kDa Heat Shock Research Articles

Cloning, prokaryotic expression, and functional characterization of a novel 70-kDa heat shock protein (DnaK) from Bacillus persicus

The dnaK gene of Bacillus persicus strain B48(T) was cloned, sequenced, and functionally characterized in the present study. The gene was 1836 bp in length, encoding a polypeptide of 611 amino acid residues. The 3D structure of protein predicted by I-TASSER represented the similarity of the overall structures of DnaK from B. persicus strain B48(T) and human protein Hsp70 (BiP) with a homology of 89 %. Based on the results, refolding heat-denatured carbonic anhydrase increased significantly up to 80 % in the presence of the purified recombinant DnaK. In addition, salt resistance experiments demonstrated a 2.7-fold increase in the survival of recombinant E. coli BL21–DnaK in the presence of 0.4 M NaCl for 60 h compared to that of the control cells. Further, Cd2+ failed to affect DnaK refolding function, while Hg2+ ion reflected a biphasic effect (inhibiting and stimulating at lower and higher than 100 nM concentrations, respectively). Finally, DnaK from B. persicus can potentially be used for improving the functional properties of enzymes and proteins through increasing folding activity and enhancing stress tolerance.

Origin and Evolution of the Human Bcl2-Associated Athanogene-1 (BAG-1).

Molecular chaperones, particularly the 70-kDa heat shock proteins (Hsp70s), are key orchestrators of the cellular stress response. To perform their critical functions, Hsp70s require the presence of specific co-chaperones, which include nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. BAG-1 is one of these proteins that function in a wide range of cellular processes, including apoptosis, protein refolding, and degradation, as well as tumorigenesis. However, the origin of BAG-1 proteins and their evolution between and within species are mostly uncharacterized. This report investigated the macro- and micro-evolution of BAG-1 using orthologous sequences and single nucleotide polymorphisms (SNPs) to elucidate the evolution and understand how natural variation affects the cellular stress response. We first collected and analyzed several BAG-1 sequences across animals, plants, and fungi; mapped intron positions and phases; reconstructed phylogeny; and analyzed protein characteristics. These data indicated that BAG-1 originated before the animals, plants, and fungi split, yet most extant fungal species have lost BAG-1. Furthermore, although BAG-1’s structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional specialization within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP density is unequally distributed within the BAG1 gene, and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the BAG domain region, which is an indication of positive selection. To further explore this notion, we performed several biochemical assays and found that only one out of five mutations tested altered the major co-chaperone properties of BAG-1. These data collectively suggest that although the co-chaperone functions of BAG-1 are highly conserved and can probably tolerate several radical mutations, BAG-1 might have acquired specialized and potentially unexplored functions during the evolutionary process.

Pollen development in cotton (Gossypium hirsutum) is highly sensitive to heat exposure during the tetrad stage.

The development of gametes in plants is acutely susceptible to heatwaves as brief as a few days, adversely affecting pollen maturation and reproductive success. Pollen in cotton (Gossypium hirsutum) was differentially affected when tetrad and binucleate stages were exposed to heat, revealing new insights into the interaction between heat and pollen development. Squares were tagged and exposed to 36/25°C (day/night, moderate heat) or 40/30°C (day/night, extreme heat) for 5 days. Mature pollen grains and leaves were collected for physiological and proteomic responses. While photosynthetic competence was not compromised even at 40°C, leaf tissues became leakier. In contrast, pollen grains were markedly smaller after the tetrad stage was exposed to 40°C and boll production was reduced by 65%. Sugar levels in pollen grains were elevated after exposure to heat, eliminating carbohydrate deficits as a likely cause of poor reproductive capacity. Proteomic analysis of pure pollen samples revealed a particularly high abundance of 70-kDa heat shock (Hsp70s) and cytoskeletal proteins. While short-term bursts of heat had a minor impact on leaves, male gametophyte development was profoundly damaged. Cotton acclimates to maxima of 36°C at both the vegetative and reproductive stages but 5-days exposure to 40°C significantly impairs reproductive development.

Neem secretory cells: developmental cytology and indications of cell autotoxicity.

