medRxiv (Cold Spring Harbor Laboratory), Oct 20, 2020
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
International Journal of Antimicrobial Agents, 2021
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in s... more The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in situ immune complex formation by accumulated immunoglobulins and/or antigens bound to long-lived structural proteins that have undergone excessive nonenzymatic glycosylation. In this report, we have tested this hypothesis using nonenzymatically glycosylated collagen. Binding of both albumin and IgG averaged four times the amount bound to unmodified collagen. Both albumin and IgG (anti-BSA) bound to nonenzymatically glycosylated collagen retained their ability to form immune complexes in situ with free antibody and antigen.
Background: Members of swarming bacterial consortia compete for nutrients but also use a cooperat... more Background: Members of swarming bacterial consortia compete for nutrients but also use a cooperation mechanism called quorum sensing (QS) that relies on chemical signals as well as other secreted products (''public goods'') necessary for swarming. Deleting various genes of this machinery leads to cheater mutants impaired in various aspects of swarming cooperation. Methodology/Principal Findings: Pairwise consortia made of Pseudomonas aeruginosa, its QS mutants as well as B. cepacia cells show that a interspecies consortium can ''combine the skills'' of its participants so that the strains can cross together barriers that they could not cross alone. In contrast, deleterious mutants are excluded from consortia either by competition or by local population collapse. According to modeling, both scenarios are the consequence of the QS signalling mechanism itself. Conclusion/Significance: The results indirectly explain why it is an advantage for bacteria to maintain QS systems that can cross-talk among different species, and conversely, why certain QS mutants which can be abundant in isolated niches, cannot spread and hence remain localized.
In order to obtain an accurate description of the protein interior, we describe a simple and fast... more In order to obtain an accurate description of the protein interior, we describe a simple and fast algorithm that measures the depth of each atom in a protein (dpx), defined as its distance (Å) from the closest solvent accessible atom. The program reads a PDB file containing the atomic solvent accessibility in the B-factor field, and writes a file in the same format, where the Bfactor field now contains the dpx value. Output structure files can be thus directly displayed with molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB View and colored according to dpx values.
Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure ... more Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequencedependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in proteincoding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http://www.icgeb.trieste.it/dna).
The WWW servers at http://www.icgeb.trieste.it/dna/ are dedicated to the analysis of user-submitt... more The WWW servers at http://www.icgeb.trieste.it/dna/ are dedicated to the analysis of user-submitted DNA sequences; plot.it creates parametric plots of 45 physicochemical, as well as statistical, parameters; bend.it calculates DNA curvature according to various methods. Both programs provide 1D as well as 2D plots that allow localisation of peculiar segments within the query. The server model.it creates 3D models of canonical or bent DNA starting from sequence data and presents the results in the form of a standard PDB file, directly viewable on the user's PC using any molecule manipulation program. The recently established introns server allows statistical evaluation of introns in various taxonomic groups and the comparison of taxonomic groups in terms of length, base composition, intron type etc. The options include the analysis of splice sites and a probability test for exon-shuffling.
bioRxiv (Cold Spring Harbor Laboratory), Jul 1, 2022
Our goal was to develop a platform, CovidOutcome2, capable of predicting disease severity from vi... more Our goal was to develop a platform, CovidOutcome2, capable of predicting disease severity from viral mutation profiles using automated machine learning (autoML) and deep neural networks applied to the available large corpus of sequenced SARS-CoV2 genomes. CovidOutcome2 accepts either usersubmitted genomes or user defined mutation combinations as the input. The output is a predicted severity score plus a list of identified, annotated mutations and their functional effects in VCF format. The best model performance is a ROC-AUC 0.899 for the model including patient age and ROC-AUC 0.83 for the model without patient age.
