified and characterized in Coix. Phylogenetic analysis indicated that ClHSP20s were grouped into 11 subfamilies. The duplicated event analysis demonstrated that tandem duplication and segment duplication events played crucial roles in promoting the expansion of the ClHSP20 gene family. Synteny analysis showed that Coix shared the highest homology in 36 HSP20 gene pairs with wheat, followed by 22, 19, 15, and 15 homologous gene pairs with maize, sorghum, barley, and rice, respectively. The expression profile analysis showed that almost all ClHSP20 genes had different expression levels in at least one tissue. Furthermore, 22 of the 32 ClHSP20 genes responded to heat stress, with 11 ClHSP20 genes being significantly upregulated and 11 ClHSP20 genes being significantly downregulated. Furthermore, 13 of the 32 ClHSP20 genes responded to drought stress, with 6 ClHSP20 genes being significantly upregulated and 5 ClHSP20 genes being significantly downregulated. CONCLUSIONS: Thirty-two ClHSP20 genes were identified and characterized in the genome of Coix. Tandem and segmental duplication were identified as having caused the expansion of the ClHSP20 gene family. The expression patterns of the ClHSP20 genes suggested that they play a critical role in growth, development, and response to heat and drought stress. The current study provides a theoretical basis for further research on ClHSP20s and will facilitate the functional characterization of ClHSP20 genes.

Heat shock protein 20 suppresses breast carcinogenesis by inhibiting the MAPK and AKT signaling pathways.

Heat shock protein (HSP) 20 belongs to the small HSP family and exhibits diverse functions, including tumor suppression, in addition to being a molecular chaperon, which is the classical property of HSPs. The present study aimed to examine the association between HSP20 expression and breast cancer (BC) progression in patients, and to explore the possible role of HSP20 in malignant phenotypes of BC cells. A series of experiments, including reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8 and flow cytometry, were performed. Data from Gene Expression Omnibus and Kaplan-Meier Plotter revealed that HSP20 expression was significantly downregulated in BC tissues, and patients with BC with lower HSP20 expression exhibited poorer recurrence-free survival. The data revealed that HSP20 was closely associated with the pathological tumor stage (P=0.015) and pathological tumor node metastasis (P=0.031) of patients with BC. Additionally, HSP20 expression was markedly decreased in BC cell lines. Exogenous overexpression of HSP20 inhibited proliferation and accelerated apoptosis of BC cells. These cells exhibited decreased migration and invasion when HSP20 was overexpressed. Furthermore, HSP20 overexpression suppressed the MAPK and AKT signaling pathways, as evidenced by the reduced phosphorylation levels of AKT, ERK, JNK and p38. Knockdown of HSP20 exerted the opposite effects. Notably, the AKT agonist, SC79, and the ERK agonist, LM22B-10, reversed the decrease in cell proliferation and migration induced by HSP20 overexpression. Overall, the data suggest that the decreased expression of HSP20 in BC tissues may be associated with disease progression. HSP20 also attenuated the malignant phenotype of BC cells and the inhibition of MAPK and AKT signaling may be associated with this effect. Therefore, HSP20 may be a potential prognostic marker or a candidate therapeutic target for BC.

Genome-wide identification, characterization and expression of HSP 20 gene family in dove.

