Fetal growth patterns in Beckwith-Wiedemann syndrome.

We provide data on fetal growth pattern on the molecular subtypes of Beckwith-Wiedemann syndrome (BWS): IC1 gain of methylation (IC1-GoM), IC2 loss of methylation (IC2-LoM), 11p15.5 paternal uniparental disomy (UPD), and CDKN1C mutation. In this observational study, gestational ages and neonatal growth parameters of 247 BWS patients were compared by calculating gestational age-corrected standard deviation scores (SDS) and proportionality indexes to search for differences among IC1-GoM (n = 21), UPD (n = 87), IC2-LoM (n = 147), and CDKN1C mutation (n = 11) patients. In IC1-GoM subgroup, weight and length are higher than in other subgroups. Body proportionality indexes display the following pattern: highest in IC1-GoM patients, lowest in IC2-LoM/CDKN1C patients, intermediate in UPD ones. Prematurity was significantly more prevalent in the CDKN1C (64%) and IC2-LoM subgroups (37%). Fetal growth patterns are different in the four molecular subtypes of BWS and remarkably consistent with altered gene expression primed by the respective molecular mechanisms. IC1-GoM cases show extreme macrosomia and severe disproportion between weight and length excess. In IC2-LoM/CDKN1C patients, macrosomia is less common and associated with more proportionate weight/length ratios with excess of preterm birth. UPD patients show growth patterns closer to those of IC2-LoM, but manifest a body mass disproportion rather similar to that seen in IC1-GoM cases.

Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate.

A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribution of uPA in tumor cells and tissues suggest that the uPA/uPAR interaction may be important in regulating extracellular proteolysis-dependent processes (e.g., invasion, tissue destruction). Phorbol myristate acetate (PMA), an inducer of U937 cell differentiation to macrophage-like cells, elicits a time- and concentration-dependent increase in the number of uPAR molecules as shown by binding, cross-linking, and immunoprecipitation studies. The effect of PMA is blocked by cycloheximide. Overall, the data indicate that PMA increases the synthesis of uPA. PMA treatment also causes a decrease in the affinity of the uPAR for uPA, thus uncovering another way of regulating the interaction between uPA and uPAR. In addition, the PMA treatment causes a modification of migration of the cross-linked receptor in mono- and bidimensional gel electrophoresis.

Environment specific substitution tables for thermophilic proteins.

BACKGROUND: Thermophilic organisms are able to live at high temperatures ranging from 50 to > 100 degrees C. Their proteins must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive. Subtle differences between thermophilic and mesophilic molecules can be found when sequences or structures from homologous proteins are compared, but often these differences are family-specific and few general rules have been derived. The availability of complete genome sequences has now made it feasible to perform a large-scale comparison between mesophilic and thermophilic proteins, the latter of which primarily come from archaeal genomes although a few complete genomes of thermophilic eubacteria are also available. RESULTS: We compared mesophilic proteins with their thermophilic counterparts of archaeal or eubacterial origins independently. This was based on the assumption that in these two kingdoms, different mechanisms may have been exploited for the adaptation of proteins at high temperatures. We derived the environment specific amino acid compositions of thermophilic proteins from 10 archaeal and seven eubacterial genomes, by aligning a large number of sequences from thermophilic proteins with their close mesophilic homologues of known three-dimensional (3D) structure. We further analysed environment specific substitutions, which lead from mesophilic proteins to either archaeal or eubacterial thermophilic proteins. CONCLUSION: Our comparisons were based on homology-based structural predictions for a large number of thermophilic proteins. We demonstrated that thermal adaptation in the archaeal and eubacterial kingdoms is achieved in different ways. The main differences concern the usage of Gln, Ile and positively charged amino acids. In particular archaeal organisms appeared to have acquired thermostability by substituting non-charged polar amino acids (such as Gln) with Glu and Lys, and non-polar amino acids with Ile on the surface of proteins.

Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes.

Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized. The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli. The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum. From this analysis it turned out that indole-3-glycerol-phosphate synthase from S. solfataricus can be considered a model for studying thermostable TIM-barrel enzymes. Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S. solfataricus are discussed in relation to the thermostability of the enzyme.

Expression of a hyperthermophilic aspartate aminotransferase in Escherichia coli.

