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Background Beckwith–Wiedemann syndrome (BWS) and Pseudohypoparathyroidism type 1B (PHP1B) are imprinting disorders (ID) caused by deregulation of the imprinted gene clusters located at 11p15.5 and 20q13.32, respectively. In both of these diseases a subset of the patients is affected by multi-locus imprinting disturbances (MLID). In several families, MLID is associated with damaging variants of maternal-effect genes encoding protein components of the subcortical maternal complex (SCMC). However, frequency, penetrance and recurrence risks of these variants are still undefined. In this study, we screened two cohorts of BWS patients and one cohort of PHP1B patients for the presence of MLID, and analysed the positive cases for the presence of maternal variants in the SCMC genes by whole exome-sequencing and in silico functional studies. Results We identified 10 new cases of MLID associated with the clinical features of either BWS or PHP1B, in which segregate 13 maternal putatively damaging missense variants of the SCMC genes. The affected genes also included KHDC3L that has not been associated with MLID to date. Moreover, we highlight the possible relevance of relatively common variants in the aetiology of MLID. Conclusion Our data further add to the list of the SCMC components and maternal variants that are involved in MLID, as well as of the associated clinical phenotypes. Also, we propose that in addition to rare variants, common variants may play a role in the aetiology of MLID and imprinting disorders by exerting an additive effect in combination with rarer putatively damaging variants. These findings provide useful information for the molecular diagnosis and recurrence risk evaluation of MLID-associated IDs in genetic counselling.

Fabry disease is one of the most common lysosomal storage disorders caused by mutations in the gene encoding lysosomal &alpha, galactosidase A (&alpha, Gal A) and resultant accumulation of glycosphingolipids. The sugar mimetic 1-deoxygalactonojirimycin (DGJ), an orally available pharmacological chaperone, was clinically approved as an alternative to intravenous enzyme replacement therapy. The decision as to whether a patient should be treated with DGJ depends on the genetic variant within the &alpha, galactosidase A encoding gene (GLA). A good laboratory practice (GLP)-validated cell culture-based assay to investigate the biochemical responsiveness of the variants is currently the only source available to obtain pivotal information about susceptibility to treatment. Herein, variants were defined amenable when an absolute increase in enzyme activity of &ge, 3% of wild type enzyme activity and a relative increase in enzyme activity of &ge, 1.2-fold was achieved following DGJ treatment. Efficacy testing was carried out for over 1000 identified GLA variants in cell culture. Recent data suggest that about one-third of the variants comply with the amenability criteria. A recent study highlighted the impact of inter-assay variability on DGJ amenability, thereby reducing the power of the assay to predict eligible patients. This prompted us to compare our own &alpha, galactosidase A enzyme activity data in a very similar in-house developed assay with those from the GLP assay. In an essentially retrospective approach, we reviewed 148 GLA gene variants from our former studies for which enzyme data from the GLP study were available and added novel data for 30 variants. We also present data for 18 GLA gene variants for which no data from the GLP assay are currently available. We found that both differences in experimental biochemical data and the criteria for the classification of amenability cause inter-assay discrepancy. We conclude that low baseline activity, borderline biochemical responsiveness, and inter-assay discrepancy are alarm signals for misclassifying a variant that must not be ignored. Furthermore, there is no solid basis for setting a minimum response threshold on which a clinical indication with DGJ can be justified.