We present a method, called equivalence learning, which applies a two-class classification approa... more We present a method, called equivalence learning, which applies a two-class classification approach to object-pairs defined within a multi-class scenario. The underlying idea is that instead of classifying objects into their respective classes, we classify object pairs either as equivalent (belonging to the same class) or non-equivalent (belonging to different classes). The method is based on a vectorisation of the similarity between the objects and the application of a machine learning algorithm (SVM, ANN, LogReg, Random Forests) to learn the differences between equivalent and non-equivalent object pairs, and define a unique kernel function that can be obtained via equivalence learning. Using a small dataset of archaeal, bacterial and eukaryotic 3-phosphoglycerate-kinase sequences we found that the classification performance of equivalence learning slightly exceeds those of several simple machine learning algorithms at the price of a minimal increase in time and space requirements.
Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 2012
Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a l... more Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four α-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil-helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil-helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.
The origin of the genetic code is still not fully understood, despite considerable progress in th... more The origin of the genetic code is still not fully understood, despite considerable progress in the last decade. Far from being a frozen complete accident, the canonical genetic code is full of patterns that seem to open a window on its evolutionary history. In this chapter we rethink the hypothesis that the primary selective force in favour of the emergence of genetic coding was the added value by amino acids to the RNA world in the form of increased catalytic potential. We identify a novel pattern in the genetic code suggesting that the catalytic propensity of amino acids has considerably shaped its structure. This suggestion complements older ideas arguing in favour of a driving force to build the smallest stable oligopeptide structures, such as hairpins (β-turns stabilized by small β-sheets). We outline experiments to test some of the proposals. 1 Introduction As Crick et al. (1976) noted, 'the origin of protein synthesis is a notoriously difficult problem'. This remark refers to protein synthesis in translation using the genetic code. When thinking about difficult evolutionary transitions (cf. Maynard Smith and Szathmáry, 1995) it is rewarding to break down the problem into steps that are more readily soluble by evolution and easier to understand for us. The idea
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a tran... more Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large ex... more Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large extracellular region and a 125-residue cytoplasmic tail bearing a C-terminal PDZ recognition motif (1213 RMEYIV 1218). Binding of Jagged-1 intracellular region to the PDZ domain of afadin, a protein located at cell-cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the PDZ-binding motif. Chemical shift mapping of AF6_PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged-1, J1_ic, is mainly disordered in solution, and chemical shift mapping of J1_ic in the presence of AF6_PDZ showed that binding is not coupled to folding. Binding studies on a series of 24-residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position-2 of the PDZ-binding motif decreases its affinity for AF6_PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ-binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6_PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton.
Protein Engineering Design & Selection, Sep 1, 2001
The three-dimensional structure of the amaranth α-amylase inhibitor (AAI) adopts a knottin fold o... more The three-dimensional structure of the amaranth α-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to α-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution.
Ligands to Notch receptors [1,2] are type I membrane spanning proteins, all sharing a poorly char... more Ligands to Notch receptors [1,2] are type I membrane spanning proteins, all sharing a poorly characterized N-terminal region and a Delta ⁄ Serrate ⁄ Lag-2 domain, which are required for receptor binding, a series of tandem epidermal growth factor-like repeats, a transmembrane segment, and a unique cytoplasmic tail of 100-200 amino acids [3]. Five different ligands to Notch receptors have been identified in mammals, three orthologs (Delta-1,-3 and-4) of Drosophila Delta and two orthologs (Jagged-1 and-2) of Drosophila Serrate. Although the molecular mechanisms of ligand specificity are still unclear, evidence from in vivo studies suggests that each ligand exerts nonredundant effects. Gene knockout of Jagged-1 [4] or Delta-1 [5], heterozygous deletion of Delta-4 [6] or homozygous mutants in Jagged-2 [7] all lead to severe developmental defects and embryonic lethality in mice. There is no significant sequence similarity shared among the Keywords membrane ⁄ cytoplasm interface; regulated intramembrane proteolysis; SDS micelles; phospholipid vesicles; in-cell NMR
The cytoplasmic tail of Notch ligands drives endocytosis, mediates association with proteins impl... more The cytoplasmic tail of Notch ligands drives endocytosis, mediates association with proteins implicated in the organization of cell-cell junctions and, through regulated intra-membrane proteolysis, is released from the membrane as a signaling fragment. We survey these findings and discuss the role of Notch ligands intracellular region in bidirectional signaling and possibly in signal modulation in mammals.