Davidia involucrata is a significant living fossil with high abiotic stress tolerance. Although heat shock protein 20 (HSP20) has already been linked to heat stress, nothing is known about HSP20 family protein activities in D. involucrata. The functional dynamics of the D. involucrata HSP20 (DiHSP20) gene family were identified and characterized using a thorough genome-wide investigation. From the genome of D. involucrata, a total of 42 HSP20 genes were identified, which are distributed across 16 chromosomes. The DiHSP20 proteins were grouped into seven separate subfamilies by our phylogenetic analysis, which was validated by the conserved motif composition and gene structure studies. Segmental duplication events were shown to play a crucial role in the expansion of the DiHSP20 gene family. Synteny analysis revealed that 19 DiHSP20 genes of D. involucrata shared a syntenic connection with Arabidopsis genes, 39 with C. acuminata genes, and just 6 with O. sativa genes. Additionally, heat stress differently enhanced the expression levels of D. involucrata HSP20 genes. After 1 hour of heat treatment, the expression levels of most DiHSP20 genes, particularly DiHSP20-7, DiHSP20-29, DiHSP20-30, DiHSP20-32, and DiHSP20-34, were dramatically increased, suggestted that they might be employed as heat tolerance candidate genes. Overall, these findings add to our knowledge of the HSP20 family genes and provide helpful information for breeding heat stress resistance in D. involucrata.

Genome-Wide Identification and Characterization of Heat Shock Protein 20 Genes in Maize.

Maize is an important cereal crop worldwide and is sensitive to abiotic stresses in fluctuant environments that seriously affect its growth, yield, and quality. The small heat shock protein (HSP20) plays a crucial role in protecting plants from abiotic stress. However, little is known about HSP20 in maize (ZmHSP20). In this study, 44 ZmHSP20s were identified, which were unequally distributed over 10 chromosomes, and 6 pairs of ZmHSP20s were tandemly presented. The gene structure of ZmHSP20s was highly conserved, with 95% (42) of the genes having no more than one intron. The analysis of the cis-element in ZmHSP20s promoter demonstrated large amounts of elements related to hormonal and abiotic stress responses, including abscisic acid (ABA), high temperature, and hypoxia. The ZmHSP20s protein had more than two conserved motifs that were predictably localized in the cytoplasm, nucleus, endoplasmic reticulum, peroxisome, mitochondria, and plasma. Phylogenetic analysis using HSP20s in Arabidopsis, rice, maize, and Solanum tuberosum indicated that ZmHSP20s were classified into 11 categories, of which each category had unique subcellular localization. Approximately 80% (35) of ZmHSP20 were upregulated under heat stress at the maize seedling stage, whereas the opposite expression profiling of 10 genes under 37 and 48 °C was detected. A total of 20 genes were randomly selected to investigate their expression under treatments of ABA, gibberellin (GA), ethylene, low temperature, drought, and waterlogging, and the results displayed that more than half of these genes were downregulated while ZmHSP20-3, ZmHSP20-7, ZmHSP20-24, and ZmHSP20-44 were upregulated under 1 h treatment of ethylene. A yeast-one-hybrid experiment was conducted to analyze the binding of four heat stress transcription factors (ZmHSFs) with eight of the ZmHSP20s promoter sequences, in which ZmHSF3, ZmHSF13, and ZmHSF17 can bind to most of these selected ZmHSP20s promoters. Our results provided a valuable resource for studying HSP20s function and offering candidates for genetic improvement under abiotic stress.

Hsp20 Promotes Endothelial Progenitor Cell Angiogenesis via Activation of PI3K/Akt Signaling Pathway under Hypoxia.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. METHODS: Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. RESULTS: CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. CONCLUSION: In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

Comprehensive Analysis of the Hsp20 Gene Family in Canavalia rosea Indicates Its Roles in the Response to Multiple Abiotic Stresses and Adaptation to Tropical Coral Islands.