Beckwith-Wiedemann syndrome (BWS) is characterized by cancer predisposition, overgrowth and highly variable association of macroglossia, abdominal wall defects, nephrourological anomalies, nevus flammeus, ear malformations, hypoglycemia, hemihyperplasia, and organomegaly. BWS molecular defects, causing alteration of expression or activity of the genes regulated by two imprinting centres (IC) in the 11p15 chromosomal region, are also heterogeneous. In this paper we define (epi)genotype-phenotype correlations in molecularly confirmed BWS patients. The characteristics of 318 BWS patients with proven molecular defect were compared among the main four molecular subclasses: IC2 loss of methylation (IC2-LoM, n=190), IC1 gain of methylation (IC1-GoM, n=31), chromosome 11p15 paternal uniparental disomy (UPD, n=87), and cyclin-dependent kinase inhibitor 1C gene (CDKN1C) variants (n=10). A characteristic growth pattern was found in each group; neonatal macrosomia was almost constant in IC1-GoM, postnatal overgrowth in IC2-LoM, and hemihyperplasia more common in UPD (P

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and post-natal growth retardation, dysmorphic facial features and body asymmetry. About 50% of the patients carry (epi)genetic alterations involving chromosomes 7 or 11.The high proportion of patients with unidentified molecular etiology suggests the involvement of other genes. Interestingly, SRS patients share clinical features with the 12q14 microdeletion syndrome, characterized by several deletions with a 2.6 Mb region of overlap. Among the genes present in this interval, high mobility AT-hook 2 (HMGA2) appears to be the most likely cause of the growth deficiency, due to its described growth control function. To define the role of HMGA2 in SRS, we looked for 12q14 chromosome imbalances and HMGA2 mutations in a cohort of 45 patients with growth retardation and SRS-like phenotype but no 11p15 (epi)mutations or maternal uniparental disomy of chromosome 7 (matUPD7). We identified a novel 7 bp intronic deletion in HMGA2 present in heterozygosity in the proband and her mother both displaying the typical features of SRS. We demonstrated that the deletion affected normal splicing, indicating that it is a likely cause of HMGA2 deficiency. This study provides the first evidence that a loss-of-function mutation of HMGA2 can be associated with a familial form of SRS. We suggest that HMGA2 mutations leading to haploinsufficiency should be investigated in the SRS patients negative for the typical 11p15 (epi)mutations and matUPD7.

Fabry disease is caused by a deficiency of lysosomal alpha galactosidase and has a very large genotypic and phenotypic spectrum. Some patients who carry hypomorphic mutations can benefit from oral therapy with a pharmacological chaperone. The drug requires a very precise regimen because it is a reversible inhibitor of alpha-galactosidase. We looked for molecules that can potentiate this pharmacological chaperone, among drugs that have already been approved for other diseases. We tested candidate molecules in fibroblasts derived from a patient carrying a large deletion in the gene GLA, which were stably transfected with a plasmid expressing hypomorphic mutants. In our cell model, three drugs were able to potentiate the action of the pharmacological chaperone. We focused our attention on one of them, acetylsalicylic acid. We expect that acetylsalicylic acid can be used in synergy with the Fabry disease pharmacological chaperone and prolong its stabilizing effect on alpha-galactosidase.

Ambroxol (ABX) is a mucolytic agent used for the treatment of respiratory diseases. Bioactivity has been demonstrated as an enhancement effect on lysosomal acid β-glucosidase (β-Glu) activity in Gaucher disease (GD). The positive effects observed have been attributed to a mechanism of action similar to pharmacological chaperones (PCs), but an exact mechanistic description is still pending. The current study uses cell culture and in vitro assays to study the effects of ABX on β-Glu activity, processing, and stability upon ligand binding. Structural analogues bromohexine, 4-hydroxybromohexine, and norbromohexine were screened for chaperone efficacy, and in silico docking was performed. The sugar mimetic isofagomine (IFG) strongly inhibits β-Glu, while ABX exerts its inhibitory effect in the micromolar range. In GD patient fibroblasts, IFG and ABX increase mutant β-Glu activity to identical levels. However, the characteristics of the banding patterns of Endoglycosidase-H (Endo-H)-digested enzyme and a substantially lower half-life of ABX-treated β-Glu suggest different intracellular processing. In line with this observation, IFG efficiently stabilizes recombinant β-Glu against thermal denaturation in vitro, whereas ABX exerts no significant effect. Additional β-Glu enzyme activity testing using Bromohexine (BHX) and two related structures unexpectedly revealed that ABX alone can refunctionalize β-Glu in cellula. Taken together, our data indicate that ABX has little in vitro ability to act as PC, so the mode of action requires further clarification.

