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Biomedical subjects

Catherine H Schein

Publications and source records attributed to Catherine H Schein.

At least 19 recordsLinked to original sources
PubMed2006

Recombinant production and structural studies of the Aplysia water-borne protein pheromone enticin indicates it has a novel disulfide stabilized fold.

Enticin is one of three Aplysia proteins released during egg laying that act in concert with the pheromone attractin to attract other Aplysia and stimulate mating behavior. Whereas the enticin cDNA predicts a 69-residue mature protein, enticin isolated from the albumen gland was found to be posttranslationally processed in vivo by cleavage at Arg(50) residue to generate a smaller 49-residue mature peptide. The Arg(50) cleavage site is conserved in enticin from both Aplysia californica and Aplysia brasiliana. In order to generate sufficient enticin for structural studies, recombinant full-length protein was produced in a soluble form in Escherichia coli using a cold shock promoter-based protein expression system. The enticin cDNA was cloned into the bacterial vector pCold III, and efficiently expressed, as determined by amino acid microsequence and immunoblot analyses. Recombinant enticin, which contained an additional N-terminal 13-residue translation-enhancing element, was purified by reversed-phase HPLC and compared to enticin isolated from the albumen gland. The three disulfide bonds in enticin were characterized by endoproteinase Glu-C proteolysis followed by mass spectrometric characterization of the fragments. The cysteine pairing, for both recombinant and native enticin, was I-II, III-IV, and V-VI, confirming that the protein produced in the bacterial system was correctly folded. The circular dichroism spectrum of the recombinant protein indicated it was predominantly alpha-helical. While this was consistent with fold recognition server results indicating a fold for enticin similar to that of attractin, the disulfide bonding pattern differs. A model for enticin was prepared based on its helical structure and these disulfide constraints.

Amino Acid Sequence↗
PubMed2006

Novel, structure-based mechanism for uridylylation of the genome-linked peptide (VPg) of picornaviruses.

The VPg peptide, which is found in poliovirus infected cells either covalently bound to the 5'-end of both plus and minus strand viral RNA, or in a uridylylated free form, is essential for picornavirus replication. Combining experimental structure and mutation results with molecular modeling suggests a new mechanism for VPg uridylylation, which assigns an additional function, that of scaffold, to the polymerase. The polarity of the NMR structure of VPg is complementary to the binding site on the surface of poliovirus polymerase determined previously by mutagenesis. Docking VPg at this position places the reactive tyrosinate close to the 5'-end of Poly(A)7 RNA when this is bound with its 3'-end in the active site of the polymerase. The triphosphate tail of a UTP moiety, base paired with the 5'-end of the RNA, projects back over the Tyr3-OH and is held in position by conserved positively charged side-chains of VPg. Other conserved residues mediate binding to the polymerase surface and serve as ligands for metal ion catalyzed transphosphorylation. Additional viral proteins or a second polymerase molecule may aid in stabilizing the components of the reaction. In the model complex, VPg can direct its own uridylylation before entering the polymerase active site.

Amino Acid Sequence↗
PubMed2006

Determining functionally important amino acid residues of the E1 protein of Venezuelan equine encephalitis virus.

A new method for predicting interacting residues in protein complexes, InterProSurf, was applied to the E1 envelope protein of Venezuelan equine encephalitis (VEEV). Monomeric and trimeric models of VEEV-E1 were constructed with our MPACK program, using the crystal structure of the E1 protein of Semliki forest virus as a template. An alignment of the E1 sequences from representative alphavirus sequences was used to determine physical chemical property motifs (likely functional areas) with our PCPMer program. Information on residue variability, propensity to be in protein interfaces, and surface exposure on the model was combined to predict surface clusters likely to interact with other viral or cellular proteins. Mutagenesis of these clusters indicated that the predictions accurately detected areas crucial for virus infection. In addition to the fusion peptide area in domain 2, at least two other surface areas play an important role in virus infection. We propose that these may be sites of interaction between the E1-E1 and E1-E2 subdomains of the envelope proteins that are required to assemble the functional unit. The InterProSurf method is, thus, an important new tool for predicting viral protein interactions. These results can aid in the design of new vaccines against alphaviruses and other viruses.

Amino Acids↗
PubMed2006

NMR structure of the viral peptide linked to the genome (VPg) of poliovirus.

