The lamina-associated polypeptide 2 (LAP2) genes of zebrafish and chicken: no LAP2alpha isoform is synthesised by non-mammalian vertebrates

The mammalian lamina-associated polypeptide 2 (LAP2) gene encodes six isoforms (LAP2alpha, beta, delta, epsilon, gamma, zeta) that are synthesised from alternatively spliced mRNAs. The mammalian LAP2alpha is one of the predominant isoforms and localised in the nucleoplasm whereas LAP2beta, delta, epsilon, and gamma are integral membrane proteins of the inner nuclear membrane. We have analysed the LAP2 gene structure of the zebrafish Danio rerio as an attractive lower vertebrate model organism. The zebrafish LAP2 (ZLAP2) gene without regulatory sequences spans approximately 19 kb of genomic DNA. It contains 15 exons that encode the isoforms ZLAP2beta, gamma, and omega which are localised in the inner nuclear membrane. By radiation hybrid mapping, we have located the gene onto linkage group 4 between EST markers fc01g04 (213.97cR) and fb49f01 (215.69cR). The identification of a chicken genomic clone comprising the complete coding region of the avian LAP2 gene enabled us to compare the LAP2 gene structure amongst vertebrates. In contrast to the mammalian LAP2 gene, the zebrafish and the chicken sequences do not encode for an alpha-isoform. In parallel we searched for an alpha-isoform in birds using polyclonal and monoclonal LAP2 antibodies specific for the common evolutionary conserved aminoterminal domain present in all isoforms. We detected LAP2beta as the predominant isoform but no LAP2alpha in tissues of 10-day-old chicken embryos and cultured chicken fibroblasts thus confirming the genomic analysis. The comparison of each zebrafish and chicken LAP2 exon with the corresponding exons of the human LAP2 gene demonstrates that the degree of identity at the amino acid level is much higher between the human and chicken than between the human and zebrafish sequences. By Blast search with the nucleotide and amino acid sequences of the human LAP2alpha, we did not find any significant homologies in databases of the zebrafish and chicken sequences. Our data suggest that LAP2alpha is a novelty of mammals.     

Heterooligomeric complexes formed by human small heat shock proteins HspB1 (Hsp27) and HspB6 (Hsp20)

Formation of heterooligomeric complexes of human small heat shock proteins (sHsp) HspB6 (Hsp20) and HspB1 (Hsp27) was analyzed by means of native gel electrophoresis, analytical ultracentrifugation, chemical cross-linking and size-exclusion chromatography. HspB6 and HspB1 form at least two different complexes with apparent molecular masses 100–150 and 250–300 kDa, and formation of heterooligomeric complexes is temperature dependent. These complexes are highly mobile, easily exchange their subunits and are interconvertible. The stoichiometry of HspB1 and HspB6 in both complexes is close to 1/1 and smaller complexes are predominantly formed at low, whereas larger complexes are predominantly formed at high protein concentration. Formation of heterooligomeric complexes does not affect the chaperone-like activity of HspB1 and HspB6 if insulin or skeletal muscle F-actin was used as model protein substrates. After formation of heterooligomeric complexes the wild type HspB1 inhibits the rate of phosphorylation of HspB6 by cAMP-dependent protein kinase. The 3D mutant mimicking phosphorylation of HspB1 also forms heterooligomeric complexes with HspB6, but is ineffective in inhibition of HspB6 phosphorylation. Inside of heterooligomeric complexes HspB6 inhibits phosphorylation of HspB1 by MAPKAP2 kinase. Thus, in heterooligomeric complexes HspB6 and HspB1 mutually affect the structure of each other and formation of heterooligomeric complexes might influence diverse processes depending on small heat shock proteins.     