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.

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.

Fabry disease is caused by mutations in the GLA gene and is characterized by a large genotypic and phenotypic spectrum. Missense mutations pose a special problem for graduating diagnosis and choosing a cost-effective therapy. Some mutants retain enzymatic activity, but are less stable than the wild type protein. These mutants can be stabilized by small molecules which are defined as pharmacological chaperones. The first chaperone to reach clinical trial is 1-deoxygalactonojirimycin, but others have been tested in vitro. Residual activity of GLA mutants has been measured in the presence or absence of pharmacological chaperones by several authors. Data obtained from transfected cells correlate with those obtained in cells derived from patients, regardless of whether 1-deoxygalactonojirimycin was present or not. The extent to which missense mutations respond to 1-deoxygalactonojirimycin is variable and a reference table of the results obtained by independent groups that is provided with this paper can facilitate the choice of eligible patients. A review of other pharmacological chaperones is provided as well. Frequent mutations can have residual activity as low as one-fourth of normal enzyme in vitro. The reference table with residual activity of the mutants facilitates the identification of non-pathological variants.

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.

Many mutations responsible of Fabry disease destabilize lysosomal alpha-galactosidase, but retain the enzymatic activity. These mutations are associated to a milder phenotype and are potentially curable with a pharmacological therapy either with chaperones or with drugs that modulate proteostasis. We demonstrate the effectiveness of molecular dynamics simulations to correlate the genotype to the severity of the disease. We studied the relation between protein flexibility and residual enzymatic activity of pathological missense mutants in the cell. We found that mutations occurring at flexible sites are likely to retain activity in vivo. The usefulness of molecular dynamics for diagnostic purposes is not limited to lysosomal galactosidase because destabilizing mutations are widely encountered in other proteins, too, and represent a large share of all the ones associated to human diseases., Highlights • Residual alpha-galactosidase activity may relate to mild phenotype in Fabry disease. • Molecular dynamics identifies flexible residues in lysosomal alpha-galactosidase. • Mutations at flexible sites tend to maintain residual alpha-galactosidase activity.

A promising strategy for the treatment of genetic diseases, pharmacological chaperone therapy, has been proposed recently. It exploits small molecules which can be administered orally, reach difficult tissues such as the brain and have low cost. This strategy has a vast field of application. In order to make drug development as fast as possible, it is important to exploit drug repositioning. We evaluated the impact and limitations of this approach for rare diseases and we provide a shortcut in finding drugs for off-target usage. Electronic supplementary material The online version of this article (doi:10.1186/s13023-015-0273-2) contains supplementary material, which is available to authorized users.

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.

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of the genome structure of the Paracentrotus lividus (sea urchin) DNA methyltransferase (DNA MTase) gene showed the presence of an open reading frame, named METEX, in intron 7 of the gene. METEX expression is developmentally regulated, showing no correlation with DNA MTase expression. In fact, DNA MTase transcripts are present at high concentrations in the early developmental stages, while METEX is expressed at late stages of development. Two METEX cDNA clones (Met1 and Met2) that are different in the 3′ end have been isolated in a cDNA library screening. The putative translated protein from Met2 cDNA clone showed similarity with Escherichia coli endonuclease III on the basis of sequence and predictive three-dimensional structure. The protein, overexpressed in E. coli and purified, had functional properties similar to the endonuclease specific for apurinic/apyrimidinic (AP) sites on the basis of the lyase activity. Therefore the open reading frame, present in intron 7 of the P. lividus DNA MTase gene, codes for a functional AP endonuclease designated SuAP1.

