Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

Insights into hemoglobin assembly through in vivo mutagenesis of α-hemoglobin stabilizing protein

α-Hemoglobin stabilizing protein (AHSP) is believed to facilitate adult Hemoglobin A assembly and protect against toxic free α-globin subunits. Recombinant AHSP binds multiple forms of free α-globin to stabilize their structures and inhibit precipitation. However, AHSP also stimulates autooxidation of αO(2) subunit and its rapid conversion to a partially unfolded bishistidyl hemichrome structure. To investigate these biochemical properties, we altered the evolutionarily conserved AHSP proline 30 in recombinantly expressed proteins and introduced identical mutations into the endogenous murine Ahsp gene. In vitro, the P30W AHSP variant bound oxygenated α chains with 30-fold increased affinity. Both P30W and P30A mutant proteins also caused decreased rates of αO(2) autooxidation as compared with wild-type AHSP. Despite these abnormalities, mice harboring P30A or P30W Ahsp mutations exhibited no detectable defects in erythropoiesis at steady state or during induced stresses. Further biochemical studies revealed that the AHSP P30A and P30W substitutions had minimal effects on AHSP interactions with ferric α subunits. Together, our findings indicate that the ability of AHSP to stabilize nascent α chain folding intermediates prior to hemin reduction and incorporation into adult Hemoglobin A is physiologically more important than AHSP interactions with ferrous αO(2) subunits.     

The potassium channel KcsA: a model protein in studying membrane protein oligomerization and stability of oligomeric assembly?

Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein–protein and protein–lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein–lipid interaction between anionic phospholipids and “hot spots” on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel.     

The phylogenesis of protein α-amylase inhibitors from wheat seed and the speciation of polyploid wheats

Summary Protein -amylase inhibitors extracted with water from seeds of a number of Triticum and Aegilops species were characterized according to their molecular weights and action specificities towards human salivary and Tenebrio molitor L. -amylases. Four inhibitor peaks, with molecular weights 60000, 44000, 22000 and 11000, active towards the two amylases have been detected. Another inhibitor peak with molecular weight 11000, only active towards the insect -amylase, has been found in several species tested. Triticum urartu showed only the 22000 inhibitor peak, while other diploid Triticum species did not exhibit any inhibitory activity. All the diploid Aegilops species tested contained -amylase inhibitors and the inhibitor patterns differed greatly even for closely related species. In general, tetraploid Triticum species (turgidun and timopheevi) exhibited amylase inhibitor patterns of higher complexity than diploid Triticum and Aegilops species.The relationships existing among the amylase inhibitor patterns of the Triticinae species tested are consistent with the hypothesis of the polyphyletic origin of tetraploid wheats by Sarkar and Stebbins (1956) and suggest that the amylase inhibitors from diploid species all derive from common ancestral genes.     

Expression of core-binding factor α1 and osteocalcin in fluoride-treated fibroblasts and osteoblasts

To study the effects and importance of fluoride on FBs in the development of extraperiosteal calcification and the ossification of skeletal fluorosis, the presence of the osteogenic phenotype, which is indicated by the expression of core-binding factor α1 (Cbfa1) and osteocalcin (OCN), in an FB cell line (L929) and in osteoblasts (OBs) exposed to fluoride was determined. Fibroblasts and osteoblasts were exposed to different concentrations of fluoride (0, 0.0001, 0.001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻). By using RT-PCR and ELISA, the mRNA levels of Cbfa1 and OCN were measured at 48 h, and the protein levels of Cbfa1 and OCN were measured at 2, 4, 24, 48 and 72 h. The data demonstrated the following: (1) The Cbfa1 protein level in fluoride-treated fibroblasts clearly increased at 48 h in the groups treated with 0.0001, 0.001, 0.1, 1.0 and 20.0 mg/L F⁻. The Cbfa1 protein level of the group treated with 10 mg/L F⁻ at 72 h was higher than that of the control group. The level of Cbfa1 mRNA in the fibroblasts was much higher at 48 h in the group treated with 10.0 mg/L F⁻ than in the control group. (2) The OCN protein level in fluoride-treated fibroblasts was significantly higher than that of the control group in the 0.0001, 0.1, 1.0, 10.0 and 20.0 mg/L F⁻ groups at 2 h, and in the 0.001 and 0.1 F⁻ groups at 4 h. A slightly higher level of OCN mRNA in fluoride-treated fibroblasts was also found in the 1.0 and 20.0 mg/L F⁻ groups compared to the control group. (3) The expressions of Cbfa1 and OCN in osteoblasts treated with the same experimental conditions as the fibroblasts were up-regulated by fluoride following the same trend as in the fibroblasts. Our results showed an increase in the expression of Cbfa1 and OCN in fibroblasts and osteoblasts exposed to fluoride and suggested that the osteogenic function of fibroblasts induced by fluoride could play an important role in the development of extraperiosteal ossification during skeletal fluorosis.     

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α

Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.     

