Biogenesis of the yeast frataxin homolog Yfh1p. Tim44-dependent transfer to mtHsp70 facilitates folding of newly imported proteins in mitochondria.

Tim44 is an essential component of the mitochondrial inner membrane protein import machinery. In this study we asked if Tim44 is of relevance in intramitochondrial protein folding. We investigated the role of Tim44 in the biogenesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia. After inactivation of Tim44, binding of mitochondrial heat shock protein (mtHsp)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked. Residual amounts of imported Yfh1p showed an increased tendency to aggregate. To further characterize the functions of Tim44 in the matrix, we imported dihydrofolate reductase (DHFR) as a model protein. Depletion of Tim44 allowed import of DHFR, although folding of the newly imported DHFR was delayed. Moreover, the depletion of Tim44 caused a strongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide. Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicating that mtHsp70-substrate complexes formed independently of Tim44 differ from the complexes that form under the control of Tim44. We conclude that Tim44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding.     

Inhibition by [corrected] ursolic acid of [corrected] calcium-induced mitochondrial permeability transition and release of two proapoptotic proteins

The possible inhibition by [corrected] ursolic acid (UA) of [corrected] mitochondrial permeability transition (MPT) in mouse liver was investigated to identify the mechanisms underlying the hepatoprotective effect of UA. The effect of UA on liver MPT induced by Ca2+ was assessed by measuring changes in mitochondrial volume, mitochondrial membrane potential (MMP), release of matrix Ca2+, and transfer of cytochrome c (Cyt c) and apoptosis-inducing factor (AIF) from the intermembrane space to the cytoplasm. The results showed that obvious mitochondrial swelling, loss of MMP, and release of matrix Ca2+ occurred after the addition of 50 microM Ca2+. However, preincubation with 20, 50 or 100 microg ml(-1) UA significantly blocked the above changes. Addition of 100 microg ml(-1) UA inhibited on mitochondrial swelling by 73.2% after 5 min, while the MMP dissipating and Ca2+ releasing were, respectively, suppressed by 59.3% and 54.1% after 3 min. In addition, Western blot analysis showed Cyt c and AIF transferred from mitochondrial pellet to the supernatant after the addition of 50 microM Ca2+, but the process was significantly inhibited by various concentrations of UA. The results suggest that the mechanisms underlying the hepatoprotection of UA may be related to its direct inhibitory action on MPT.     

Contribution of the conserved amino acids of the melanocortin-4 receptor in [corrected] [Nle4,D-Phe7]-alpha-melanocyte-stimulating [corrected] hormone binding and signaling

Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for alpha-melanocortin-stimulating hormone (alpha-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for alpha-MSH binding and signaling. alpha-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and alpha-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter alpha-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for alpha-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu(100) in TM2, Asp(122), Asp(126) in TM3 and Trp(258), Phe(261), His(264) in TM6 are involved in alpha-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for alpha-MSH binding and signaling.     

Echinococcus granulosus: in vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels.

Martinez, J., Perez-Serrano, J., Bernadina, W. E., Rodriguez-Caabeiro, F. 1999 Echinococcus granulosus: In vitro effects of ivermectin and praziquantel on hsp60 and hsp70 levels. Experimental Parasitology93, 171-180. Organisms or cells exposed to injurious stresses such as heat shock or chemicals respond by increased (or altered) expression of heat-shock proteins (HSPs). Conversely, an earlier exposure to stress can prepare cells to cope with a subsequent more severe stress. In the present study, protoscolices of Echinococcus granulosus were subjected to several anthelmintic treatments, involving storage of the protoscolices for 18, 30, and 50 h with 0.1 mg/ml of ivermectin (IV), praziquantel (PZ), and a combination of each with albendazole (ALB). The organisms were analyzed for the effects of drug treatment on cell integrity and on levels of hsp60 and hsp70 production. Drug efficacy was evaluated by microscopy and by protein content measurement. Hsp60 and hsp70 were detected by Western blotting and incubation with anti-hsp60 and anti-hsp70 antibody, respectively, and quantitation of these proteins was obtained using image analysis. Incubation with IV alone produced the most damage to the protoscolices as indicated by viability loss, decreased protein content, and altered hsp60 and hsp70 levels; incubation with IV + ALB produced less damage as manifested by fewer changes in the aforementioned damage parameters but PZ and PZ + ALB, in this context, were poor anthelmintics. Exposure of protoscolices to thermal stress prior to anthelmintic treatment, in most cases, increased drug efficacy. It is concluded that in the E. granulosus model system drug efficacy is associated with decreased levels of hsp70 expression and increased levels of hsp60 expression.     

