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The data presented in this article are connected to our research article entitled “D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo” (Furlan et al., 2018). These data further extend our understanding of the inhibitory effects of D2A-Ala peptide. Dose-response curve using a wide range of concentrations of D2A-Ala shows that this peptide has no effects per se on proliferation of rat smooth muscle cells (RSMC). However, D2A-Ala dose-dependently inhibits epidermal growth factor (EGF)-induced RSMC proliferation. Kinetics lasting up to seven days revealed that D2A-Ala peptide completely blocked EGF-promoted RSMC proliferation. Moreover, D2A-Ala peptide inhibited invasion of HT 1080 cells towards RSMC. Keywords: Peptide, Urokinase receptor, Cell proliferation, Cell invasion

Type I disorders of glycosylation (CDG), the most frequent of which is phosphomannomutase 2 (PMM2-CDG), are a group of diseases causing the incomplete N-glycosylation of proteins. PMM2-CDG is an autosomal recessive disease with a large phenotypic spectrum, and is associated with mutations in the PMM2 gene. The biochemical analysis of mutants does not allow a precise genotype&ndash, phenotype correlation for PMM2-CDG. PMM2 is very tolerant to missense and loss of function mutations, suggesting that a partial deficiency of activity might be beneficial under certain circumstances. The patient phenotype might be influenced by variants in other genes associated with the type I disorders of glycosylation in the general population.

D2A-Ala is a synthetic peptide that has been created by introducing mutations in the original D2A sequence, 130 IQEGEEGRPKDDR 142 of human urokinase receptor (uPAR). In vitro, D2A-Ala peptide displays strong anti- tumoural properties inhibiting EGF-induced chemotaxis, invasion and proliferation of a human fi brosarcoma cell line, HT 1080, and a human colorectal adenocarcinoma cell line, HT 29. D2A-Ala exerts its e ff ects by preventing EGF receptor (EGFR) phosphorylation. To test D2A-Ala in vivo, this peptide was PEGylated generating polyethyleneglycol (PEG)-D2A-Ala peptide. PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunode fi cient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala signi fi cantly prevents tumour growth decreasing size, weight and density of tumours. The most e ffi cient doses of the peptide were 5 and 10 mg/kg, thereby relevant for possible development of the peptide into a drug against cancer in particular tumours ex- pressing EGFR.

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvβ3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.

Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. Funding was provided by Telethon - Italy (Grant no. GGP12108). This work was partially supported by the Fundación Séneca del Centro de Coordinación de la Investigación de la Región de Murcia under Project 18946/JLI/13. Powered@NLHPC: This research was partially supported by the supercomputing infrastructure of the NLHPC (ECM-02). Farmacia Medicina

The most frequent disorder of glycosylation, PMM2-CDG, is caused by a deficiency of phosphomannomutase activity. In humans two paralogous enzymes exist, both of them require mannose 1,6-bis-phosphate or glucose 1,6-bis-phosphate as activators, but only phospho-mannomutase1 hydrolyzes bis-phosphate hexoses. Mutations in the gene encoding phosphomannomutase2 are responsible for PMM2-CDG. Although not directly causative of the disease, the role of the paralogous enzyme in the disease should be clarified. Phosphomannomutase1 could have a beneficial effect, contributing to mannose 6-phosphate isomerization, or a detrimental effect, hydrolyzing the bis-phosphate hexose activator. A pivotal role in regulating mannose-1phosphate production and ultimately protein glycosylation might be played by inosine monophosphate that enhances the phosphatase activity of phosphomannomutase1. In this paper we analyzed human phosphomannomutases by conventional enzymatic assays as well as by novel techniques such as 31P-NMR and thermal shift assay. We characterized a triple mutant of phospomannomutase1 that retains mutase and phosphatase activity, but is unable to bind inosine monophosphate.

Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments. © 2015 The Authors Drug Testing and Analysis Published by John Wiley & Sons Ltd.