The neem tree (Azadirachta indica A.Juss.) contains a range of biologically active compounds-mainly triterpenoids produced in single secretory cells, which are distributed among all plant parts. Neem secretions are toxic to animal cells, triggering autolytic mechanisms that culminate in cell disruption. However, little is known about the self-toxicity of these secretions to the cells that produce them. We carried out an anatomical, histochemical, and ultrastructural investigation of neem's single secretory cells in the shoot apex and in young leaves. We evaluated the morphological changes as possible evidences of stress reactions to their own secretions. The subcellular apparatus involved in synthesis and compartmentation was consistent with hydrophilic and lipophilic secretions. Polymorphic plastids devoid of thylakoids and abundant smooth endoplasmic reticulum in the later stages of differentiation are comparable with previous reports on neem cotyledons with regard to terpenoid synthesis. However, secretions were compartmentalized within autophagic vacuoles and periplasmic spaces instead of in terpenoid vesicles. Cellular swelling, increased vesiculation, dilatation of endoplasmic reticulum cisternae, mitochondrial hypertrophy in the cristolysis process, autolytic vacuoles, and vacuolar degeneration culminating in protoplast autolysis are all consistent with early indications of autotoxicity. The signaling stress reaction mechanism was expressed as cytoplasmic deposits of calcium salt and by the expression of a 70-kDa heat-shock protein. The morphological and histochemical changes in the secreting cells are comparable with those described in animal cells exposed to neem oil. Our data provide evidence of cell damage and signaling reactions linked to these cells' own secretions before autolysis.

Characterization of the Relationship between the Chaperone and Lipid-Binding Functions of the 70-kDa Heat-Shock Protein, HspA1A.

HspA1A, a molecular chaperone, translocates to the plasma membrane (PM) of stressed and cancer cells. This translocation results in HspA1A’s cell-surface presentation, which renders tumors radiation insensitive. To specifically inhibit the lipid-driven HspA1A’s PM translocation and devise new therapeutics it is imperative to characterize the unknown HspA1A’s lipid-binding regions and determine the relationship between the chaperone and lipid-binding functions. To elucidate this relationship, we determined the effect of phosphatidylserine (PS)-binding on the secondary structure and chaperone functions of HspA1A. Circular dichroism revealed that binding to PS resulted in minimal modification on HspA1A’s secondary structure. Measuring the release of inorganic phosphate revealed that PS-binding had no effect on HspA1A’s ATPase activity. In contrast, PS-binding showed subtle but consistent increases in HspA1A’s refolding activities. Furthermore, using a Lysine-71-Alanine mutation (K71A; a null-ATPase mutant) of HspA1A we show that although K71A binds to PS with affinities similar to the wild-type (WT), the mutated protein associates with lipids three times faster and dissociates 300 times faster than the WT HspA1A. These observations suggest a two-step binding model including an initial interaction of HspA1A with lipids followed by a conformational change of the HspA1A-lipid complex, which accelerates the binding reaction. Together these findings strongly support the notion that the chaperone and lipid-binding activities of HspA1A are dependent but the regions mediating these functions do not overlap and provide the basis for future interventions to inhibit HspA1A’s PM-translocation in tumor cells, making them sensitive to radiation therapy.

Central role of 70-kDa heat shock protein in adaptation of plants to drought stress

The 70-kDa heat shock proteins (HSP70s) are a conserved class of chaperones that play critical roles during the normal life cycle of plants. HSP70s are particularly involved in the regulation of biotic and abiotic stress responses. In this paper, the potential roles of this protein were investigated. A reverse genetic approach was employed for transient silencing of hsp70 gene in tomato (Solanum lycopersicum L.) to evaluate different growth and physiological parameters under normal conditions and during the response to drought stress. A combined ANOVA (analysis of variance) and HCA (hierarchical clustering analysis) showed that hsp70 silencing led to severe growth retardation and mortality, significant membrane damage and leakage, decline in relative water content, low rate of pigment accumulation, and reduced antioxidant enzyme activity under normal and drought stress conditions. Among the different parameters, proline was the only trait that was unaffected by gene silencing and accumulated by similar amounts to that of nonsilent plants. In conclusion, HSP70 played critical roles in maintaining the cellular homeostasis of plants during adaptation to drought and under normal plant life conditions. It was speculated that proline was, to some extent, involved in improving the loss of protein folding or function resulting from HSP70 deficiency, and played a crucial role in the adaptation of plants on exposure to stress.