OnD-CRF: predicting order and disorder in proteins using [corrected] conditional random fields

MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/     

Cytoplasmic chaperones in precursor targeting to mitochondria: the role of MSF and hsp 70

Despite extensive study since the early 1980s, the mechanism by which newly synthesized protein precursors are unfolded in the cytoplasm and targeted correctly to the mitochondrial surface prior to translocation through the mitochondrial membranes is understood poorly. Recently, an N-ethylmaleimide (NEM)-sensitive cytoplasmic factor called mitochondrial import stimulation factor (MSF), which catalyses the ATP-dependent unfolding of precursor proteins, was described. Unlike the more general chaperone proteins of the hsp70 families, MSF not only unfolds proteins but also targets the unfolded precursor proteins to the mitochondria. Here, Mihara and Omura summarize what is known about MSF and speculate on how it, and other cytoplasmic factors, may be involved in mitochondrial import.     

Nucleotide binding states of hsp70 and hsp90 during sequential steps in the process of glucocorticoid receptor.hsp90 heterocomplex assembly

A minimal system of five purified proteins, hsp90, hsp70, Hop, hsp40, and p23, assembles glucocorticoid receptor (GR).hsp90 heterocomplexes and causes the simultaneous opening of the steroid binding cleft to access by steroid. The first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR, which primes the receptor for subsequent ATP-dependent activation by hsp90, Hop, and p23 (Morishima, Y., Murphy, P. J. M., Li, D. P., Sanchez, E. R., and Pratt, W. B. (2000) J. Biol. Chem. 275, 18054-18060). Here we have examined the nucleotide-bound states of the two essential chaperones in each step. We show that it is the ATP-bound state of hsp70 that interacts initially with the GR. After rapid priming and washing, the primed GR.hsp70 complex rapidly binds hsp90 in the second step reaction in a nucleotide-independent manner. The rate-limiting step is the ATP-dependent opening of the steroid binding cleft after hsp90 binding. This activating step requires the N-terminal ATP-binding site of hsp90, but we cannot establish any role for a C-terminal ATP-binding site in steroid binding cleft opening. The reported specific inhibitors of the C-terminal ATP site on hsp90 inhibit the generation of steroid binding, but they have other effects in this multiprotein system that could explain the inhibition.     

Characterization and inducibility of hsp 70 proteins in the male mouse germ line

The properties and inducibility of the heat shock protein 70 (hsp 70) gene products were examined during differentiation of mouse testicular cells by one and two-dimensional gel electrophoresis and immunoblotting. Low levels of the 72- and 73-kD heat shock proteins normally found in mouse cell lines were detected in the mouse testis. A novel isoform with a relative molecular mass of 73 kD (called 73T) was also observed, in the presence or absence of heat shock. 73T was shown to be produced by germ cells since it was not detected in testes from mutant mice devoid of germ cells. Furthermore, 73T was found only in adult mouse testicular cells, not in testes from animals that lack meiotic germ cells. 73T was synthesized in enriched cell populations of both meiotic prophase and postmeiotic cells, but was not inducible by in vitro heat shock. In the adult testis, low levels of the bona fide 72-kD heat-inducible (hsp72) were induced in response to elevated temperatures. In contrast, in testes from animals in which only somatic cells and premeiotic germ cells were present, there was a substantial induction of hsp 72. It is suggested that hsp 72 is inducible in the somatic compartment and possibly in the premeiotic germ cells, but not in germ cells which have entered meiosis and which are expressing members of the hsp 70 gene family in a developmentally regulated fashion.     

Isolation from poultry litter and characterization [corrected] of Staphylococcus spp. capable of growth in high phosphate conditions [corrected

AIMS: To isolate and characterize micro-organisms from poultry litter capable of growing under phosphate concentrations typical of poultry litter. METHODS AND RESULTS: Poultry litter extracts were plated onto brain-heart infusion medium (BHI) containing an additional 0.75 mol l(-1) phosphate (BHI-P). Colonies were screened for the presence of inclusion granules with five being selected for further study. All strains displayed identical biochemical characteristics consistent with Staphylococcus spp. and grouped with Staphylococcus spp. by comparative 16S rDNA analysis. Thus all five strains were identified as such. All strains displayed elevated intracellular phosphate levels when cultured in BHI-P broth (0.417-0.600 microg phosphate mg(-1) protein) vs BHI broth (0.075-0.093 microg phosphate mg(-1) protein). When grown using an austere semi-defined medium or BHI-P, Staph. sp. #7 displayed similar elevated intracellular phosphate levels compared with growth in BHI. CONCLUSIONS: Poultry litter contains novel Staphylococcus spp. capable of robust growth when exposed to phosphate levels comparable with that typically found in poultry litter. Data suggest intracellular phosphate levels in these strains increase in response to increasing phosphate in the medium or austere medium conditions. Intracellular phosphate did not reach levels comparable with known hyper-accumulating micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest poultry litter possesses a resident microflora that thrives and accumulates intracellular phosphate in response to high phosphate conditions.     