Heat shock protein 20 (Hsp20) is a major family of heat shock proteins that mainly function as molecular chaperones and are markedly accumulated in cells when organisms are subjected to environmental stress, particularly heat. Canavalia rosea is an extremophile halophyte with good adaptability to environmental high temperature and is widely distributed in coastal areas or islands in tropical and subtropical regions. In this study, we identified a total of 41 CrHsp20 genes in the C. rosea genome. The gene structures, phylogenetic relationships, chromosome locations, and conserved motifs of each CrHsp20 or encoding protein were analyzed. The promoters of CrHsp20s contained a series of predicted cis-acting elements, which indicates that the expression of different CrHsp20 members is regulated precisely. The expression patterns of the CrHsp20 family were analyzed by RNA sequencing both at the tissue-specific level and under different abiotic stresses, and were further validated by quantitative reverse transcription PCR. The integrated expression profiles of the CrHsp20s indicated that most CrHsp20 genes were greatly upregulated (up to dozens to thousands of times) after 2 h of heat stress. However, some of the heat-upregulated CrHsp20 genes showed completely different expression patterns in response to salt, alkaline, or high osmotic stresses, which indicates their potential specific function in mediating the response of C. rosea to abiotic stresses. In addition, some of CrHsp20s were cloned and functionally characterized for their roles in abiotic stress tolerance in yeast. Taken together, these findings provide a foundation for functionally characterizing Hsp20s to unravel their possible roles in the adaptation of this species to tropical coral reefs. Our results also contribute to the understanding of the complexity of the response of CrHsp20 genes to other abiotic stresses and may help in future studies evaluating the functional characteristics of CrHsp20s for crop genetic improvement.

Heat Shock Protein 20 Gene Superfamilies in Red Algae: Evolutionary and Functional Diversities.

Heat shock protein 20 (Hsp20) genes play important roles in plant growth, development, and response to environmental stress. However, the Hsp20 gene family has not yet been systematically investigated, and its function in red algae (Rhodophyta) remains poorly understood. Herein, we characterized Hsp20 gene families in red algae by studying gene structure, conserved motifs, phylogenetic relationships, chromosome location, gene duplication, cis-regulatory elements, and expression profiles. In this study, 97 Hsp20 genes were identified using bioinformatic methods and classified into 13 subfamilies based on phylogenetic relationships. Phylogenetic analysis revealed that Hsp20 genes might have a polyphyletic origin and a complex evolutionary pattern. Gene structure analysis revealed that most Hsp20 genes possessed no introns, and all Hsp20 genes contained a conserved α-crystalline domain in the C-terminal region. Conserved motif analysis revealed that Hsp20 genes belonging to the same subfamily shared similar motifs. Gene duplication analysis demonstrated that tandem and segmental duplication events occurred in these gene families. Additionally, these gene families in red algae might have experienced strong purifying selection pressure during evolution, and Hsp20 genes in Pyropia yezoensis, Pyropia haitanensis, and Porphyra umbilicalis were highly evolutionarily conserved. The cis-elements of phytohormone-, light-, stress-responsive, and development-related were identified in the red algal Hsp20 gene promoter sequences. Finally, using Py. yezoensis, as a representative of red algae, the Hsp20 gene expression profile was explored. Based on the RNA-seq data, Py. yezoensis Hsp20 (PyyHsp20) genes were found to be involved in Py. yezoensis responses against abiotic and biotic stresses and exhibited diverse expression patterns. Moreover, PyyHsp20 is involved in Py. yezoensis growth and development and revealed spatial and temporal expression patterns. These results provide comprehensive and valuable information on Hsp20 gene families in red algae and lay a foundation for their functional characterization. In addition, our study provides new insights into the evolution of Hsp20 gene families in red algae and will help understand the adaptability of red algae to diverse environments.

Pumpkin (Cucurbita moschata) HSP20 Gene Family Identification and Expression Under Heat Stress.

Pumpkin (Cucurbita moschata) is an important cucurbit vegetable crop that has strong resistance to abiotic stress. While heat shock protein 20 (HSP20) has been implicated in vegetable response to heat stress, little is known regarding activity of HSP20 family proteins in C. moschata. Here, we performed a comprehensive genome-wide analysis to identify and characterize the functional dynamics of the Cucurbita moschata HSP20 (CmoHSP20) gene family. A total of 33 HSP20 genes distributed across 13 chromosomes were identified from the pumpkin genome. Our phylogenetic analysis determined that the CmoHSP20 proteins fell into nine distinct subfamilies, a division supported by the conserved motif composition and gene structure analyses. Segmental duplication events were shown to play a key role in expansion of the CmoHSP20 gene family. Synteny analysis revealed that 19 and 18 CmoHSP20 genes were collinear with those in the cucumber and melon genomes, respectively. Furthermore, the expression levels of pumpkin HSP20 genes were differentially induced by heat stress. The transcript level of CmoHSP20-16, 24 and 25 were down-regulated by heat stress, while CmoHSP20-7, 13, 18, 22, 26 and 32 were up-regulated by heat stress, which could be used as heat tolerance candidate genes. Overall, these findings contribute to our understanding of vegetable HSP20 family genes and provide valuable information that can be used to breed heat stress resistance in cucurbit vegetable crops.

Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells.

BACKGROUND: Heat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed. AIM: To evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration. METHODS: We used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism. RESULTS: This study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSION: We found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.

Transcriptional Responses of the Heat Shock Protein 20 (Hsp20) and 40 (Hsp40) Genes to Temperature Stress and Alteration of Life Cycle Stages in the Harmful Alga Scrippsiella trochoidea (Dinophyceae).

The small heat shock protein (sHsp) and Hsp40 are Hsp members that have not been intensively investigated but are functionally important in most organisms. In this study, the potential roles of a Hsp20 (StHsp20) and a Hsp40 (StHsp40) in dinoflagellates during adaptation to temperature fluctuation and alteration of different life stages were explored using the representative harmful algal blooms (HABs)-causative dinoflagellate species, Scrippsiella trochoidea. We isolated the full-length cDNAs of the two genes via rapid amplification of cDNA ends (RACE) and tracked their differential transcriptions via real-time qPCR. The results revealed StHsp20 and StHsp40 exhibited mRNA accumulation patterns that were highly similar in response to heat stress but completely different toward cold stress, which implies that the mechanisms underlying thermal and cold acclimation in dinoflagellates are regulated by different sets of genes. The StHsp20 was probably related to the heat tolerance of the species, and StHsp40 was closely involved in the adaptation to both higher and lower temperature fluctuations. Furthermore, significantly higher mRNA abundance of StHsp40 was detected in newly formed resting cysts, which might be a response to intrinsic stress stemmed from encystment. This finding also implied StHsp40 might be engaged in resting cyst formation of S. trochoidea. Our findings enriched the knowledge about possible cross-talk of different Hsp members in dinoflagellates and provided clues to further explore the molecular underpinnings underlying resting cyst production and broad temperature tolerance of this group of HABs contributors.

Inducible expression of heat shock protein 20 protects airway epithelial cells against oxidative injury involving the Nrf2-NQO-1 pathway.

BACKGROUND: Heat shock protein (HSP) 20 is a molecular chaperone that exerts multiple protective functions in various kinds of tissues. However, the expression of HSP20 and its specific functions in airway epithelial cells (AECs) remain elusive. RESULTS: In current study, we first confirmed the inducible expression of HSP20 in mouse AECs and in a human bronchial epithelial cell line BEAS-2B cells, under different oxidant stressors. Then by establishing a HSP20-abundant mouse model with repeated low-level-ozone exposures and stimulating this model with a single high-level ozone exposure, we found that the HSP20 abundance along with its enhanced phosphorylation potentially contributed to the alleviation of oxidative injuries, evidenced by the decreases in the bodyweight reduction, the BAL neutrophil accumulation, the AECs shedding, and the BAL concentrations of albumin and E-cadherin. The biological function of HSP20 and its molecular mechanisms were further investigated in BEAS-2B cells that were transfected with HSP20-, unphosphorylatable HSP20(Ala) or empty vector plasmids prior to the stimulation of H2O2, of which its oxidant capacity has been proved to be similar with those of ozone in an air-liquid culture system. We found that the H2O2-induced intracellular ROS level and the early cell apoptosis were attenuated in the HSP20- but not HSP20(Ala)- transfected cells. The intracellular expression of NQO-1 (mRNA and protein) and the intranuclear content of Nrf2 were significantly increased in the HSP20- transfected cells but not in the HSP20(Ala)- and empty vector-transfected cells after the stimulation of H2O2. CONCLUSIONS: The inducible expression of HSP20 in AECs by oxidative stress exerts protective roles against oxidative damages, which may involve the activation of the Nrf2-NQO-1 pathway.