Heat Shock Protein 70 as a Double Agent Acting Inside and Outside the Cell: Insights into Autoimmunity.

Heat shock proteins (Hsp) are a diverse group of constitutive and/or stress-induced molecules that are categorized into several classes on the basis of their molecular weight. Mammalian Hsp have been mostly regarded as intracellular chaperones that mediate a range of essential cellular functions, including proper folding of newly synthesized polypeptides, refolding of denatured proteins, protein transport, and stabilization of native proteins’ structures. The well-characterized and highly evolutionarily conserved, stress-inducible 70-kDa heat shock protein (Hsp70), is a key molecular chaperone that is overexpressed in the cell in response to stress of various origin. Hsp70 exhibits an immunosuppressive activity via, e.g., downregulation of the nuclear factor-kappa B (NF-κB) activation, and pharmacological induction of Hsp70 can ameliorate the autoimmune arthritis development in animal models. Moreover, Hsp70 might be passively or actively released from the necrotic or stressed cells, respectively. Highly immunogenic extracellular Hsp70 has been reported to impact both the innate and adaptive immune responses, and to be implicated in the autoimmune reaction. In addition, preclinical studies revealed that immunization with highly conserved Hsp70 peptides could be regarded as a potential treatment target for autoimmune arthritis, such as the rheumatoid arthritis, via induction of antigen-specific regulatory T helper cells (also called Treg). Here, a dual role of the intra- and extracellular Hsp70 is presented in the context of the autoimmune reaction.

Dietary peppermint (Mentha piperita) powder affects growth performance, hematological indices, skin mucosal immune parameters, and expression of growth and stress-related genes in Caspian roach (Rutilus caspicus).

Peppermint is a popular herbal medicine due to its several pharmaceutical applications. In this study, peppermint powder was used as a feed additive to evaluate growth performance, hematological parameters, protein profile of skin mucus, and immune parameters, as well as growth hormone (GH), insulin-like growth factor (IGF), and 70kDa heat shock protein (HSP70) gene expression in Caspian roach (Rutilus caspicus). The fingerlings (average weight of 2.40 ± 0.12g) were fed with diet containing 0 (control), 2, 3, and 4g/kg peppermint for 8weeks. The addition of peppermint significantly enhanced the growth parameters and decreased the food conversion ratio. Hematological indices of fish fed with peppermint-supplemented diets were significantly different from the control group (P < 0.05). Soluble protein, alkaline phosphatase, and lysozyme enzyme activity in mucus samples showed an incremental trend by increasing the peppermint levels in the diet. Evaluation of mucosal immunity indicated a remarkable difference between the protein profile in treatments fed with peppermint-supplemented diets and the control group. A new protein band (approximately 27kDa) was also found in the skin mucus of fish fed with the diet containing 4g/kg peppermint, and the highest band density was observed in this treatment. The highest IGF and GH gene expression were observed in 4g/kg peppermint treatment. There was a significant difference in HSP70 expression between the fish fed with peppermint powder and the control group (P < 0.05). Overall, the results showed that dietary oral administration of peppermint at 4g/kg of feed can act as a growth promoter and immunostimulant.