The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity. A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly. The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus. The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts.

The active site of Sulfolobus solfataricus aspartate aminotransferase.

Aspartate aminotransferase from the archaebacterium Sulfolobus solfataricus binds pyridoxal 5' phosphate, via an aldimine bond, with Lys-241. This residue has been identified by reducing the enzyme in the pyridoxal form with sodium cyanoboro[3H]hydride and sequencing the specifically labeled peptic peptides. The amino acid sequence centered around the coenzyme binding site is highly conserved between thermophilic aspartate aminotransferases and differs from that found in mesophilic isoenzymes. An alignment of aspartate aminotransferase from Sulfolobus solfataricus with mesophilic isoenzymes, attempted in spite of the low degree of similarity, was confirmed by the correspondence between pyridoxal 5' phosphate binding residues. Using this alignment it was possible to insert the archaebacterial aspartate aminotransferase into a subclass, subclass I, of pyridoxal 5' phosphate binding enzymes comprising mesophilic aspartate aminotransferases, tyrosine aminotransferases and histidinol phosphate aminotransferases. These enzymes share 12 invariant amino acids most of which interact with the coenzyme or with the substrates. Some enzymes of subclass I and in particular aspartate aminotransferase from Sulfolobus solfataricus, lack a positively charged residue, corresponding to Arg-292, which in pig cytosolic aspartate aminotransferase interacts with the distal carboxylate of the substrates (and determines the specificity towards dicarboxylic acids). It was confirmed that aspartate aminotransferase from Sulfolobus solfataricus does not possess any arginine residue exposed to chemical modifications responsible for the binding of omega-carboxylate of the substrates. Furthermore, it has been found that aspartate aminotransferase from Sulfolobus solfataricus is fairly active when alanine is used as substrate and that this activity is not affected by the presence of formate. The KM value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.

An extremely thermostable aromatic aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a strictly anaerobic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C by the fermentation of peptides. Cell-free extracts were found to contain two distinct aromatic aminotransferases (ArAT, EC 2.6.1.57), one of which was purified to electrophoretic homogeneity. P. furiosus ArAT is a homodimer with a subunit M(r) value of 44,000 +/- 1000. Using 2-ketoglutarate as the amino acceptor, the purified enzyme catalyzed the pyridoxal 5'-phosphate (PMP)-dependent transamination of phenylalanine, tyrosine and tryptophan with respective kcat values of 253, 72 and 62 (s-1 at 80 degrees C) under saturating conditions. The Km values for all three amino acids were between 1.1 and 2.1 mM and the optimum temperature for catalysis was above 95 degrees C. The melting point for the pure enzyme was also above 95 degrees C as determined by the change in ellipticity at 220 nm. Irreversible denaturation of the pure enzyme was not apparent after 6 h at 80 degrees C in the presence of PMP and 2-ketoglutarate and the time required for a 50% loss in activity at 95 degrees C was approx. 16 h. This decreased to approx. 12 h if cofactor and substrate were not added. In contrast, the apoenzyme (lacking PMP) lost most (70%) of its activity (measured after reconstitution) after 6 h at 80 degrees C, indicating that both PMP and 2-ketoglutarate stabilize the enzyme at extreme temperatures. Although few ArATs have been characterized to date, the molecular properties and substrate specificity of P. furiosus ArAT more resemble those of the ArAT from Escherichia coli than those of the analogous enzyme from rat liver. Moreover, the P. furiosus enzyme is by far the most thermostable aminotransferase of any type to be purified so far.

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions.

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions.

The interconversion of isoforms of seminal ribonuclease: modelling key intermediates and trypsin effects.

The stepwise tryptic degradation of the interconverting quaternary isoforms of seminal ribonuclease has been analysed by structural modelling, based on the experimental results obtained by treating the dimeric protein with trypsin. The results of the analysis were compared with those obtained applying to the action of trypsin on seminal ribonuclease a recently proposed predictive algorithm for limited proteolysis. The attention was focussed on the MxM form of the protein, in which the two subunits swap their N-terminal ends interconverting at equilibrium with the M=M form with no interchange between subunits. The analysis led to the identification of a key intermediate in the interconversion pathway, and to the resolution of the apparent contradiction between prediction and actual experimental data.