Phosphomannomutase 2 (PMM2) deficiency represents the most frequent type of congenital disorders of glycosylation. For this disease there is no cure at present. The complete loss of phosphomannomutase activity is probably not compatible with life and people affected carry at least one allele with residual activity. We characterized wild-type PMM2 and its most common hypomorphic mutant, p.F119L, which is associated with a severe phenotype of the disease. We demonstrated that active species is the dimeric enzyme and that the mutation weakens the quaternary structure and, at the same time, affects the activity and the stability of the enzyme. We demonstrated that ligand binding stabilizes both proteins, wild-type and F119L-PMM2, and promotes subunit association in vitro. The strongest effects are observed with glucose-1,6-bisphosphate (Glc-1,6-P2) or with monophosphate glucose in the presence of vanadate. This finding offers a new approach for the treatment of PMM2 deficiency. We propose to enhance Glc-1,6-P2 concentration either acting on the metabolic pathways that control its synthesis and degradation or exploiting prodrugs that are able to cross membranes.

The hyperthermophilic archaeon (formerly archaebacterium) Thermococcus litoralis grows at temperatures up to 98 degrees C using peptides and proteins as the sole sources of carbon and nitrogen. Cell-free extracts of the organism contained two distinct types of aromatic aminotransferases (EC 2.6.1.57) which were separated and purified to electrophoretic homogeneity. Both enzymes are homodimers with subunit masses of approximately 47 kDa and 45 kDa. Using 2-oxoglutarate as the amino acceptor, each catalyzed the pyridoxal-5'-phosphate-dependent transamination of the three aromatic amino acids but showed virtually no activity towards aspartic acid, alanine, valine or isoleucine. From the determination of Km and kcat values using 2-oxoglutarate, phenylalanine, tyrosine and tryptophan as substrates, both enzymes were shown to be highly efficient at transaminating phenylalanine (kcat/Km approximately 400 s-1 mM-1); the 47-kDa enzyme showed more activity towards tyrosine and tryptophan compared to the 45-kDa one. Kinetic analyses indicated a two-step mechanism with a pyridoxamine intermediate. Both enzymes were virtually inactive at 30 degrees C and exhibited maximal activity between 95-100 degrees C. They showed no N-terminal sequence similarity with each other (approximately 30 residues), nor with the complete amino acid sequences of aromatic aminotransferases from Escherichia coli and rat liver. The catalytic properties of the two enzymes are distinct from bacterial aminotransferases, which have broad substrate specificities, but are analogous to two aromatic aminotransferases which play a biosynthetic role in a methanogenic archaeon. In contrast, it is proposed that one or both play a catabolic role in proteolytic T. litoralis in which they generate glutamate and an arylpyruvate. These serve as substrates for glutamate dehydrogenase and indolepyruvate ferredoxin oxidoreductase in a novel pathway for the utilization of aromatic amino acids.

Background The pharmacological chaperones therapy is a promising approach to cure genetic diseases. It relies on substrate competitors used at sub-inhibitory concentration which can be administered orally, reach difficult tissues and have low cost. Clinical trials are currently carried out for Fabry disease, a lysosomal storage disorder caused by inherited genetic mutations of alpha-galactosidase. Regrettably, not all genotypes respond to these drugs. Results We collected the experimental data available in literature on the enzymatic activity of ninety-six missense mutants of lysosomal alpha-galactosidase measured in the presence of pharmacological chaperones. We associated with each mutation seven features derived from the analysis of 3D-structure of the enzyme, two features associated with their thermo-dynamic stability and four features derived from sequence alone. Structural and thermodynamic analysis explains why some mutants of human lysosomal alpha-galactosidase cannot be rescued by pharmacological chaperones: approximately forty per cent of the non responsive cases examined can be correctly associated with a negative prognostic feature. They include mutations occurring in the active site pocket, mutations preventing disulphide bridge formation and severely destabilising mutations. Despite this finding, prediction of mutations responsive to pharmacological chaperones cannot be achieved with high accuracy relying on combinations of structure- and thermodynamic-derived features even with the aid of classical and state of the art statistical learning methods. We developed a procedure to predict responsive mutations with an accuracy as high as 87%: the method scores the mutations by using a suitable position-specific substitution matrix. Our approach is of general applicability since it does not require the knowledge of 3D-structure but relies only on the sequence. Conclusions Responsiveness to pharmacological chaperones depends on the structural/functional features of the disease-associated protein, whose complex interplay is best reflected on sequence conservation by evolutionary pressure. We propose a predictive method which can be applied to screen novel mutations of alpha galactosidase. The same approach can be extended on a genomic scale to find candidates for therapy with pharmacological chaperones among proteins with unknown tertiary structures.