Proteomic Analysis of Lipid Droplets in Sesamum indicum

We attempted to identify the total proteome in sesame lipid droplets. Results from two-dimensional electrophoresis showed 139 protein spots in lipid droplet samples. Each spot was isolated, digested with trypsin, and applied to liquid chromatography–tandem mass spectrometry (Q-Tof Premier). As a result, 103 spots were identified. Although oleosin, caleosin, and steroleosin are known major components of the lipid droplet, many other proteins were also found in the lipid droplet. In addition to the three major proteins, TAG factor protein, glyceraldehyde-3-phosphate dehydrogenase, F1 ATPase, 70-kDa heat shock protein, seed maturation protein PM24, and 11S globulin precursor isoforms 3 and 4 were found in the lipid droplet. Three types of oleosins, 15-, 15.5-, and 17-kDa were present in the sesame lipid droplet, and the 15.5-kDa oleosin had high homology with oleosin from Coffea canephora. It has been shown by acid phosphatase treatment that oleosin proteins contain phosphate groups. Protein disulfide-isomerase 2 precursor, calreticulin-1, and BiP, which are known as marker proteins of the endoplasmic reticulum, were found as the components of the lipid droplet. Immunoconfocal microscopy was used to show that 11S globulin precursor isoform 3 and 4 were indeed localized in the lipid droplet. The presence of 11S globulin in the lipid droplets suggested a new mechanism for the lipid droplet formation.

FLOURY ENDOSPERM11-2 encodes plastid HSP70-2 involved with the temperature-dependent chalkiness of rice (Oryza sativa L.) grains.

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature-sensitive floury endosperm11-2 (flo11-2) mutant was isolated from ion beam-irradiated rice of 1116 lines. The flo11-2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole-exome sequencing of the flo11-2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid-localized 70-kDa heat shock protein2 (cpHSP70-2). The cpHSP70-2 of the flo11-2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif5 of the ATPase domain. Transgenic flo11-2 mutants that express the wild-type cpHSP70-2 showed significantly lower chalkiness than the flo11-2 mutant. Moreover, the accumulation level of cpHSP70-2 was negatively correlated with the chalky ratio, indicating that cpHSP70-2 is a causal gene for the chalkiness of the flo11-2 mutant. The intrinsic ATPase activity of recombinant cpHSP70-2 was lower by 23% at Vmax for the flo11-2 mutant than for the wild type. The growth of DnaK-defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild-type DnaK. The results indicate that the lowered cpHSP70-2 function is involved with the chalkiness of the flo11-2 mutant.

Host expression system modulates recombinant Hsp70 activity through post-translational modifications.

The use of model organisms for recombinant protein production results in the addition of model-specific post-translational modifications (PTMs) that can affect the structure, charge, and function of the protein. The 70-kDa heat shock proteins (Hsp70) were originally described as intracellular chaperones, with ATPase and foldase activity. More recently, new extracellular activities of Hsp70 proteins (e.g. as immunomodulators) have been identified. While some studies indicate an inflammatory potential for extracellular Hsp70 proteins, others suggest an immunosuppressive activity. We hypothesized that the production of recombinant Hsp70 in different expression systems would result in the addition of different PTMs, perhaps explaining at least some of these opposing immunological outcomes. We produced and purified Mycobacterium tuberculosis DnaK from two different systems, Escherichia coli and Pichia pastoris, and analyzed by mass spectrometry the protein preparations, investigating the impact of PTMs in an in silico and in vitro perspective. The comparisons of DnaK structures in silico highlighted that electrostatic and topographical differences exist that are dependent upon the expression system. Production of DnaK in the eukaryotic system dramatically affected its ATPase activity, and significantly altered its ability to downregulate MHC II and CD86 expression on murine dendritic cells (DCs). Phosphatase treatment of DnaK indicated that some of these differences related specifically to phosphorylation. Altogether, our data indicate that PTMs are an important characteristic of the expression system, with differences that impact interactions of Hsps with their ligands and subsequent functional activities.

Evaluating bivalve cytoprotective responses and their regulatory pathways in a climate change scenario