Human heat shock protein 70 (Hsp70) as a peripheral membrane protein

While a significant fraction of heat shock protein 70 (Hsp70) is membrane associated in lysosomes, mitochondria, and the outer surface of cancer cells, the mechanisms of interaction have remained elusive, with no conclusive demonstration of a protein receptor. Hsp70 contains two Trps, W90 and W580, in its N-terminal nucleotide binding domain (NBD), and the C-terminal substrate binding domain (SBD), respectively. Our fluorescence spectroscopy study using Hsp70 and its W90F and W580F mutants, and Hsp70-?SBD and Hsp70-?NBD constructs, revealed that binding to liposomes depends on their lipid composition and involves both NBD and SBD.     

The Hsp70 chaperone system maintains high concentrations of active proteins and suppresses ATP consumption during heat shock

Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures.     

Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70s as a case study

Background

Heat shock proteins (Hsps) perform a fundamental role in protecting plants against abiotic stresses. Although researchers have made great efforts on the functional analysis of individual family members, Hsps have not been fully characterized in rice (Oryza sativa L.) and little is known about their interactors.

Results

In this study, we combined orthology-based approach with expression association data to screen rice Hsps for the expression patterns of which strongly correlated with that of heat responsive probe-sets. Twenty-seven Hsp candidates were identified, including 12 small Hsps, six Hsp70s, three Hsp60s, three Hsp90s, and three clpB/Hsp100s. Then, using a combination of interolog and expression profile-based methods, we inferred 430 interactors of Hsp70s in rice, and validated the interactions by co-localization and function-based methods. Subsequent analysis showed 13 interacting domains and 28 target motifs were over-represented in Hsp70s interactors. Twenty-four GO terms of biological processes and five GO terms of molecular functions were enriched in the positive interactors, whose expression levels were positively associated with Hsp70s. Hsp70s interaction network implied that Hsp70s were involved in macromolecular translocation, carbohydrate metabolism, innate immunity, photosystem II repair and regulation of kinase activities.

Conclusions

Twenty-seven Hsps in rice were identified and 430 interactors of Hsp70s were inferred and validated, then the interacting network of Hsp70s was induced and the function of Hsp70s was analyzed. Furthermore, two databases named Rice Heat Shock Proteins (RiceHsps) and Rice Gene Expression Profile (RGEP), and one online tool named Protein-Protein Interaction Predictor (PPIP), were constructed and could be accessed at http://bioinformatics.fafu.edu.cn/.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-344) contains supplementary material, which is available to authorized users.     

谷氨酸对嗜铬细胞瘤细胞热休克蛋白70 mRNA的诱导分析

热休克反应普遍存在于从细菌到人的整个生物界。除热外,多种应激原都可引起热休克蛋白的诱导。至于神经递质是否能够诱导热休克蛋白的表达,目前并不清楚。本文以诱导型热休克蛋白（ｈｓｐ）７０的ｃＤＮＡ为探针,运用Ｎｏｒｔｈｅｒｎ ｂｌｏｔ的方法,在嗜铬细胞瘤细胞（ＰＣ１２）上,分析了谷氨酸和乙酰胆碱对ｈｓｐ７０ ｍＲＮＡ的诱导作用。在此基础上又初步分析了起作用的递质受体。结果表明：在一定的浓度（５０￣５００     

In situ expression of heat shock proteins,Hsc73, Hsj2 and Hsp86 in the developing tooth germ of mouse lower first molar

This study examined the detailed gene expression pattern of three different heat shock proteins (HSPs), Hsc73, Hsj2, and Hsp86, by means of an in situ hybridization method. Hsc73, Hsj2, and Hsp86 were shown in our previous study to be differentially expressed in the mouse embryonic mandible at day 10.5 (E10.5) gestational age. These HSP genes showed similar expression patterns during development of the mouse lower first molar. HSPs-expressing cells were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5, and then were slightly localized at E12 in an area where the tooth germ of the lower first molar is estimated to be formed. A strong expression of HSPs was observed in the tooth germ at E13.5. At the cap stage, HSPs were expressed in the enamel organ and dental papilla. At the bell stage, HSPs were distinctly expressed in the inner enamel epithelium and dental papilla cells facing the inner enamel epithelial layer, which later differentiate into ameloblasts and odontoblasts, respectively. This study is the first report in which Hsc73, Hsj2, and Hsp86 were distinctly expressed in the developing tooth germ, thus suggesting these HSPs are related to the development and differentiation of odontogenic cells.     

Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.     

CUEDC2 interacts with heat shock protein 70 and negatively regulates its chaperone activity

Recently studies have revealed that CUEDC2, a CUE domain-containing protein, plays critical roles in many biological processes, such as cell cycle, inflammation and tumorigenesis. In this study, to further explore the function of CUEDC2, we performed affinity purification combined with mass spectrometry analysis to identify its interaction proteins, which led to the identification of heat shock protein 70 (HSP70). We confirmed the interaction between CUEDC2 and HSP70 in vivo by co-immunoprecipitation assays. Mapping experiments revealed that CUE domain was required for their binding, while the PBD and CT domains of HSP70, mediated the interaction with CUEDC2. The intracellular Luciferase refolding assay indicated that CUEDC2 could inhibit the chaperone activity of HSP70. Together, our results identify HSP70 as a novel CUEDC2 interaction protein and suggest that CUEDC2 might play important roles in regulating HSP70 mediated stress responses.     

Expression of small heat shock proteins HspB2, HspB8, Hsp20 and cvHsp in different tissues of the perinatal developing pig

Recently, we have described the developmental expression of the small heat shock proteins (sHsps) Hsp27/HspB1 and alphaB-crystallin/HspB5 in different tissues of pigs from almost full-term foetuses to three years old adults (P. Tallot, J. F. Grongnet, J. C. David, Biol. Neonate, 83, 281-288, 2003). The data described in this report extends this study to four other members of the sHsp family (Hsp20/HspB6, cvHsp/HspB7, MKBP/HspB2 and HspB8). We studied expression of these proteins in porcine lens, brain, heart, liver, kidney, lung, skeletal muscle, stomach, and colon, and found a ubiquitous expression of Hsp20 and HspB8 as earlier reported for Hsp27 and alphaB-crystallin. In contrast, cvHsp and HspB2 expression is essentially restricted to heart and muscle. During development, the sHsps tend to (temporarily) increase in stomach, liver, lung, kidney, hippocampus, and striatum, while expression in heart is more or less constant, and a large variation is found in sHsp expression patterns in skeletal muscle. In cerebellum and cortex a temporary decrease of Hsp20 and HspB8 is observed directly after birth. The major impact of this study is that each tissue seems to have a unique profile of sHsp expression, which varies during development and may reflect the need of a particular tissue to maintain at all stages an optimal chaperoning machinery to protect against physiological stress.     

Blockade of islet amyloid polypeptide fibrillation and cytotoxicity by the secretory chaperones 7B2 and proSAAS

The deposition of fibrillated human islet β-cell peptide islet amyloid polypeptide (hIAPP) into amyloid plaques is characteristic of the pathogenesis of islet cell death during type 2 diabetes. We investigated the effects of the neuroendocrine secretory proteins 7B2 and proSAAS on hIAPP fibrillation in vitro and on cytotoxicity. In vitro, 21-kDa 7B2 and proSAAS blocked hIAPP fibrillation. Structure–function studies showed that a central region within 21-kDa 7B2 is important in this effect and revealed the importance of the N-terminal region of proSAAS. Both chaperones blocked the cytotoxic effects of exogenous hIAPP on Rin5f cells; 7B2 generated by overexpression was also effective. ProSAAS and 7B2 may perform a chaperone role as secretory anti-aggregants in normal islet cell function and in type 2 diabetes.     

The dual behavior of heat shock protein 70 and asymmetric dimethylarginine in relation to serum CRP levels in type 2 diabetes

Background

Experimental evidence suggests that heat shock proteins (HSP) and asymmetric dimethylarginine (ADMA) are induced in the state of chronic inflammation and stress conditions. They are both inhibitors of nitric oxide synthase (NOS). The aim of this study was to evaluate the correlation between ADMA and HSP70, in patients with type 2 diabetes with respect to serum levels of C reactive protein (CRP).