VPgs are essential for replication of picornaviruses, which cause diseases such as poliomyelitis, foot and mouth disease, and the common cold. VPg in infected cells is covalently linked to the 5' end of the viral RNA, or, in a uridylylated form, free in the cytoplasm. We show here the first solution structure for a picornaviral VPg, that of the 22-residue peptide from poliovirus serotype 1. VPg in buffer is inherently flexible, but a single conformer was obtained by adding trimethylamine N-oxide (TMAO). TMAO had only minor effects on the TOCSY spectrum. However, it increased the amount of structured peptide, as indicated by more peaks in the NOESY spectrum and an up to 300% increase in the ratio of normalized NOE cross peak intensities to that in buffer. The data for VPg in TMAO yielded a well defined structure bundle with 0.6 A RMSD (versus 6.6 A in buffer alone), with 10-30 unambiguous constraints per residue. The structure consists of a large loop region from residues 1 to 14, from which the reactive tyrosinate projects outward, and a C-terminal helix from residues 18 to 21 that aligns the sidechains of conserved residues on one face. The structure has a stable docking position at an area on the poliovirus polymerase crystal structure identified as a VPg binding site by mutagenesis studies. Further, UTP and ATP dock in a base-specific manner to the reactive face of VPg, held in place by residues conserved in all picornavirus VPgs.

Adenosine Triphosphate↗
PubMed2005

Newly identified water-borne protein pheromones interact with attractin to stimulate mate attraction in Aplysia.

The water-borne protein attractin is a potent sex pheromone involved in forming and maintaining mating and egg-laying aggregations in the marine mollusk Aplysia. Binary blends of attractin and either enticin, temptin, or seductin, three other Aplysia protein pheromones, stimulate mate attraction. The four pheromones are thought to act in concert during egg-laying. The new data presented here show that: (1) the water-borne odor of non-laying Aplysia brasiliana further increases the attractiveness of attractin and of eggs in T-maze bioassays. This suggests that individual Aplysia release additional factors that enhance the effects of attractin, enticin, temptin, and seductin during egg-laying; (2) the N-terminal region of enticin aligns well with the conserved epidermal growth factor (EGF)-like domain of mammalian reproductive proteins known as fertilins, which may mediate intercellular adhesion interactions between eggs and sperm; (3) temptin, according to fold recognition servers, may also have an EGF-like fold. Enticin and temptin also have conserved metal binding sequences that may play a role in their signaling behavior. These results suggest that aspects of mammalian egg-sperm interactions (fertilins) may have evolved from pheromonal signaling mechanisms. We also review the structure, expression, localization, release, and behavioral actions of attractin, enticin, temptin, and seductin.

Amino Acid Sequence↗
PubMed2005

Common physical-chemical properties correlate with similar structure of the IgE epitopes of peanut allergens.

Although many sequences and linear IgE epitopes of allergenic proteins have been identified and archived in databases, structural and physicochemical discriminators that define their specific properties are lacking. Current bioinformatics tools for predicting the potential allergenicity of a novel protein use methods that were not designed to compare peptides. Novel tools to determine the quantitative sequence and three-dimensional (3D) relationships between IgE epitopes of major allergens from peanut and other foods have been implemented in the Structural Database of Allergenic Proteins (SDAP; http://fermi.utmb.edu/SDAP/). These peptide comparison tools are based on five-dimensional physicochemical property (PCP) vectors. Sequences from SDAP proteins similar in their physicochemical properties to known epitopes of Ara h 1 and Ara h 2 were identified by calculating property distance (PD) values. A 3D model of Ara h 1 was generated to visualize the 3D structure and surface exposure of the epitope regions and peptides with a low PD value to them. Many sequences similar to the known epitopes were identified in related nut allergens, and others were within the sequences of Ara h 1 and Ara h 2. Some of the sequences with low PD values correspond to other known epitopes. Regions with low PD values to one another in Ara h 1 had similar predicted structure, on opposite sides of the internal dimer axis. The PD scale detected epitope pairs that are similar in structure and/or reactivity with patient IgE. The high immunogenicity and IgE reactivity of peanut allergen proteins might be due to the proteins' arrays of similar antigenic regions on opposite sides of a single protein structure.

2S Albumins, Plant↗
PubMed2005

Structural basis for epitope sharing between group 1 allergens of cedar pollen.