Pompe disease is a lysosomal storage disorder (LSD) caused by mutations in the gene that encodes acid α-glucosidase (GAA). Recently, small molecule pharmacological chaperones have been shown to increase protein stability and cellular levels for mutant lysosomal enzymes and have emerged as a new therapeutic strategy for the treatment of LSDs. In this study, we characterized the pharmacological chaperone 1-deoxynojirimycin (DNJ) on 76 different mutant forms of GAA identified in Pompe disease. DNJ significantly increased enzyme activity and protein levels for 16 different GAA mutants in patient-derived fibroblasts and in transiently transfected COS-7 cells. Additionally, DNJ increased the processing of these GAA mutants to their mature lysosomal forms, suggesting facilitated trafficking through the secretory pathway. Immunofluorescence microscopy studies showed increased colocalization of GAA with the lysosomal marker LAMP2 after incubation with DNJ, confirming increased lysosomal trafficking. Lastly, a GAA structural model was constructed based on the related eukaryotic glucosidase maltase-glucoamylase. The mutated residues identified in responsive forms of GAA are located throughout most of the structural domains, with half of these residues located in two short regions within the catalytic domain. Taken together, these data support further evaluation of DNJ as a potential treatment for Pompe disease in patients that express responsive forms of GAA. Hum Mutat 30:1–10, 2009. © 2009 Wiley-Liss, Inc.

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.

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.

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 λ 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. The sequence data have been deposited on the EMBL Sequence Data Bank (accession No Z25466).

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.

1. 1. Complementary DNA encoding cytosolic aspartate aminotransferase was isolated from an adult bovine heart library. 2. 2. The amino add 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.

The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspAT Ss ), was expressed in Escherichia coli and the enzyme purified to homogencity. 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 AspAT Ss are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same K m values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspAT Ss is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus . The protocol described may be used to produce thermostable archaebacterial enzymes in mesophilic hosts.

The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (> 60-degrees-C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75-degrees-C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of k(cat) is non-linear as well.

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[ 3 H]hydride and sequencing the specifically labeled peptic peptides. The aminoacidic 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 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 ω-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 K M value of the thermophilic aspartate aminotransferase towards alanine is at least one order of magnitude lower than that of the mesophilic analogue enzymes.

Urokinase-type plasminogen activator (u-PA) is a key enzyme involved in migration and invasiveness of cells, both in cancer and in several normal physiological processes (Reich, 1978; Dana et al., 1985; Saksela and Rifkin, 1988; Blasi and Verde, 1990). The application of modem biochemical and molecular biological techniques has identified some of the steps at which u-PA activity is regulated. A large number of studies have dealt with the biological roles of u-PA catalyzed plasminogen activation. These can be summarized as follows: (1) u-PA-dependent proteolysis can take place on cells with surface-bound reactants; physiologically, this may well be the most relevant form of plasminogen activation; (2) the plasminogen activating system and its regulation are complex and several molecules are known to be involved (activators, substrate, inhibitors, receptors), although other, as yet unidentified, components are also implicated and (3) on a biochemical basis the u-PA system exploited by cancer cells appears to be qualitatively identical to that used in normal

Background Thermophilic organisms are able to live at high temperatures ranging from 50 to > 100°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.

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.

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.

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.

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.

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C, The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling, Gel filtration analysis indicated that SsAspAT unfolds with an N-2 reversible arrow 2D mechanism, In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure, At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism, Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the Delta G degrees(H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter bn, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and Delta C-p is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT, In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network

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.