Ac-HSP20 Is Associated With the Infectivity and Encystation of Acanthamoeba castellanii.

Acanthamoeba castellanii is a pathogenic and opportunistic free-living amoeba that causes Acanthamoeba keratitis (AK) and granulomatous amebic encephalitis (GAE) in immunocompromised individuals. The biological and pathogenic characterizations behind this opportunistic protozoan is not fully understood. This study aimed to determine the biological functions of heat shock protein (HSP)-20 of A. castellanii (Ac-HSP20) involved in the maintenance of life cycle and the infectivity of A. castellanii. Immunoscreening A. castellanii cDNA library with A. castellanii infected rabbit sera identified three positive clones, one of them was a putative heat shock protein (Ac-HSP20). The recombinant 23 kDa Ac-HSP20 protein (rAc-HSP20) was successfully expressed in Escherichia coli BL21 (DE3) and purified using metal affinity chromatography. The rabbits immunized with rAc-HSP20 produced high titer antibody (1:25,600). Immunolocalization with the antibody identified the expression of native Ac-HSP20 on the surface of both A. castellanii trophozoites and cysts. Further, Western blot with antibody identified that the expression of native Ac-HSP20 was 7.5 times higher in cysts than in trophozoites. Blocking Ac-HSP20 on the membrane of trophozoites with specific antibody or silencing Ac-hsp20 gene transcription by siRNA inhibited their transformation into cysts at the early stage but returned to normal at the late stage by stimulating the transcription of Ac-hsp20. Incubation of trophozoites with anti-Ac-HSP20 IgG increased macrophage-involved phagocytosis to the protozoa and inhibited trophozoite infectivity on the cornea of rabbits compared with that without antibody. Our study provides that Ac-HSP20 is a surface antigen involved in the encystation and infectivity of A. castellanii and thus an important target for vaccine and drug development.

M918: A Novel Cell Penetrating Peptide for Effective Delivery of HIV-1 Nef and Hsp20-Nef Proteins into Eukaryotic Cell Lines.

BACKGROUND: HIV-1 Nef protein is a possible attractive target in the development of therapeutic HIV vaccines including protein-based vaccines. The most important disadvantage of protein-based vaccines is their low immunogenicity which can be improved by heat shock proteins (Hsps) as an immunomodulator, and cell-penetrating peptides (CPPs) as a carrier. METHODS: In this study, the HIV-1 Nef and Hsp20-Nef proteins were generated in E.coli expression system for delivery into the HEK-293T mammalian cell line using a novel cell-penetrating peptide, M918, in a non-covalent fashion. The size, zeta potential and morphology of the peptide/protein complexes were studied by scanning electron microscopy (SEM) and Zeta sizer. The efficiency of Nef and Hsp20-Nef transfection using M918 was evaluated by western blotting in HEK-293T cell line. RESULTS: The SEM data confirmed the formation of discrete nanoparticles with a diameter of approximately 200-250 nm and 50-80 nm for M918/Nef and M918/Hsp20-Nef, respectively. The dominant band of ~ 27 kDa and ~ 47 kDa was detected in the transfected cells with the Nef/ M918 and Hsp20-Nef/ M918 nanoparticles at a molar ratio of 1:20 using anti-HIV-1 Nef monoclonal antibody. These bands were not detected in the un-transfected and transfected cells with Nef or Hsp20- Nef protein alone indicating that M918 could increase the penetration of Nef and Hsp20-Nef proteins into the cells. CONCLUSION: These data suggest that M918 CPP can be used to enter HIV-1 Nef and Hsp20-Nef proteins inside mammalian cells efficiently as a promising approach in HIV-1 vaccine development.