Background: Mutations in phosphomannomutase2 cause glycosylation disorder, a disease without a cure that will largely benefit from accurate ligand-bound models. Results: We obtained two models of phospomannomutase2 bound to glucose 1,6-bisphosphate and validated them with limited proteolysis. Conclusion: Ligand binding induces a large conformational transition in PMM2. Significance: We produce and validate closed-form models of PMM2 that represent a starting point for rational drug discovery., The most common glycosylation disorder is caused by mutations in the gene encoding phosphomannomutase2, producing a disease still without a cure. Phosphomannomutase2, a homodimer in which each chain is composed of two domains, requires a bisphosphate sugar (either mannose or glucose) as activator, opening a possible drug design path for therapeutic purposes. The crystal structure of human phosphomannomutase2, however, lacks bound substrate and a key active site loop. To speed up drug discovery, we present here the first structural model of a bisphosphate substrate bound to human phosphomannomutase2. Taking advantage of recent developments in all-atom simulation techniques in combination with limited and site-directed proteolysis, we demonstrated that α-glucose 1,6-bisphosphate can adopt two low energy orientations as required for catalysis. Upon ligand binding, the two domains come close, making the protein more compact, in analogy to the enzyme in the crystals from Leishmania mexicana. Moreover, proteolysis was also carried out on two common mutants, R141H and F119L. It was an unexpected finding that the mutant most frequently found in patients, R141H, although inactive, does bind α-glucose 1,6-bisphosphate and changes conformation.

Cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) negatively regulates cellular proliferation and it has been shown that loss-of-function mutations in the imprinted CDKN1C gene (11p15.5) are associated with the overgrowth disorder Beckwith–Wiedemann syndrome (BWS). With recent reports of gain-of-function mutations of the PCNA domain of CDKN1C in growth-retarded patients with IMAGe syndrome or Silver–Russell syndrome (SRS), its key role for growth has been confirmed. Thereby, the last gap in the spectrum of molecular alterations in 11p15.5 in growth-retardation and overgrowth syndromes could be closed. Recent functional studies explain the strict association of CDKN1C mutations with clinically opposite phenotypes and thereby contribute to our understanding of the function and regulation of the gene in particular and epigenetic regulation in general.

Background The majority of the disease-causing mutations affect protein stability, but not functional sites and are amenable, in principle, to be treated with pharmacological chaperones. These drugs enhance the thermodynamic stability of their targets. Fabry disease, a disorder caused by mutations in the gene encoding lysosomal alpha-galactosidase, represents an excellent model system to develop experimental protocols to test the efficiency of such drugs. Methods The stability of lysosomal alpha-galactosidase under different conditions was studied by urea-induced unfolding followed by limited proteolysis and Western blotting. Results We measured the concentration of urea needed to obtain half-maximal unfolding because this parameter represents an objective indicator of protein stability. Conclusions Urea-induced unfolding is a versatile technique that can be adapted to cell extracts containing tiny amounts of wild-type or mutant proteins. It allows testing of protein stability as a function of pH, in the presence or in the absence of drugs. Results are not influenced by the method used to express the protein in transfected cells. General significance Scarce and dispersed populations pose a problem for the clinical trial of drugs for rare diseases. This is particularly true for pharmacological chaperones that must be tested on each mutation associated with a given disease. Diverse in vitro tests are needed. We used a method based on chemically induced unfolding as a tool to assess whether a particular Fabry mutation is responsive to pharmacological chaperones, but, by no means is our protocol limited to this disease., Graphical abstract, Highlights • Pharmacological chaperones stabilize the folded state of proteins. • Only some Fabry mutations can be treated with pharmacological chaperones. • Urea-induced unfolding represents a novel assay to test the efficiency of drugs. • The test with urea can be applied to a tiny amount of mutants in raw extracts. • Responsiveness of Fabry mutations to drugs can be tested with urea-induced unfolding.