The identification and characterization of IbpA,a novel α-crystallin-type heat shock protein from mycoplasma

α-Crystallin-type small heat shock proteins (sHsps) are expressed in many bacteria, animals, plants, and archaea. Among mycoplasmas (Mollicutes), predicted sHsp homologues so far were found only in the Acholeplasmataceae family. In this report, we describe the cloning and functional characterization of a novel sHsp orthologue, IbpA protein, present in Acholeplasma laidlawii. Importantly, similar to the endogenously expressed sHsp proteins, the recombinant IbpA protein was able to spontaneously generate oligomers in vitro and to rescue chemically denatured bovine insulin from irreversible denaturation and aggregation. Collectively, these data suggest that IbpA is a bona fide member of the sHsps family. The immune-electron microscopy data using specific antibodies against IbpA have revealed different intracellular localization of this protein in A. laidlawii cells upon heat shock, which suggests that IbpA not only may participate in the stabilization of individual polypeptides, but may also play a protective role in the maintenance of various cellular structures upon temperature stress.     

Purification and partial characterisation of and α-tocopherol-binding protein from rabbit heart cytosol

An -tocopherol-binding protein has been isolated and purified from rabbit heart cytosol. The purified protein had an apparent molecular mass of 14,200, as derived from SDS-PAGE. The content of the protein in rabbit heart was around 11.8 g per g of tissue. The binding of -tocopherol to the purified protein was rapid, reversible, and saturable. Neither nor tocopherol could displace the bound -tocopherol from the protein, suggesting a high specificity for -tocopherol. -Tocopherol-binding protein did not bind oleate. Transfer of -tocopherol from liposomes to mitochodria was stimulated 8-fold in the presence of the binding protein, suggesting that this protein may be involved in the intracellular transport of -tocopherol in the heart.     

Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits

β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α'- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α'/α-subunits were complexes of more than 720 kD. The α'- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α'/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α'- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.     

The role of the prion protein in the internalization of α-synuclein amyloids

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein amyloids in several regions of the brain. α-Synuclein fibrils are able to spread via cell-to-cell transfer, and once inside the cells, they can template the misfolding and aggregation of the endogenous α-synuclein. Multiple mechanisms have been shown to participate in the process of propagation: endocytosis, tunneling nanotubes and macropinocytosis. Recently, we published a research showing that the cellular form of the prion protein (PrP^C) acts as a receptor for α-synuclein amyloid fibrils, facilitating their internalization through and endocytic pathway. This interaction occurs by a direct interaction between the fibrils and the N-terminal domain of PrP^C. In cell lines expressing the pathological form of PrP (PrP^Sc), the binding between PrP^C and α-synuclein fibrils prevents the formation and accumulation of PrP^Sc, since PrP^C is no longer available as a substrate for the pathological conversion templated by PrP^Sc. On the contrary, PrP^Sc deposits are cleared over passages, probably due to the increased processing of PrP^C into the neuroprotective fragments N1 and C1. Starting from these data, in this work we present new insights into the role of PrP^C in the internalization of protein amyloids and the possible therapeutic applications of these findings.     

M?ssbauer studies of adrenodoxin. The mechanism of electron transfer in a hydroxylase iron–sulphur protein

1. Mössbauer spectra were measured of adrenodoxin purified from porcine adrenal glands. They show similarities to the spectra of the plant ferredoxins. All of these proteins contain two atoms of iron and two of inorganic sulphide per molecule, and on reduction accept one electron. 2. As with the plant ferredoxins the adrenodoxin for these measurements was enriched with ⁵⁷Fe by reconstitution of the apo-protein, and subsequently was carefully purified and checked by a number of methods to ensure that it was in the same conformation as the native protein and contained no extraneous iron. 3. The Mössbauer spectra of oxidized adrenodoxin at temperatures from 4.2°K to 197°K show that the iron atoms are probably high-spin Fe³⁺, and in similar environments, and experience little or no magnetic field from the electrons. 4. Mössbauer spectra of reduced adrenodoxin showed magnetic hyperfine structure at all temperatures from 1.7°K to 244°K, in contrast with the reduced plant ferredoxins, which showed it only at lower temperatures. This is a consequence of a longer electron-spin relaxation time in reduced adrenodoxin. 5. At 4.2°K in a small magnetic field the spectrum of reduced adrenodoxin shows a sixline Zeeman pattern due to Fe³⁺ superimposed upon a combined magnetic and quadrupole spectrum due to Fe²⁺. 6. In a large magnetic field (30kG) each hyperfine pattern is further split into two. Analysis of these spectra at 4.2°K and 1.7°K shows that the effective fields at the Fe³⁺ and Fe²⁺ nuclei are in opposite directions. This agrees with the proposal, first made for the ferredoxins, that the iron atoms are antiferromagnetically coupled. 7. In accord with the model for the ferredoxins, it is proposed that the oxidized adrenodoxin contains two high-spin Fe³⁺ atoms which are antiferromagnetically coupled; on reduction one iron atom becomes high-spin Fe²⁺.     