medRxiv (Cold Spring Harbor Laboratory), Oct 20, 2020
doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by pee... more doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
International Journal of Antimicrobial Agents, 2021
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in s... more The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in situ immune complex formation by accumulated immunoglobulins and/or antigens bound to long-lived structural proteins that have undergone excessive nonenzymatic glycosylation. In this report, we have tested this hypothesis using nonenzymatically glycosylated collagen. Binding of both albumin and IgG averaged four times the amount bound to unmodified collagen. Both albumin and IgG (anti-BSA) bound to nonenzymatically glycosylated collagen retained their ability to form immune complexes in situ with free antibody and antigen.
Background: Members of swarming bacterial consortia compete for nutrients but also use a cooperat... more Background: Members of swarming bacterial consortia compete for nutrients but also use a cooperation mechanism called quorum sensing (QS) that relies on chemical signals as well as other secreted products (''public goods'') necessary for swarming. Deleting various genes of this machinery leads to cheater mutants impaired in various aspects of swarming cooperation. Methodology/Principal Findings: Pairwise consortia made of Pseudomonas aeruginosa, its QS mutants as well as B. cepacia cells show that a interspecies consortium can ''combine the skills'' of its participants so that the strains can cross together barriers that they could not cross alone. In contrast, deleterious mutants are excluded from consortia either by competition or by local population collapse. According to modeling, both scenarios are the consequence of the QS signalling mechanism itself. Conclusion/Significance: The results indirectly explain why it is an advantage for bacteria to maintain QS systems that can cross-talk among different species, and conversely, why certain QS mutants which can be abundant in isolated niches, cannot spread and hence remain localized.
In order to obtain an accurate description of the protein interior, we describe a simple and fast... more In order to obtain an accurate description of the protein interior, we describe a simple and fast algorithm that measures the depth of each atom in a protein (dpx), defined as its distance (Å) from the closest solvent accessible atom. The program reads a PDB file containing the atomic solvent accessibility in the B-factor field, and writes a file in the same format, where the Bfactor field now contains the dpx value. Output structure files can be thus directly displayed with molecular graphics programs like RASMOL, MOLMOL, Swiss-PDB View and colored according to dpx values.
Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure ... more Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequencedependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in proteincoding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http://www.icgeb.trieste.it/dna).
The WWW servers at http://www.icgeb.trieste.it/dna/ are dedicated to the analysis of user-submitt... more The WWW servers at http://www.icgeb.trieste.it/dna/ are dedicated to the analysis of user-submitted DNA sequences; plot.it creates parametric plots of 45 physicochemical, as well as statistical, parameters; bend.it calculates DNA curvature according to various methods. Both programs provide 1D as well as 2D plots that allow localisation of peculiar segments within the query. The server model.it creates 3D models of canonical or bent DNA starting from sequence data and presents the results in the form of a standard PDB file, directly viewable on the user's PC using any molecule manipulation program. The recently established introns server allows statistical evaluation of introns in various taxonomic groups and the comparison of taxonomic groups in terms of length, base composition, intron type etc. The options include the analysis of splice sites and a probability test for exon-shuffling.
bioRxiv (Cold Spring Harbor Laboratory), Jul 1, 2022
Our goal was to develop a platform, CovidOutcome2, capable of predicting disease severity from vi... more Our goal was to develop a platform, CovidOutcome2, capable of predicting disease severity from viral mutation profiles using automated machine learning (autoML) and deep neural networks applied to the available large corpus of sequenced SARS-CoV2 genomes. CovidOutcome2 accepts either usersubmitted genomes or user defined mutation combinations as the input. The output is a predicted severity score plus a list of identified, annotated mutations and their functional effects in VCF format. The best model performance is a ROC-AUC 0.899 for the model including patient age and ROC-AUC 0.83 for the model without patient age.