Genome-Wide Analysis of Watermelon HSP20s and Their Expression Profiles and Subcellular Locations under Stresses.

Watermelon (Citrullus lanatus L.), which is an economically important cucurbit crop that is cultivated worldwide, is vulnerable to various adverse environmental conditions. Small heat shock protein 20s (HSP20s) are the most abundant plant HSPs and they play important roles in various biotic and abiotic stress responses. However, they have not been systematically investigated in watermelon. In this study, we identified 44 watermelon HSP20 genes and analyzed their gene structures, conserved domains, phylogenetic relationships, chromosomal distributions, and expression profiles. All of the watermelon HSP20 proteins have a conserved the α-crystallin (ACD) domain. Half of the ClHSP20s arose through gene duplication events. Plant HSP20s were grouped into 18 subfamiles and a new subfamily, nucleo-cytoplasmic XIII (CXIII), was identified in this study. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter regions of the watermelon HSP20 genes. Different from that in other species, half of the watermelon HSP20s were repressed by heat stress. Plant HSP20s displayed diverse responses to different virus infections and most of the ClHSP20s were generally repressed by Cucumber green mottle mosaic virus (CGMMV). Some ClHSP20s exhibited similar transcriptional responses to abscisic acid, melatonin, and CGMMV. Subcellular localization analyses of six selected HSP20- green fluorescence protein fusion proteins revealed diverse subcellular targeting. Some ClHSP20 proteins were affected by CGMMV, as reflected by changes in the size, number, and distribution of fluorescent granules. These systematic analyses provide a foundation for elucidating the physiological functions and biological roles of the watermelon HSP20 gene family.

The oligomeric plasticity of Hsp20 of Sulfolobus acidocaldarius protects environment-induced protein aggregation and membrane destabilization.

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that rescue misfolded proteins from irreversible aggregation during cellular stress. Many such sHsps exist as large polydisperse species in solution, and a rapid dynamic subunit exchange between oligomeric and dissociated forms modulates their function under a variety of stress conditions. Here, we investigated the structural and functional properties of Hsp20 from thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. To provide a framework for investigating the structure-function relationship of Hsp20 and understanding its dynamic nature, we employed several biophysical and biochemical techniques. Our data suggested the existence of a ~24-mer of Hsp20 at room temperature (25 °C) and a higher oligomeric form at higher temperature (50 °C-70 °C) and lower pH (3.0-5.0). To our surprise, we identified a dimeric form of protein as the functional conformation in the presence of aggregating substrate proteins. The hydrophobic microenvironment mainly regulates the oligomeric plasticity of Hsp20, and it plays a key role in the protection of stress-induced protein aggregation. In Sulfolobus sp., Hsp20, despite being a non-secreted protein, has been reported to be present in secretory vesicles and it is still unclear whether it stabilizes substrate proteins or membrane lipids within the secreted vesicles. To address such an issue, we tested the ability of Hsp20 to interact with membrane lipids along with its ability to modulate membrane fluidity. Our data revealed that Hsp20 interacts with membrane lipids via a hydrophobic interaction and it lowers the propensity of in vitro phase transition of bacterial and archaeal lipids.

Development of HCV Therapeutic Vaccines Using Hp91 Peptide and Small Heat Shock Protein 20 as an Adjuvant.