Multicentric osteolysis, nodulosis and arthropathy (MONA) is a rare autosomal recessive disorder. To date, 13 mutations of the matrix metalloproteinase 2 (MMP2) gene have been detected in 26 patients with MONA and other osteolytic syndromes. Here, we describe the molecular and functional analysis of a novel MMP2 mutation in two adult Italian siblings with MONA. Both siblings displayed palmar-plantar subcutaneous nodules, tendon retractions, limb arthropathies, osteolysis in the toes and pigmented fibrous skin lesions. Molecular analysis identified a homozygous MMP2 missense mutation in exon 8 c.1228G>C (p.G410R), not detected in 260 controls and predicted by several bioinformatic tools to be pathogenic. By protein modelling, the mutant residue was predicted to affect the main chain conformation of the catalytic domain. Gelatin zymography, the gold standard test for MMP2 function, of serum-free conditioned medium from G410R-MMP2-expressing human embryonic kidney (HEK) cells, showed a complete loss of gelatinolytic activity. The novel mutation is located in the catalytic domain, as are 3 (p.E404K, p.V400del and p.G406D) of the other 13 MMP2 mutations described to date; however, p.G410R underlies a phenotype that is only partially overlapping that of other MMP2 exon 8 mutation carriers. Our results further delineate the complexity of genotype-phenotype correlations in MONA, broaden the repertoire of reported MMP2 mutation and enhance the comprehension of the protein motifs crucial for MMP2 catalytic activity.

Fabry_CEP is a user-friendly web-application designed to help clinicians Choose Eligible Patients for the therapy with pharmacological chaperones. It provides a database and a predictive tool to evaluate the responsiveness of lysosomal alpha-galactosidase mutants to a small molecule drug, namely 1-Deoxy-galactonojirimycin. The user can introduce any missense/nonsense mutation in the coding sequence, learn whether it is has been tested and gain access to appropriate reference literature. In the absence of experimental data structural, functional and evolutionary analysis provides a prediction and the probability that a given mutation is responsive to the drug.

Background: The interaction between proteins and ligands occurs at pockets that are often lined by conserved amino acids. These pockets can represent the targets for low molecular weight drugs. In order to make the research for new medicines as productive as possible, it is necessary to exploit "in silico" techniques, high throughput and fragment-based screenings that require the identification of druggable pockets on the surface of proteins, which may or may not correspond to active sites. Results: We developed a tool to evaluate the conservation of each pocket detected on the protein surface by CastP. This tool was named DrosteP because it recursively searches for optimal input sequences to be used to calculate conservation. DrosteP uses a descriptor of statistical significance, Poisson p-value, as a target to optimize the choice of input sequences. To benchmark DrosteP we used monomeric or homodimer human proteins with known 3D-structure whose active site had been annotated in UniProt. DrosteP is able to detect the active site with high accuracy because in 81% of the cases it coincides with the most conserved pocket. Comparing DrosteP with analogous programs is difficult because the outputs are different. Nonetheless we could assess the efficacy of the recursive algorithm in the identification of active site pockets by calculating conservation with the same input sequences used by other programs. We analyzed the amino-acid composition of conserved pockets identified by DrosteP and we found that it differs significantly from the amino-acid composition of non conserved pockets. Conclusions: Several methods for predicting ligand binding sites on protein surfaces, that combine 3D-structure and evolutionary sequence conservation, have been proposed. Any method relying on conservation mainly depends on the choice of the input sequences. DrosteP chooses how deeply distant homologs must be collected to evaluate conservation and thus optimizes the identification of active site pockets. Moreover it recognizes conserved pockets other than those coinciding with the sites annotated in UniProt that might represent useful druggable sites. The distinctive amino-acid composition of conserved pockets provides useful hints on the fundamental principles underlying protein-ligand interaction. Availability: http://www.icb.cnr.it/project/drosteppy/

At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes. © The Author 2012. Published by Oxford University Press.