Role of Hsc70 binding cycle in CFTR folding and endoplasmic reticulum-associated degradation

The Hsp/c70 cytosolic chaperone system facilitates competing pathways of protein folding and degradation. Here we use a reconstituted cell-free system to investigate the mechanism and extent to which Hsc70 contributes to these co- and posttranslational decisions for the membrane protein cystic fibrosis transmembrane conductance regulator (CFTR). Hsc70 binding to CFTR was destabilized by the C-terminal domain of Bag-1 (CBag), which stimulates client release by accelerating ADP-ATP exchange. Addition of CBag during CFTR translation slightly increased susceptibility of the newly synthesized protein to degradation, consistent with a profolding function for Hsc70. In contrast, posttranslational destabilization of Hsc70 binding nearly completely blocked CFTR ubiquitination, dislocation from the endoplasmic reticulum, and proteasome-mediated cleavage. This effect required molar excess of CBag relative to Hsc70 and was completely reversed by the CBag-binding subdomain of Hsc70. These results demonstrate that the profolding role of Hsc70 during cotranslational CFTR folding is counterbalanced by a dominant and essential role in posttranslational targeting to the ubiquitin-proteasome system. Moreover, the degradative outcome of Hsc70 binding appears highly sensitive to the duration of its binding cycle, which is in turn governed by the integrated expression of regulatory cochaperones.     

Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.     

Nonnative interactions in coupled folding and binding processes of intrinsically disordered proteins

Proteins function by interacting with other molecules, where both native and nonnative interactions play important roles. Native interactions contribute to the stability and specificity of a complex, whereas nonnative interactions mainly perturb the binding kinetics. For intrinsically disordered proteins (IDPs), which do not adopt rigid structures when being free in solution, the role of nonnative interactions may be more prominent in binding processes due to their high flexibilities. In this work, we investigated the effect of nonnative hydrophobic interactions on the coupled folding and binding processes of IDPs and its interplay with chain flexibility by conducting molecular dynamics simulations. Our results showed that the free-energy profiles became rugged, and intermediate states occurred when nonnative hydrophobic interactions were introduced. The binding rate was initially accelerated and subsequently dramatically decreased as the strength of the nonnative hydrophobic interactions increased. Both thermodynamic and kinetic analysis showed that disordered systems were more readily affected by nonnative interactions than ordered systems. Furthermore, it was demonstrated that the kinetic advantage of IDPs ("fly-casting" mechanism) was enhanced by nonnative hydrophobic interactions. The relationship between chain flexibility and protein aggregation is also discussed.     

Effect of ATP on the Release of hsp 70 and hsp 40 from the Nucleus in Heat-Shocked HeLa Cells

We have recently found a novel 40-kDa heat-shock protein (hsp 40) in mammalian and avian cells and reported that the N-terminal amino acid sequence of mammalian hsp 40 has homology with the bacterial DnaJ heat-shock protein. Also, hsp 40 has been shown to be translocated from the cytoplasm into the nuclei/nucleoli by heat shock and colocalized with hsc 70 (p73) in the nucleoli of exactly the same cells. We here investigated the effect of ATP on the release of hsp 70 (both constitutive p73 and inducible p72) and hsp 40 from the nuclei/nucleoli of heat-shocked HeLa cells which were permeabilized with Nonidet-P40 using immunoflourescence and immunoblotting. Hsp 70 in the nucleoli was released by the addition of ATP but not by ADP, GTP, nonhydrolyzable ATP, nor high salt buffer. In contrast, hsp 40 was not released from the nucleoli with any of these treatments or any combination of these treatments. Thus, hsp 40 might dissociate spontaneously from the nucleoli after hsp 70 has been released in an ATP-dependent manner. Using cell fractionation methods, we showed that while the majority of hsp 40 is localized in the cytoplasm, a small portion of it is located in the microsome fraction in non-heat-shocked control cells and in cells which recovered from heat shock.     