Temperature is a relevant abiotic factor affecting physiological performance and distribution of marine animals in natural environments. The changes in global seawater temperatures make it necessary to understand how molecular mechanisms operate under the cumulative effects of global climate change and chemical pollution to promote/hamper environmental acclimatization. Marine mussels are excellent model organisms to infer the impacts of those anthropogenic threats on coastal ecosystems. In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed to different concentrations of the metal copper (Cu as CuCl2: 2.5, 5, 10, 20, 40 μg/L) or the antibiotic oxytetracycline (OTC: 0.1, 1, 10, 100, 1000 μg/L) at increasing seawater temperatures (16 °C, 20 °C, 24 °C). Transcriptional modulation of a 70-kDa heat shock protein (HSP70) and of the ABC transporter P-glycoprotein (P-gp, encoded by the ABCB gene) was assessed along with the cAMP/PKA signaling pathway regulating both gene expressions. At the physiological temperature of mussels (16 °C), Cu and OTC induced bimodal changes of cAMP levels and PKA activities in gills of exposed animals. A correlation between OTC- or Cu- induced changes of PKA activity and expression of hsp70 and ABCB was observed. Temperature increases (up to 24 °C) altered ABCB and hsp70 responses to the pollutants and disrupted their relationship with cAMP/PKA modulation, leading to loss of correlation between the biological endpoints. On the whole, the results indicate that temperature may impair the effects of inorganic and organic chemicals on the cAMP/PKA signaling pathway of mussels, in turn altering key molecular mediators of physiological plasticity and cytoprotection.

HSP70 Multi-Functionality in Cancer.

The 70-kDa heat shock proteins (HSP70s) are abundantly present in cancer, providing malignant cells selective advantage by suppressing multiple apoptotic pathways, regulating necrosis, bypassing cellular senescence program, interfering with tumor immunity, promoting angiogenesis and supporting metastasis. This direct involvement of HSP70 in most of the cancer hallmarks explains the phenomenon of cancer “addiction” to HSP70, tightly linking tumor survival and growth to the HSP70 expression. HSP70 operates in different states through its catalytic cycle, suggesting that it can multi-function in malignant cells in any of these states. Clinically, tumor cells intensively release HSP70 in extracellular microenvironment, resulting in diverse outcomes for patient survival. Given its clinical significance, small molecule inhibitors were developed to target different sites of the HSP70 machinery. Furthermore, several HSP70-based immunotherapy approaches were assessed in clinical trials. This review will explore different roles of HSP70 on cancer progression and emphasize the importance of understanding the flexibility of HSP70 nature for future development of anti-cancer therapies.

Abstract WMP74: Cofilin-Actin Rod Formation in Experimental Stroke is Attenuated by Therapeutic Hypothermia and Overexpression of the Inducible 70-kDa Heat Shock Protein (Hsp70)

Background and Purpose: Cofilin-actin rods are covalently linked aggregates of cofilin -1 and actin. Under ischemic conditions, these rods have been observed in neuronal processes, but their significance is unknown. Here, we explored a potential role of these rods in two different models of neuroprotection from experimental stroke—therapeutic hypothermia and the 70-kDa heat shock protein (Hsp70). Methods: Male C57/BL6 mice were subjected to distal middle cerebral artery occlusion (dMCAO), and treated with hypothermia. Cooling (31°C, 2hrs) was begun at the onset of dMCAO. A neuroprotective effect of hypothermia was validated by functional assessments and infarct volume measurement. Cofilin-actin rod formation was assessed by histological analysis at 4 and 24hrs after dMCAO. Its expression was analyzed in three different regions, infarct core (the center of the infarct), penumbra (area salvaged by intervention and a fixed distance from the midline), and ischemic borderzone (border of ischemic lesion). In addition, Hsp70-overexpressing transgenic (Tg) mice and Hsp70-deficient (Ko) mice were also subjected to dMCAO, and cofilin-actin rod expression was assessed in same manner. Results: As shown previously by our lab, both hypothermia and Hsp70 Tg mice had smaller lesion sizes and improved neurological outcomes compared to normothermic and wildtype (Wt) mice. Hsp70 Ko mice had larger lesion sizes and worsened neurological outcomes. Following dMCAO, cofilin-actin rods were increased, but were reduced by hypothermia in the ischemic core (24hrs, p&lt;0.05), penumbra (4 and 24hrs, p&lt;0.05), and ischemic borderzone (4 and 24hrs, p&lt;0.05). Among Hsp70 Tg mice, cofilin-actin rod formation was decreased in the ischemic borderzone (4 and 24hrs, p&lt;0.05), while Hsp70 Ko mice showed increased rod formation in the penumbra (4 and 24hrs, p&lt;0.05). Conclusions: Cofilin-actin rod formation was suppressed under conditions of improved neurological outcome, and increased under circumstances where outcome was worsened. This suggests that cofilin-actin rods may act to participate in or exacerbate ischemic pathology, and warrants further study as a potential therapeutic target.