The group 1 allergens are a major cause of cedar pollen hypersensitivity in several geographic areas. Allergens from several taxa have been shown to cross-react. The goal of these studies was to compare the structural features of the shared and unique epitopes of the group 1 allergen from mountain cedar (Jun a 1) and Japanese cedar (Cry j 1). An array of overlapping peptides from the sequence of Jun a 1 and a panel of monoclonal anti-Cry j 1 antibodies were used to identify the IgE epitopes recognized by cedar-sensitive patients from Texas and Japan. IgE from Japanese patients reacted with peptides representing one of the two linear epitopes within the highly conserved beta-helical core structure and both epitopes within less ordered loops and turns near the N- and C-termini of Jun a 1. A three-dimensional (3D) model of the Cry j 1, based on the crystal structure of Jun a 1, indicated a similar surface exposure for the four described epitopes of Jun a 1 and the homologous regions of Cry j 1. The monoclonal antibodies identified another shared epitope, which is most likely conformational and a unique Cry j 1 epitope that may be the previously recognized glycopeptide IgE epitope. Defining the structural basis for shared and unique epitopes will help to identify critical features of IgE epitopes that can be used to develop mimotopes or identify allergen homologues for vaccine development.

Adolescent↗
PubMed2005

Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses.

BACKGROUND: Flaviviruses, which include Dengue (DV) and West Nile (WN), mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies. RESULTS: Stereophysicochemical variability plots (SVPs), 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs) near escape mutant positions. The analysis showed 1) that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2) two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion. CONCLUSION: The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines.

Amino Acid Motifs↗
PubMed2005

Molego-based definition of the architecture and specificity of metal-binding sites.

Decomposing proteins into "molegos," building blocks that are conserved in sequence and 3D-structure, can identify functional elements. To demonstrate the specificity of the decomposition method, the PCPMer program suite was used to numerically define physical chemical property motifs corresponding to the molegos that make up the metal-containing active sites of three distinct enzyme families, from the dimetallic phosphatases, DNase 1 related nucleases/phosphatases, and dioxygenases. All three superfamilies bind metal ions in a beta-strand core region but differ in the number and type of ions needed for activity. The motifs were then used to automatically identify proteins in the ASTRAL40 database that contained similar motifs. The proteins with the highest PCPMer score in the database were primarily metal-binding enzymes that were related in function to those in the alignment used to generate the PCPMer motif lists. The proteins that contained motifs similar to the dioxygenases differed from those found with PCP-motifs for phosphatases and nucleases. Relatively few metal-binding enzymes were detected when the search was done with PCP-motifs defined for interleukin-1 related proteins, which have a beta-strand core but do not bind metal ions. While the box architecture was constant in each superfamily, the specificity for the metal ion preferred for enzymatic activity is determined by the pattern of carbonyl, hydroxyl or imadazole groups in key positions in the molegos. These results have implications for the design of metal-binding enzymes, and illustrate the ability of the PCPMer approach to distinguish, at the sequence level, structural and functional elements.

Binding Sites↗
PubMed2005

Molluscan attractins, a family of water-borne protein pheromones with interspecific attractiveness.

The marine mollusk Aplysia releases the water-borne pheromone attractin during egg laying. This small protein stimulates the formation and maintenance of mating and egg-laying aggregations. Attractin has been characterized from five Aplysia species: A. californica, A. brasiliana, A. fasciata, A. vaccaria, and A. depilans. We describe here the isolation of attractin from Bursatella leachii, and show that it belongs to the same protein family. The pattern of residue conservation, especially the six invariant cysteines, suggests that all of these attractins have a common fold. The nuclear magnetic resonance solution structure of A. californica attractin contains two antiparallel alpha-helices, the second of which contains the heptapeptide sequence IEECKTS that has been implicated in attractin function. Synthetic peptides containing this IEECKTS region are attractive, and mutating surface exposed charged residues within this region of attractin abolishes attractin activity. This suggests that the second helix is an essential part of the receptor-binding interface. In contrast to the peptide pheromonal attractants in amphibians, which are species specific, the attractins are, to our knowledge, the first water-borne peptide or protein pheromone family in invertebrates and vertebrates that are not species specific.

Amino Acid Sequence↗
PubMed2004

Structural and functional analysis of Aplysia attractins, a family of water-borne protein pheromones with interspecific attractiveness.