BACKGROUND: Hepatitis C Virus (HCV) is a major cause of chronic liver disease in the world. Many studies showed that T-cell responses to HCV Nonstructural Protein 3 (NS3) play an important role in clearing acute HCV infections, thus NS3 can be considered as a suitable candidate antigen for development of a HCV therapeutic vaccine. OBJECTIVE: We used Hp91 peptide and small heat shock protein 20 as an adjuvant for enhancement of NS3-specific humoral and cellular immunity in mice. METHOD: In this study, various NS3 DNA and protein constructs were generated in eukaryotic and prokaryotic expression systems, and their potency in eliciting humoral and cellular immune responses was compared using small Heat shock protein 20 (Hsp20), the High Mobility Group Box 1 protein (HMGB1)-derived peptide (Hp91), and Freund's adjuvant in a BALB/c mouse model. RESULTS: Our results indicated that both the Hsp20 conjugated with NS3 protein and Hp91 significantly enhanced the levels of IgG2a, IgG2b and IFN-γ directed toward Th1 responses compared to other groups. Moreover, the immunostimulatory properties of Hp91 were significantly higher than Hsp20 and Freund's adjuvant in various immunization strategies. Mice immunized by NS3 protein formulated with Freund's adjuvant, and also mixed with Hp91 peptide showed higher secretion of Granzyme B than other groups. CONCLUSION: Hp91 peptide could develop NS3-specific B- and T-cell immune responses as a promising HCV therapeutic vaccine candidate in future.

Exercise Rehabilitation Attenuates Cognitive Deficits in Rats with Traumatic Brain Injury by Stimulating the Cerebral HSP20/BDNF/TrkB Signalling Axis.

Physical exercise (PE) is an effective method for improving cognitive function among patients with traumatic brain injury (TBI). We previously demonstrated that PE with an infrared-sensing running wheel (ISRW) system provides strong neuroprotection in an experimental animal model of stroke. In this study, we used fluid percussion injury in rats to simulate mild TBI. For rats, we used both passive avoidance learning and the Y-maze tests to evaluate cognitive function. We investigated whether PE rehabilitation attenuated cognitive deficits in rats with TBI and determined the contribution of hippocampal and cortical expression of heat shock protein 20 (HSP20) to PE-mediated cognitive recovery. In addition to increasing hippocampal and cortical expression of HSP20, brain-derived neurotrophic factor (BDNF), and the tropomyosin receptor kinase B (TrkB) ratio, PE rehabilitation significantly attenuated brain contusion and improved cognitive deficits in the rat model. Furthermore, reducing hippocampal and cortical expression of HSP20 with an intracerebral injection of pSUPER hsp20 small interfering RNA significantly diminished the PE-induced overexpression of hippocampal and cortical BDNF and the TrkB ratio and also reversed the beneficial effect of PE in reducing neurotrauma and the cognitive deficits. A positive Pearson correlation was found between HSP20 and BDNF, as well as between HSP20 and TrkB, in the hippocampal and cortical tissues. We thus conclude that post-ischaemic ISRW exercise rehabilitation attenuates cognitive deficits, as well as brain contusions, in TBI rats by stimulating the cerebral HSP20/BDNF/TrkB signalling axis.

Alteration of heat shock protein 20 expression in preeclamptic patients and its effect in vascular and coagulation function.

Preeclampsia (PE) is a pregnancy-specific, multi-system disorder and the leading cause of maternal and perinatal morbidity and mortality in obstetrics worldwide. Excessive vasoconstriction and dysregulated coagulation function are closely associated with PE. Heat shock protein 20 (HSP20) is ubiquitously expressed under normal physiological conditions and has important roles in vascular dilatation and suppression of platelet aggregation. However, the role of HSP20 in the pathogenesis of PE remains unclear. In this study, we collected chorionic plate resistance arteries (CPAs) and serum from 118 healthy pregnant women and 80 women with PE and detected the levels of HSP20 and its phosphorylated form. Both HSP20 and phosphorylated HSP20 were downregulated in CPAs from women with PE. Comparison of the vasodilative ability of CPAs from the two groups showed impaired relaxation responses to acetyl choline in preeclamptic vessels. In addition to the reduced HSP20 in serum from women with PE, the platelet distribution width and mean platelet volume were also decreased, and the activated partial thromboplastin time and thromboplastin time were elevated.With regard to the vital roles of HSP20 in mediating vasorelaxation and coagulation function, the decreased HSP20 might contribute to the pathogenesis of PE.