Methods

We quantified serum HSP70, ADMA and CRP in 80 newly-diagnosed patients with type 2 diabetes plus 80 age-, sex and BMI-matched healthy controls. The patients and controls were also stratified into groups of high and low CRP levels (cut-point: 2.5 mg/ml).

Results

Patients with type 2 diabetes had significantly higher serum HSP70 (0.52 [0.51–0.66] vs. 0.27 [0.26–0.36], p < 0.001), ADMA (0.86 [0.81–0.92] vs. 0.72 [0.71–0.85], p < 0.05) and CRP (2.9 [1.7–3.4] vs. 1.6[1.2–2.3], p < 0.05) compared with healthy controls. Serum HSP70 and ADMA levels were significantly correlated in patients with high CRP levels (r = 0.89, p < 0.01), whereas there were no correlation in patients with low CRP (r = − 0.37, p = 0.07) and controls. This correlation was significant (r = 0.77, p < 0.001) in patients with high CRP and also in patients with low CRP levels (r = − 0.51, p < 0.05), after multiple adjustments for LDL and HDL levels.

Discussion

We showed that, in a state of high inflammation; serum levels of ADMA parallel the HSP70 levels. However in low inflammation, they are negatively correlated. The duality in HSP70 and ADMA correlation may be related to the duality of NOS function in low and high CRP levels.     

RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.     

Structure of the Bone Morphogenetic Protein Receptor ALK2 and Implications for Fibrodysplasia Ossificans Progressiva

Bone morphogenetic protein (BMP) receptor kinases are tightly regulated to control development and tissue homeostasis. Mutant receptor kinase domains escape regulation leading to severely degenerative diseases and represent an important therapeutic target. Fibrodysplasia ossificans progressiva (FOP) is a rare but devastating disorder of extraskeletal bone formation. FOP-associated mutations in the BMP receptor ALK2 reduce binding of the inhibitor FKBP12 and promote leaky signaling in the absence of ligand. To establish structural mechanisms of receptor regulation and to address the effects of FOP mutation, we determined the crystal structure of the cytoplasmic domain of ALK2 in complex with the inhibitors FKBP12 and dorsomorphin. FOP mutations break critical interactions that stabilize the inactive state of the kinase, thereby facilitating structural rearrangements that diminish FKBP12 binding and promote the correct positioning of the glycine-serine-rich loop and αC helix for kinase activation. The balance of these effects accounts for the comparable activity of R206H and L196P. Kinase activation in the clinically benign mutant L196P is far weaker than R206H but yields equivalent signals due to the stronger interaction of FKBP12 with R206H. The presented ALK2 structure offers a valuable template for the further design of specific inhibitors of BMP signaling.     

Small heat shock proteins prevent aggregation of citrate synthase and bind to the N-terminal region which is absent in thermostable forms of citrate synthase

Citrate synthase (CS) is often used in chaperone assays since this thermosensitive enzyme aggregates at moderately increased temperatures. Small heat shock proteins (sHsps) are molecular chaperones specialized in preventing the aggregation of other proteins, termed substrate proteins, under conditions of transient heat stress. To investigate the mechanism whereby sHsps bind to and stabilize a substrate protein, we here used peptide array screening covering the sequence of porcine CS (P00889). Strong binding of sHsps was detected to a peptide corresponding to the most N-terminal α-helix in CS (amino acids Leu₁₃ to Gln₂₇). The N-terminal α-helices in the CS dimer intertwine with the C-terminus in the other subunit and together form a stem-like structure which is protruding from the CS dimer. This stem-like structure is absent in thermostable forms of CS from thermophilic archaebacteria like Pyrococcus furiosus and Sulfolobus solfatacarium. These data therefore suggest that thermostabilization of thermosensitive CS by sHsps is achieved by stabilization of the C- and N-terminae in the protruding thermosensitive softspot, which is absent in thermostable forms of the CS dimer.