Detection and molecular characterization of Cryptosporidium species in wild-caught pet spiny-tailed lizards.

Uromastyx is a genus of the herbivorous agamid lizards, also known as spiny-tailed lizards or mastigures, which are found in parts of Africa and the Middle East. Currently, several species of this genus are available in the international pet trade in Japan. In this study, two imported wild-caught spiny-tailed lizards (Arabian blue mastigure, Uromastyx ornata philbyi, and Sudan mastigure, Uromastyx dispar flavifasciata) were diagnosed with a Cryptosporidium (Apicomplexa: Cryptosporidiidae) infection based on the presence of the oocysts in the rectal feces using sucrose flotation and light microscopy examination at a local animal hospital in Tokyo, Japan. One of the lizards had died, and histopathological examination revealed enteritis with the Cryptosporidium parasite. Sequence analyses using the small subunit ribosomal RNA, actin, and 70-kDa heat shock protein genes indicated that the lizards had contracted a novel variant of C. avium that commonly infects avian species.

Pro-inflammatory mediators in vaginal fluid and short cervical length in pregnancy.

We hypothesized that elevated vaginal levels of matrix metalloproteinase-8 (MMP-8), interleukin-8 (IL-8) and the 70kDa heat shock protein (hsp70), compounds involved in inflammatory responses, correlated with a short cervix in pregnant women. This prospective cohort study used a convenience sample of 64 women in their early third trimester with a singleton pregnancy. A short cervical length was present in 35 women (54.7 %). Vaginal fluid was tested for levels of MMP-8, IL-8 and hsp70 by enzyme-linked immunosorbent assay (ELISA). A receiver operating charasteristic (ROC) analysis was used to calculate the area under the curve (AUC) for each mediator in predicting short cervical length. MMP-8 (109 vs 29.6 ng/ml, p=0.014), IL-8 (689 vs 330 pg/ml, p=0.007) and hsp70 (4.4 vs 2.9 ng/ml, p=0.036) were all elevated in vaginal samples from women with a short cervix. In addition, there was a negative association between the concentration of each compound in vaginal fluid and cervical length p≤0.026). The vaginal IL-8 concentration had the highest negative correlation with a short cervix (AUC=0.7, p=0.007). MMP-8, hsp70 and IL-8 contribute to a pro-inflammatory cervico-vaginal milieu that weakens cervical integrity and leads to a shortening in cervical length (Tab. 4, Fig. 1, Ref. 27).

Arylquin 1, a potent Par-4 secretagogue, induces lysosomal membrane permeabilization-mediated non-apoptotic cell death in cancer cells.

Arylquin 1, a small-molecule prostate-apoptosis-response-4 (Par-4) secretagogue, targets vimentin to induce Par-4 secretion. Secreted Par-4 binds to its receptor, 78-kDa glucose-regulated protein (GRP78), on the cancer cell surface and induces apoptosis. In the present study, we investigated the molecular mechanisms of arylquin 1 in cancer cell death. Arylquin 1 induces morphological changes (cell body shrinkage and cell detachment) and decreases cell viability in various cancer cells. Arylquin 1-induced cell death is not inhibited by apoptosis inhibitors (z-VAD-fmk, a pan-caspase inhibitor), necroptosis inhibitors (necrostatin-1), and paraptosis inhibitors. Furthermore, arylquin 1 significantly induces reactive oxygen species levels, but antioxidants [N-acetyl-l-cysteine and glutathione ethyl ester] do not inhibit arylquin 1-induced cell death. Furthermore, Par-4 knock-down by small interfering RNA confers no effect on cytotoxicity in arylquin 1-treated cells. Interestingly, arylquin 1 induces lysosomal membrane permeabilization (LMP), and cathepsin inhibitors and overexpression of 70-kDa heat shock protein (HSP70) markedly prevent arylquin 1-induced cell death. Therefore, our results suggest that arylquin 1 induces non-apoptotic cell death in cancer cells through the induction of LMP.