Synthesis of [N]glutamate from [N]h(4) and [N]glycine by mitochondria isolated from pea and corn shoots

Metabolically competent mitochondria were isolated from pea and corn shoots on Percoll discontinuous density gradients. Rates of synthesis of [¹⁵N]glutamate were measured by gas chromatography-mass spectrometry after the incubation of mitochondria with either 2 millimolar [¹⁵N] H₄⁺ or [¹⁵N]glycine in the presence of 1 millimolar citrate as the respiratory substrate. When [¹⁵N]H₄⁺ was provided, mitochondria isolated from light-grown pea shoots synthesized [¹⁵N]glutamate with a rate of 2.64 nanomoles per hour per milligram mitochondrial protein. Corn mitochondria produced [¹⁵N]glutamate at a rate approximately 11 times greater than the pea mitochondria. Dark treatment during growth for the last 24 hours caused a slight reduction in the rate of synthesis in both species. When [¹⁵N]glycine was used, pea mitochondria synthesized [¹⁵N]glutamate with a rate of 6.32 nanomoles per hour per milligram protein. Rapid disappearance of [¹⁵N]glycine and synthesis of [¹⁵N]serine was observed with a molar ratio of 2 glycine to 0.78 serine. The rate of glutamate synthesis was only 0.2% that of serine, due in part to the dilution of [¹⁵N]H₄⁺ by the [¹⁴N]H₄⁺ pool in the mitochondria. The majority of the [¹⁵N]H₄⁺ released from glycine appears to have been released from or remains unmetabolized in the mitochondria. Corn mitochondria showed no apparent disappearance of [¹⁵N]glycine and little synthesis of [¹⁵N]serine, indicating that our preparation originated primarily from mesophyll cells. Under our conditions of glycine/serine conversion, [¹⁵N]glutatmate was synthesized at a rate of 7% of that of [¹⁵N]serine synthesis by corn mitochondria.     

Hyper-acidic protein fusion partners improve solubility and assist correct folding of recombinant proteins expressed in Escherichia coli

High expression of recombinant proteins in Escherichia coli (E. coli) often leads to protein aggregation. One popular approach to address this problem is the use of fusion tags (or partners) that improve the solubility of the proteins in question. However, such fusion tags are not effective for all proteins. In this study, we demonstrate that the hyper-acidic protein fusion partners can largely enhance the soluble expression of target proteins recalcitrant to the efforts by using routine solubilising tags. This new type of fusion partners examined includes three extremely acidic E. coli polypeptides, i.e. yjgD, the N-terminal domain of rpoD (sigma 70 factor of RNA polymerase) and our preliminarily evaluated msyB. The target proteins used are highly aggregation-prone, including EK (the bovine enterokinase), TEV (the tobacco etch virus protease) and rbcL (the large subunit of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase). On removal in vitro and in vivo of the fusion tags by using yeast SUMO/Ulp1 reaction and TEV auto-cleavage, the resultant findings indicate the hyper-acidic fusion partners can function as intramolecular chaperones assisting in the correct folding of the target proteins.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

Saturation, competition, and specificity in interaction of heat shock proteins (hsp) gp96, hsp90, and hsp70 with CD11b+ cells

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.     

Stepwise assembly of a glucocorticoid receptor.hsp90 heterocomplex resolves two sequential ATP-dependent events involving first hsp70 and then hsp90 in opening of the steroid binding pocket

A system of five purified proteins that assembles stable glucocorticoid receptor (GR)-hsp90 heterocomplexes has been reconstituted from reticulocyte lysate. Two proteins, hsp90 and hsp70, are required for the activation of steroid binding activity that occurs with heterocomplex assembly, and three proteins, Hop, hsp40, p23, act as co-chaperones that enhance activation and assembly (Morishima, Y., Kanelakis, K. C., Silverstein, A.M., Dittmar, K. D., Estrada, L., and Pratt, W. B. (2000) J. Biol. Chem. 275, 6894-6900). Here we demonstrate that the first step in assembly is the ATP-dependent and hsp40 (YDJ-1)-dependent binding of hsp70 to the GR. After elimination of free hsp70, these preformed GR.hsp70 complexes can be activated to the steroid binding state by the hsp70 free assembly system in a second ATP-dependent step. hsp90 is required for opening of the steroid binding pocket and is converted to its ATP-dependent conformation during this second step. We predict that hsp70 in its ATP-dependent conformation binds initially to the folded receptor and is then converted to the ADP-dependent form with high affinity for hydrophobic substrate. This conversion initiates the opening of the hydrophobic steroid binding pocket such that it can now accept the hydrophobic binding form of hsp90, which in turn must be converted to its ATP-dependent conformation for the pocket to be accessible by steroid.