A cluster of imprinted genes at chromosome 11p15.5 is associated with the growth disorders, Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS). The cluster is divided into two domains with independent imprinting control regions (ICRs). We describe two maternal 11p15.5 microduplications with contrasting phenotypes. The first is an inverted and in cis duplication of the entire 11p15.5 cluster associated with the maintenance of genomic imprinting and with the SRS phenotype. The second is a 160 kb duplication also inverted and in cis, but resulting in the imprinting alteration of the centromeric domain. It includes the centromeric ICR (ICR2) and the most 5' 20 kb of the non-coding KCNQ1OT1 gene. Its maternal transmission is associated with ICR2 hypomethylation and the BWS phenotype. By excluding epigenetic mosaicism, cell clones analysis indicated that the two closely located ICR2 sequences resulting from the 160 kb duplication carried discordant DNA methylation on the maternal chromosome and supported the hypothesis that the ICR2 sequence is not sufficient for establishing imprinted methylation and some other property, possibly orientation-dependent, is needed. Furthermore, the 1.2 Mb duplication demonstrated that all features are present for correct imprinting at ICR2 when this is duplicated and inverted within the entire cluster. In the individuals maternally inheriting the 160 kb duplication, ICR2 hypomethylation led to the expression of a truncated KCNQ1OT1 transcript and to down-regulation of CDKN1C. We demonstrated by chromatin RNA immunopurification that the KCNQ1OT1 RNA interacts with chromatin through its most 5' 20 kb sequence, providing a mechanism likely mediating the silencing activity of this long non-coding RNA. © The Author 2011. Published by Oxford University Press.

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.

Genomic imprinting is an epigenetic phenomenon resulting in differential expression of maternal and paternal alleles of a subset of genes. In the mouse, mutation of imprinted genes often results in contrasting phenotypes, depending on parental origin. The overgrowth-associated Beckwith-Wiedemann syndrome (BWS) and the growth restriction-associated Silver-Russell syndrome (SRS) have been linked with a variety of epigenetic and genetic defects affecting a cluster of imprinted genes at chromosome 11p15.5. Paternally derived and maternally derived 11p15.5 duplications represent infrequent findings in BWS and SRS, respectively. Here, we report a case in which a 6.5 Mb duplication of 11p15.4-pter resulted in SRS and BWS phenotypes in a child and her mother, respectively. Molecular analyses demonstrated that the duplication involved the maternal chromosome 11p15 in the child and the paternal chromosome 11p15 in the mother. This observation provides a direct demonstration that SRS and BWS represent specular images, both at the clinical and molecular levels. © 2010 Society for Pediatric Pathology.

Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith-Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally methylated loci and that the clinical presentation of these complex cases may reflect the loci and tissues affected with the epigenetic abnormalities.European Journal of Human Genetics advance online publication, 17 December 2008; doi:10.1038/ejhg.2008.233

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 parent of origin-dependent expression of the IGF2 and H19 genes is controlled by the imprinting centre 1 (IC1) consisting in a methylation-sensitive chromatin insulator. Deletions removing part of IC1 have been found in patients affected by the overgrowth- and tumour-associated Beckwith - Wiedemann syndrome (BWS). These mutations result in the hypermethylation of the remaining IC1 region, loss of IGF2/H19 imprinting and fully penetrant BWS phenotype when maternally transmitted. We now report that 12 additional cases with IC1 hypermethylation have a similar clinical phenotype but showed neither a detectable deletion nor other mutation in the local vicinity. Likewise, no IC1 deletion was detected in 40 sporadic non-syndromic Wilms' tumours. A detailed analysis of the BWS patients showed that the hypermethylation variably affected the IC1 region and was generally mosaic. We observed that all these cases were sporadic and in at least two families affected and unaffected members shared the same maternal IC1 allele but not the abnormal maternal chromosome epigenotype. Furthermore, the chromosome with the imprinting defect derived from either the maternal grandfather or maternal grandmother. Overall, these results indicate that methylation-imprinting defects at the IGF2-H19 locus can result from inherited mutations of the IC and have high recurrence risk or arise independently from the sequence context and generally not transmitted to the progeny. Despite these differences, the epigenetic abnormalities are usually present in the patients in the mosaic form and probably acquired by post-zygotic de novo methylation. Distinguishing between these two groups of cases is important for genetic counselling. © The Author 2008. Published by Oxford University Press. All rights reserved.