The first α-helical domain of the vesicle-inducing protein in plastids 1 promotes oligomerization and lipid binding

The vesicle-inducing protein in plastids 1 (Vipp1) is an essential component for thylakoid biogenesis in cyanobacteria and chloroplasts. Vipp1 proteins share significant structural similarity with their evolutionary ancestor PspA (bacterial phage shock protein A), namely a predominantly α-helical structure, the formation of oligomeric high molecular weight complexes (HMW-Cs) and a tight association with membranes. Here, we elucidated domains of Vipp1 from Arabidopsis thaliana involved in homo-oligomerization as well as association with chloroplast inner envelope membranes. We could show that the 21 N-terminal amino acids of Vipp1, which form the first α-helix of the protein, are essential for assembly of the 2 MDa HMW-C but are not needed for formation of smaller subcomplexes. Interestingly, removal of this domain also interferes with association of the Vipp1 protein to the inner envelope. Fourier transform infrared spectroscopy of recombinant Vipp1 further indicates that Escherichia coli lipids bind tightly enough that they can be co-purified with the protein. This feature also depends on the presence of the first helix, which strongly supports an interaction of lipids with the Vipp1 HMW-C but not with smaller subcomplexes. Therefore, Vipp1 oligomerization appears to be a prerequisite for its membrane association. Our results further highlight structural differences between Vipp1 and PspA, which might be important in regard to their different function in thylakoid biogenesis and bacterial stress response, respectively.     

The stability and thermodynamic parameters of a very thermostable and calcium-independent α-amylase from a newly isolated bacterium,Anoxybacillus beppuensis TSSC-1

Anoxybacillus beppuensis TSSC-1 (GenBank Number, EU710556), a thermophilic bacterium isolated from a hot spring reservoir, was found to optimally secrete a monomeric α-amylase at 55 °C and pH 7. The enzyme was purified to homogeneity by a single-step purification on phenyl sepharose 6FF, achieving a 58% yield, 10,000 U/mg specific activity and 19.5 fold purification. The molecular weight, K_m and V_max were 43 kD, 0.5 mg ml^?1 and 3571.42 μmol ml^?1 m^?1, respectively. The enzymatic catalysis of soluble starch was optimum at 80 °C and pH 7. The thermodynamic parameters, K_d, t_1/2, ΔH*, ΔS*, E and ΔG*, were consistent. The very compact structure of the enzyme and the transitional enzyme–substrate complex resisted denaturation at extreme temperatures and alkaline pH. The K_d and t_1/2 measurements were consistent with the high thermostability and pH tolerance observed. The structural stability of the enzyme was also reflected by the values of ΔH*, ΔS*, E and ΔG*. While the enzyme did not exhibit metal ion dependency, it was resistant to chemical denaturation. The broad thermo- and pH-tolerance of this enzyme suggests potential commercial opportunities.     

The α4β1 integrin and the EDA domain of fibronectin regulate a profibrotic phenotype in dermal fibroblasts

Prompt deposition of fibronectin-rich extracellular matrix is a critical feature of normal development and the host-response to injury. Fibronectin isoforms that include the EDA and EDB domains are prominent in these fibronectin matrices. We now report using human dermal fibroblast cultures that the EDA domain of fibronectin or EDA-derived peptides modeled after the C–C′ loop promote stress fiber formation and myosin-light chain phosphorylation. These changes are accompanied by an increase in fibronectin synthesis and fibrillogenesis. These effects are blocked by pretreating cells with either siRNA or blocking antibody to the α4 integrin. Our data indicate that the interaction between the α4β1 integrin and the EDA domain of fibronectin helps to drive tissue fibrosis by promoting a contractile phenotype and an increase in fibronectin synthesis and deposition.     

Properties of allophycocyanin II and its α- and β-subunits from the thermophilic blue–green alga Mastigocladus laminosus

Purified allophycocyanin II and its subunits have been examined with respect to spectroscopic properties, sedimentation, reconstitution and isoelectric behaviour. In 0.02m-potassium phosphate buffer, pH8.0, and at a concentration of 0.25mg/ml, allophycocyanin II and its alpha- and beta-subunits show visible absorption maxima at 650, 615 and 615nm respectively, whereas the fluorescence emission maxima were determined to be at 662, 640 and 630nm respectively. The absorption difference spectrum (dilution difference) of allophycocyanin II displays maxima at 650 and 590nm with a minimum at 610nm. The c.d. spectrum of allophycocyanin II showed only one positive-ellipticity band at 635nm, and a major negative-ellipticity band at 340nm. Oxidation of allophycocyanin II, low- and high-pH solutions (pH3.0 and 11.0), various ethanol concentrations as well as dialysis against distilled water induce a spectral change leading to phycocyanin-like characteristics. In most cases these shifts are reversible. Allophycocyanin II is thermostable over a period of 60min at temperatures up to 60 degrees C. The isoelectric points of allophycocyanin II and its alpha- and beta-subunits are 4.65, 4.64 and 4.82 respectively. Estimated molecular weights from sedimentation-equilibrium analyses were 102500 for allophycocycanin II, 16000 for the alpha- and 31500 for the beta-subunit. Recombination of alpha- and beta-subunits leads to allophycocyanin II, which is indistinguishable from native allophycocyanin with respect to its spectral form, to its gel-filtration and to its electrophoretic behaviour.