We present a method, called equivalence learning, which applies a two-class classification approa... more We present a method, called equivalence learning, which applies a two-class classification approach to object-pairs defined within a multi-class scenario. The underlying idea is that instead of classifying objects into their respective classes, we classify object pairs either as equivalent (belonging to the same class) or non-equivalent (belonging to different classes). The method is based on a vectorisation of the similarity between the objects and the application of a machine learning algorithm (SVM, ANN, LogReg, Random Forests) to learn the differences between equivalent and non-equivalent object pairs, and define a unique kernel function that can be obtained via equivalence learning. Using a small dataset of archaeal, bacterial and eukaryotic 3-phosphoglycerate-kinase sequences we found that the classification performance of equivalence learning slightly exceeds those of several simple machine learning algorithms at the price of a minimal increase in time and space requirements.
Biochimica Et Biophysica Acta - Biomembranes, Jul 1, 2012
Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a l... more Human Jagged-1, one of the ligands of Notch receptors, is a transmembrane protein composed of a large extracellular region and a 125-residue cytoplasmic tail which bears a C-terminal PDZ recognition motif. To investigate the interaction between Jagged-1 cytoplasmic tail and the inner leaflet of the plasma membrane we determined, by solution NMR, the secondary structure and dynamics of the recombinant protein corresponding to the intracellular region of Jagged-1, J1_tmic, bound to negatively charged lysophospholipid micelles. NMR showed that the PDZ binding motif is preceded by four α-helical segments and that, despite the extensive interaction between J1_tmic and the micelle, the PDZ binding motif remains highly flexible. Binding of J1_tmic to negatively charged, but not to zwitterionic vesicles, was confirmed by surface plasmon resonance. To study the PDZ binding region in more detail, we prepared a peptide corresponding to the last 24 residues of Jagged-1, J1C24, and different phosphorylated variants of it. J1C24 displays a marked helical propensity and undergoes a coil-helix transition in the presence of negatively charged, but not zwitterionic, lysophospholipid micelles. Phosphorylation at different positions drastically decreases the helical propensity of the peptides and abolishes the coil-helix transition triggered by lysophospholipid micelles. We propose that phosphorylation of residues upstream of the PDZ binding motif may shift the equilibrium from an ordered, membrane-bound, interfacial form of Jagged-1 C-terminal region to a more disordered form with an increased accessibility of the PDZ recognition motif, thus playing an indirect role in the interaction between Jagged-1 and the PDZ-containing target protein.
The origin of the genetic code is still not fully understood, despite considerable progress in th... more The origin of the genetic code is still not fully understood, despite considerable progress in the last decade. Far from being a frozen complete accident, the canonical genetic code is full of patterns that seem to open a window on its evolutionary history. In this chapter we rethink the hypothesis that the primary selective force in favour of the emergence of genetic coding was the added value by amino acids to the RNA world in the form of increased catalytic potential. We identify a novel pattern in the genetic code suggesting that the catalytic propensity of amino acids has considerably shaped its structure. This suggestion complements older ideas arguing in favour of a driving force to build the smallest stable oligopeptide structures, such as hairpins (β-turns stabilized by small β-sheets). We outline experiments to test some of the proposals. 1 Introduction As Crick et al. (1976) noted, 'the origin of protein synthesis is a notoriously difficult problem'. This remark refers to protein synthesis in translation using the genetic code. When thinking about difficult evolutionary transitions (cf. Maynard Smith and Szathmáry, 1995) it is rewarding to break down the problem into steps that are more readily soluble by evolution and easier to understand for us. The idea
Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a tran... more Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions.
Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large ex... more Jagged-1, one of the five Notch ligands in man, is a membrane-spanning protein made of a large extracellular region and a 125-residue cytoplasmic tail bearing a C-terminal PDZ recognition motif (1213 RMEYIV 1218). Binding of Jagged-1 intracellular region to the PDZ domain of afadin, a protein located at cell-cell adherens junctions, couples Notch signaling with the adhesion system and the cytoskeleton. Using NMR chemical shift perturbation and surface plasmon resonance, we studied the interaction between the PDZ domain of afadin (AF6_PDZ) and a series of polypeptides comprising the PDZ-binding motif. Chemical shift mapping of AF6_PDZ upon binding of ligands of different length (6, 24, and 133 residues) showed that the interaction is strictly local and involves only the binding groove in the PDZ. The recombinant protein corresponding to the entire intracellular region of Jagged-1, J1_ic, is mainly disordered in solution, and chemical shift mapping of J1_ic in the presence of AF6_PDZ showed that binding is not coupled to folding. Binding studies on a series of 24-residue peptides phosphorylated at different positions showed that phosphorylation of the tyrosine at position-2 of the PDZ-binding motif decreases its affinity for AF6_PDZ, and may play a role in the modulation of this interaction. Finally, we show that the R1213Q mutation located in the PDZ-binding motif and associated with extrahepatic biliary atresia increases the affinity for AF6_PDZ, suggesting that this syndrome may arise from an imbalance in the coupling of Notch signaling to the cytoskeleton.
Protein Engineering Design & Selection, Sep 1, 2001
The three-dimensional structure of the amaranth α-amylase inhibitor (AAI) adopts a knottin fold o... more The three-dimensional structure of the amaranth α-amylase inhibitor (AAI) adopts a knottin fold of abcabc topology. Upon binding to α-amylase, it adopts a more compact conformation characterized by an increased number of intramolecular hydrogen bonds, a decreased volume and in addition a trans to cis isomerization of Pro20. A systematic analysis of the 3-D structural databanks revealed that similar proteins and domains share with AAI the characteristic presence of proline residues, many of which are in a cis backbone conformation. As these proteins fulfil a variety of functional roles and are expressed in very different organisms, we conclude that the structure of the knottin fold, including the propensity of the cis bond, are the result of convergent evolution.
Ligands to Notch receptors [1,2] are type I membrane spanning proteins, all sharing a poorly char... more Ligands to Notch receptors [1,2] are type I membrane spanning proteins, all sharing a poorly characterized N-terminal region and a Delta ⁄ Serrate ⁄ Lag-2 domain, which are required for receptor binding, a series of tandem epidermal growth factor-like repeats, a transmembrane segment, and a unique cytoplasmic tail of 100-200 amino acids [3]. Five different ligands to Notch receptors have been identified in mammals, three orthologs (Delta-1,-3 and-4) of Drosophila Delta and two orthologs (Jagged-1 and-2) of Drosophila Serrate. Although the molecular mechanisms of ligand specificity are still unclear, evidence from in vivo studies suggests that each ligand exerts nonredundant effects. Gene knockout of Jagged-1 [4] or Delta-1 [5], heterozygous deletion of Delta-4 [6] or homozygous mutants in Jagged-2 [7] all lead to severe developmental defects and embryonic lethality in mice. There is no significant sequence similarity shared among the Keywords membrane ⁄ cytoplasm interface; regulated intramembrane proteolysis; SDS micelles; phospholipid vesicles; in-cell NMR
The cytoplasmic tail of Notch ligands drives endocytosis, mediates association with proteins impl... more The cytoplasmic tail of Notch ligands drives endocytosis, mediates association with proteins implicated in the organization of cell-cell junctions and, through regulated intra-membrane proteolysis, is released from the membrane as a signaling fragment. We survey these findings and discuss the role of Notch ligands intracellular region in bidirectional signaling and possibly in signal modulation in mammals.
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Papers by Sándor Pongor