Occurrence of a Cryptosporidium xiaoi–like genotype in peafowl (Pavo cristatus) in China

The aim of this study was to survey the Cryptosporidium species in peafowls (Pavo cristatus) in Henan Province, China. A total of 143 fecal specimens collected from a breeding farm were tested for Cryptosporidium by nested PCR targeting the small subunit rRNA (SSU rRNA), 70-kDa heat shock protein (HSP70), and actin genes of Cryptosporidium followed by sequence analysis. Only one isolate from an asymptomatic host was obtained, and the isolate differed from a new C. xiaoi-like genotype by one nucleotide and from C. xiaoi or C. bovis at the SSU rRNA locus by six nucleotides. Likewise, the actin gene shared 99% identity with the C. xiaoi-like genotype, accompanied by four nucleotide mutations. A complete sequence of the HSP70 gene was obtained, and exhibited 96% similarity with that from C. xiaoi and differed by one nucleotide from that with the C. xiaoi-like genotype. Phylogenetic analysis of the current isolate revealed genetic relatedness to the C. xiaoi-like genotype and distinction from C. xiaoi and C. bovis. Therefore, our results provided the first documentation of avian infection with a C. xiaoi-like genotype in China and further insight into the diversity of Cryptosporidium spp. in avians.

Structural and functional analysis of the Hsp70/Hsp40 chaperone system.

As one of the most abundant and highly conserved molecular chaperones, the 70-kDa heat shock proteins (Hsp70s) play a key role in maintaining cellular protein homeostasis (proteostasis), one of the most fundamental tasks for every living organism. In this role, Hsp70s are inextricably linked to many human diseases, most notably cancers and neurodegenerative diseases, and are increasingly recognized as important drug targets for developing novel therapeutics for these diseases. Hsp40s are a class of essential and universal partners for Hsp70s in almost all aspects of proteostasis. Thus, Hsp70s and Hsp40s together constitute one of the most important chaperone systems across all kingdoms of life. In recent years, we have witnessed significant progress in understanding the molecular mechanism of this chaperone system through structural and functional analysis. This review will focus on this recent progress, mainly from a structural perspective.

Melatonin Enhances Mitophagy by Upregulating Expression of Heat Shock 70 kDa Protein 1L in Human Mesenchymal Stem Cells under Oxidative Stress.

Human mesenchymal stem cells (hMSCs) are a potent source of cell-based regenerative therapeutics used to treat patients with ischemic disease. However, disease-induced oxidative stress disrupts mitochondrial homeostasis in transplanted hMSCs, resulting in hMSC apoptosis and reducing their efficacy post-transplantation. To address this issue, we evaluated the effects of melatonin on cellular defense mechanisms and mitophagy in hMSCs subjected to oxidative stress. H2O2-induced oxidative stress increases the levels of reactive oxygen species and reduces membrane potential in hMSCs, leading to mitochondrial dysfunction and cell death. Oxidative stress also decreases the expression of 70-kDa heat shock protein 1L (HSPA1L), a molecular chaperone that assists in the recruitment of parkin to the autophagosomal mitochondrial membrane. Decreased expression of HSPA1L destabilizes parkin, thereby impairing mitophagy. Our results indicate that treating hMSCs with melatonin significantly inhibited mitochondrial dysfunction induced by oxidative stress, which decreased hMSCs apoptosis. In damaged hMSCs, treatment with melatonin increased the levels of HSPA1L, which bound to parkin. The interaction between HSPA1L and parkin increased membrane potential and levels of oxidative phosphorylation, resulting in enhanced mitophagy. Our results indicate that melatonin increased the expression of HSPA1L, thereby upregulating mitophagy and prolonging cell survival under conditions of oxidative stress. In this study, we have shown that melatonin, a readily available compound, can be used to improve hMSC-based therapies for patients with pathologic conditions involving oxidative stress.