The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG). For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.

We ascertained a large Italian family with an autosomal dominant form of non-syndromic sensorineural hearing loss with vestibular involvement. A genome-wide scan found linkage to locus DFNA11. Sequencing of the MYO7A gene in the linked region identified a new missense mutation resulting in an Ala230Val change in the motor domain of the myosin VIIA. Myosin VIIA has already been implicated in several forms of deafness, but this is the third mutation causing a dominant form of deafness, located in the myosin VIIA motor domain in a region never involved in hearing loss until now. A modelled protein structure of myosin VII motor domain provides evidence for a significant functional effect of this missense mutation.

Background Secondary structure is used in hierarchical classification of protein structures, identification of protein features, such as helix caps and loops, for fold recognition, and as a precursor to ab initio structure prediction. There are several methods available for assigning secondary structure if the three-dimensional structure of the protein is known. Unfortunately they differ in their definitions, particularly in the exact positions of the termini. Additionally, most existing methods rely on hydrogen bonding, which means that important secondary structural classes, such as isolated β-strands and poly-proline helices cannot be identified as they do not have characteristic hydrogen-bonding patterns. For this reason we have developed a more accurate method for assigning secondary structure based on main chain geometry, which also allows a more comprehensive assignment of secondary structure. Results We define secondary structure based on a number of geometric parameters. Helices are defined based on whether they fit inside an imaginary cylinder: residues must be within the correct radius of a central axis. Different types of helices (alpha, 310 or π) are assigned on the basis of the angle between successive peptide bonds. β-strands are assigned based on backbone dihedrals and with alternating peptide bonds. Thus hydrogen bonding is not required and β-strands can be within a parallel sheet, antiparallel sheet, or can be isolated. Poly-proline helices are defined similarly, although with three-fold symmetry. Conclusion We find that our method better assigns secondary structure than existing methods. Specifically, we find that comparing our methods with those of others, amino-acid trends at helix caps are stronger, secondary structural elements less likely to be concatenated together and secondary structure guided sequence alignment is improved. We conclude, therefore, that secondary structure assignments using our method better reflects physical and evolutionary characteristics of proteins. The program is available from http://www.bioinf.man.ac.uk/~lovell/segno.shtml

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.

Beckwith-Wiedeman syndrome (BWS) and Klippel-Trenaunay-Weber syndrome (KTWS) are different human disorders characterized, among other features, by tissue overgrowth. Deregulation of one or more imprinted genes located at chromosome 11p15.5, of which insulin-like growth factor 2 (IGF2) is the most likely candidate, is believed to cause BWS, whereas the etiology of KTWS is completely obscure. We report a case of BWS and a case of KTWS in a single family. The probands, sons of two sisters, showed relaxation of the maternal IGF2 imprinting, although they inherited different 11p15.5 alleles from their mothers and did not show any chromosome rearrangement. The patient with BWS also displayed hypomethylation at KvDMR1, a maternally methylated CpG island within an intron of the KvLQT1 gene. The unaffected brother of the BWS proband shared the same maternal and paternal 11p15.5 haplotype with his brother, but the KvDMR1 locus was normally methylated. Methylation of the H19 gene was normal in both the BWS and KTWS probands. Linkage between the insulin-like growth factor 2 receptor (IGF2R) gene and the tissue overgrowth was also excluded. These results raise the possibility that a defective modifier or regulatory gene unlinked to 11p15.5 caused a spectrum of epigenetic alterations in the germ line or early development of both cousins, ranging from the relaxation of IGF2 imprinting in the KTWS proband to disruption of both the imprinted expression of IGF2 and the imprinted methylation of KvDMR1 in the BWS proband. Analysis of these data also indicates that loss of IGF2 imprinting is not necessarily linked to alteration of methylation at the KvDMR1 or H19 loci and supports the notion that IGF2 overexpression is involved in the etiology of the tissue hypertrophy observed in different overgrowth disorders, including KTWS.