Nucleotide sequence of a cDNA coding for bovine mitochondrial aspartate aminotransferase.

Aspartate aminotransferase is a pyridoxal-phosphate dependent enzyme which plays a key role in cell metabolism. We describe the cloning and sequence analysis of the cDNA encoding bovine mitochondrial aspartate aminotransferase and compare the sequence with those of isoenzymes from other mammalian species. An adult bovine heart cDNA library constructed in lambda lambda gt11 was screened using two 32P-end labeled synthetic oligonucleotides. From the screening of the cDNA library two positive clones were isolated. A subclone in pEMBL18, 6B2, generated from the longest recombinant phage was further analyzed. This clone contains an insert of 2500 bp with an Open Reading Frame of 1287 bp that encodes a protein of 430 amino acids. The deduced amino acid sequence confirms previous results obtained by mass spectrometric sequencing. We calculated the percentage of amino acid identity for each protein pair and for each comparison the average number of amino acid substitution per site (Kaa); the lowest Kaa values were obtained from the comparison between the bovine and pig enzymes. This study shows that the rate of evolution of mammalian mitochondrial AspAT is lower and more constant than the equivalent cytosolic enzyme and adds to the growing body of knowledge on the evolution of the aspartate aminotransferase.

Isolation and sequencing of a new beta-galactosidase-encoding archaebacterial gene.

The gene lacS coding for a beta-galactosidase (beta Gal; EC 3.2.1.23) has been cloned from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. It encodes a polypeptide chain of 489 amino acids (aa) (56,764 Da) in good agreement with the value directly measured for the enzyme (60 +/- 2 kDa per subunit). The aa composition of the enzyme and, in particular, its peculiarly low cysteine content (one Cys per subunit) has been confirmed; at the same time, it has been observed that the very low G + C content of the S. solfataricus genome strongly influences the codon usage preferences in the lacS sequence. There appears to be no evident similarity between this and the Escherichia coli lacZ sequence, thus suggesting that the two enzymes have analogous function, but are not homologous. By comparison with the published sequences of archaebacterial promoters, terminators and ribosome-binding sites, potential regulatory sites have been identified in the flanking regions of the S. solfataricus lacS gene.

Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor PAI-1.

The receptor for urokinase plasminogen activator (uPA) has been previously shown not to internalize its ligand, but rather to focalize its activity at the cell surface, allowing a regulated cell surface plasmin dependent proteolysis. The receptor in fact binds the proenzyme pro-uPA and allows its very efficient conversion to the active two chains form. Receptor bound active uPA can also interact with its specific type 1 inhibiror (PAI-1) which is therefore able to inhibit the cell surface plasmin formation. In this paper we show that the uPA-PAI-1 complex bound to the uPA receptor is internalized and degraded. U937 cells were incubated at 4 degrees C with labeled uPA-PAI-1 (and other ligands), the temperature then raised to 37 degrees C and the fate of the ligand followed for 3 h thereafter. The uPA-PAI-1 complex was internalized into the cells (i.e. could not be dissociated by acid treatment) and thereafter degraded (i.e. appeared in the supernatant in a non TCA-precipitable form). Other ligands (free uPA, ATF and DFP-treated uPA) were not internalized nor degraded. The degradation of the uPA-PAI-1 complex is preceded by internalization and is inhibited by chloroquine, an inhibitor of lysosomal protein degradation. These data suggest the existence of a cellular cycle of uPA. After synthesis pro-uPA is secreted, bound to the receptor and activated to two chain uPA. On the surface, uPA can activate surface bound plasminogen to produce surface bound plasmin. In the presence of PAI-1 uPA activity is inhibited and plasmin production interrupted, while the uPA-PAI-1 complex is internalized and degraded.

The receptor for urokinase-plasminogen activator.

Many human cells and cell lines possess a specific receptor that binds urokinase plasminogen activator (uPA) with an affinity of about 10(-10) M. Bound enzyme is not internalized, is slowly dissociated, and retains its enzymatic activity. The amino acid sequence of uPA responsible for receptor binding is located within the first 35 aminoterminal residues, ie, in the growth factor domain. Binding, however, is not competed for by other proteins that contain the growth factor domain (including epidermal growth factor). Cells that produce uPA secrete the pro-uPA form, which subsequently binds to the receptor. A431 cells, in fact, have their receptors completely saturated with pro-uPA. It is proposed that uPA:uPA-receptor interaction plays a direct role in physiological and pathological processes that require cell migration.

Binding of single-chain prourokinase to the urokinase receptor of human U937 cells.

The single-chain form of human urokinase plasminogen activator (uPA) is the major form of the enzyme found in cells, tissues, and extracellular fluids. The protein, called pro-uPA, has high (Kd = 0.5 nM) affinity for the specific uPA receptor of U937 human monocyte-like cells. Its conversion to two-chain uPA by plasmin does not appreciably change the binding parameters. In addition, conversion of pro-uPA to uPA occurs with receptor-bound pro-uPA and does not lead to dissociation from the membrane. These data show that secreted pro-uPA can find its way to the specific surface receptor without previous conversion to the two-chain form and that, once bound, can be activated by plasmin.

Comparative studies on thermophilicity and thermostability of aspartate aminotransferases.

Aspartate aminotransferase from Sulfolobus solfataricus (AspATSs) is an extremely thermophilic and thermostable enzyme. In order to investigate the structural features which underlie thermophilicity and thermostability, two isoforms of AspATSs differing by a single amino acid residue were compared. The first isoform is the naturally occurring enzyme, whereas the second is a genetically engineered mutant. Thermophilicity, short-term and long-term thermostability of the isoenzymes were independently evaluated and the influence of a cysteine residue on the three properties was assessed.

Cloning and sequence analysis of A cDNA encoding bovine cytosolic aspartate aminotransferase.

1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library. 2. The amino acid sequence deduced for the protein (412 amino acids) is extremely similar (> 94% identity) to that of porcine cytosolic aspartate aminotransferase but interesting differences were noticed comparing the position of cysteine residues.

Cloning and sequencing of the gene coding for aspartate aminotransferase from the thermoacidophilic archaebacterium Sulfolobus solfataricus.

The gene coding for aspartate aminotransferase (EC 2.6.1.1) has been cloned from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus strain MT4. Partial sequence data obtained directly from the purified protein and from the two cyanogen-bromide-generated peptides confirm the primary structure of aspartate aminotransferase inferred from the nucleotide sequence of its gene. A comparison of the enzyme with other aminotransferases revealed an interesting similarity with tyrosine aminotransferase from rat liver (EC 2.6.1.5) and allowed some tentative assignments of the residues implied in the catalysis. The aspartate aminotransferase gene-flanking regions were compared to those of other archaebacterial genes already described in the literature with the aim of identifying potential regulatory sites.

Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor.

Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). We have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of 125I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Performed 125I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor.

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.

Stability of a thermophilic TIM-barrel enzyme: indole-3-glycerol phosphate synthase from the thermophilic archaeon Sulfolobus solfataricus.

The stability and activity of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus were studied as a function of pH and temperature. In this paper we focus on three points: (1) the long-term stability of the protein to irreversible denaturation at high temperature; (2) the short-term stability of the protein to reversible temperature-driven unfolding; and (3) the dependence of its activity on temperature. Results can be summarized as follows: (a) the same first-order kinetic constant (0.020+/-0.003 min-1) was determined at different pH values (6.5, 8.0 and 9.5) from long-term stability experiments at 80 degrees C; (b) short-term stability experiments revealed different behaviour in two different pH ranges (6.5-8.0, 8.5-9.5), suggesting that the melting temperature is higher at alkaline than at neutral pH; (c) the dependence of activity on temperature was investigated at pH 7.0 and 9.0, and a discontinuity was observed in the Arrhenius plot of kcat values at pH 9.0. We also investigated the stability in the presence of guanidinium chloride at 20 degrees C either at pH 7.0 or at pH 9.0, and we present data that indicate that the unfolding mechanism closely approaches a two-state model at pH 7.0 and a more complex mechanism at pH 9.0. Satisfactory fitting of the equilibrium unfolding transition obtained by fluorescence measurements at pH 9.0 required a model that involves a stable intermediate in addition to the native and unfolded forms. At 20 degrees C the folded conformation is more stable than the unfolded conformation by (14. 7+/-1.2) kJ/mol at pH 7.0 and by (25.5+/-1.8) kJ/mol at pH 9.0.