We report on the molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Southern Italy (Campania region). Thirty-one unrelated G6PD-deficient males were analysed at DNA level for the presence of G6PD gene mutations. Nine different G6PD variants were identified, eight of which have already been described (Mediterranean, Seattle, two different A-, Santamaria, Cassano, Union and Cosenza). G6PD Mediterranean, Santamaria, A- and Union were associated with haemolytic episodes. G6PD Seattle, which is polymorphic in several populations, Cassano and Cosenza appeared to be asymptomatic. A new variant (G6PD Neapolis) is reported here. The 467(Pro-->Arg) substitution responsible for G6PD Neapolis is discussed in the light of the current 3D model of human G6PD and in comparison with other natural mutations which occur in the proximity of residue 467.

Udgivelsesdato: 1991-Dec-11

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.

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

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.

Single-chain pro-urokinase (pro-uPA) is present both in the medium and lysate of the A431 epidermoid carcinoma cell line. Most of the cell-associated pro-uPA is on the cell surface, as shown by indirect immunofluorescence and by surface iodination. Pro-uPA is not an integral membrane protein but is bound to a specific surface receptor that is completely saturated. A mild acid treatment uncovers the surface receptors by dissociating pro-uPA. Resaturation of uncovered receptors has been studied by reincubating cells in normal medium; within 40 min, 50% of the free sites are reoccupied. Excess uPA-specific antibodies prevent rebinding of ligand to the receptors. Thus, A431 cells first secrete uPA, which then binds to the surface receptor. We propose that the synthesis of uPA and uPA receptor by the same cell may provide a pathway for the activation of the metastatic potential of malignant cells.

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.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280-289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C-terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross-linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

Background Fabry disease is a rare disorder caused by a large variety of mutations in the gene encoding lysosomal alpha-galactosidase. Many of these mutations are unique to individual families. Fabry disease can be treated with enzyme replacement therapy, but a promising novel strategy relies on small molecules, so called "pharmacological chaperones", which can be administered orally. Unfortunately only 42% of genotypes respond to pharmacological chaperones. Results A procedure to predict which genotypes responsive to pharmacological chaperones in Fabry disease has been recently proposed. The method uses a position-specific substitution matrix to score the mutations. Using this method, we have screened public databases for predictable responsive cases and selected nine representative mutations as yet untested with pharmacological chaperones. Mutant lysosomal alpha galactosidases were produced by site directed mutagenesis and expressed in mammalian cells. Seven out of nine mutations responded to pharmacological chaperones. Nineteen other mutations that were tested with pharmacological chaperones, but were not included in the training of the predictive method, were gathered from literature and analyzed in silico. In this set all five mutations predicted to be positive were responsive to pharmacological chaperones, bringing the percentage of responsive mutations among those predicted to be positive and not used to train the classifier to 86% (12/14). This figure differs significantly from the percentage of responsive cases observed among all the Fabry mutants tested so far. Conclusions In this paper we provide experimental support to an "in silico" method designed to predict missense mutations in the gene encoding lysosomal alpha galactosidase responsive to pharmacological chaperones. We demonstrated that responsive mutations can be predicted with a low percentage of false positive cases. Most of the mutations tested to validate the method were described in the literature as associated to classic or mild classic phenotype. The analysis can provide a guideline for the therapy with pharmacological chaperones supported by experimental results obtained in vitro. We are aware that our results were obtained in vitro and cannot be translated straightforwardly into benefit for patients, but need to be validated by clinical trials.