Mate attraction in Aplysia involves a long-distance water-borne signal (the protein pheromone attractin), which is released during egg laying. Aplysia californica attractin attracts species that produce closely related attractins, such as Aplysia brasiliana, whose geographic distribution does not overlap that of A. californica. This finding suggests that other mollusks release attractin-related pheromones to form and maintain breeding aggregations. We describe four additional members of the attractin family: A. brasiliana, Aplysia fasciata, Aplysia depilans (which aggregates with A. fasciata aggregations), and Aplysia vaccaria (which aggregates with A. californica aggregations). On the basis of their sequence similarity with A. californica attractin, the attractin proteins fall into two groups: A. californica, A. brasiliana, and A. fasciata (91-95% identity), and A. depilans and A. vaccaria (41-43% identity). The sequence similarity within the attractin family, the conserved six cysteines, and the compact fold of the NMR solution structure of A. californica attractin suggest a common fold for this pheromone family containing two antiparallel helices. The second helix contains the IEECKTS sequence conserved in Aplysia attractins. Mutating surface-exposed charged residues within this heptapeptide sequence abolishes attractin activity, suggesting that the second helix is an essential part of the receptor-binding interface.

Amino Acid Sequence↗
PubMed2004

Effects of backbone contacts 3' to the abasic site on the cleavage and the product binding by human apurinic/apyrimidinic endonuclease (APE1).

The mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) is a multifunctional protein that plays essential roles in DNA repair and gene regulation. We decomposed the APEs into 12 blocks of highly conserved sequence and structure (molegos). This analysis suggested that residues in molegos common to all APEs, but not to the less specific nuclease, DNase I, would dictate enhanced binding to damaged DNA. To test this hypothesis, alanine was substituted for N226 and N229, which form hydrogen bonds to the DNA backbone 3' of the AP sites in crystal structures of the APE1/DNA complex. While the cleavage rate at AP sites of both N226A and N229A mutants increased, their ability to bind to damaged DNA decreased. The ability of a double mutant (N226A/N229A) to bind damaged DNA was further decreased, while the V(max) was almost identical to that of the wild-type APE1. A double mutant at N226 and R177, a residue that binds to the same phosphate as N229, had a significantly decreased activity and substrate binding. As the affinity for product DNA was decreased in all the mutants, the enhanced reaction rate of the single mutants could be due to alleviation of product inhibition of the enzyme. We conclude that hydrogen bonds to phosphate groups 3' to the cleavage site is essential for APE1's binding to the product DNA, which may be necessary for efficient functioning of the base excision repair pathway. The results indicate that the molego analysis can aid in the redesign of proteins with altered binding affinity and activity.

Alanine↗
PubMed2004

Using property based sequence motifs and 3D modeling to determine structure and functional regions of proteins.

Homology modeling has become an essential tool for studying proteins that are targets for medical drug design. This paper describes the approach we developed that combines sequence decomposition techniques with distance geometry algorithms for homology modeling to determine functionally important regions of proteins. We show here the application of these techniques to targets of medical interest chosen from those included in the CASP5 (Critical Assessment of Techniques for Protein Structure Prediction) competition, including the dihydroneopterin aldolase from Mycobacterium tuberculosis, RNase III of Thermobacteria maritima, and the NO-transporter nitrophorin from saliva of the bedbug Cimex lectularius. Physical chemical property (PCP) motifs, identified in aligned sequences with our MASIA program, can be used to select among different alignments returned by fold recognition servers. They can also be used to suggest functions for hypothetical proteins, as we illustrate for target T188. Once a suitable alignment has been made with the template, our modeling suite MPACK generates a series of possible models. The models can then be selected according to their match in areas known to be conserved in protein families. Alignments based on motifs can improve the structural matching of residues in the active site. The quality of the local structure of our 3D models near active sites or epitopes makes them useful aids for drug and vaccine design. Further, the PCP motif approach, when combined with a structural filter, can be a potent way to detect areas involved in activity and to suggest function for novel genome sequences.

Aldehyde-Lyases↗
PubMed2003

NMR solution structure of attractin, a water-borne protein pheromone from the mollusk Aplysia californica.

Water-borne protein pheromones are essential for coordination of reproductive activities in many marine organisms. In this paper, we describe the first structure of a pheromone protein from a marine organism, that of attractin (58 residues) from Aplysia californica. The NMR solution structure was determined from TOCSY, NOESY, and DQF-COSY measurements of recombinant attractin expressed in insect cells. The sequential resonance assignments were done with standard manual procedures. Approximately 90% of the 949 unambiguous NOESY cross-peaks were assigned automatically with simultaneous three-dimensional structure calculation using our NOAH/DIAMOD/FANTOM program suite. The final bundle of energy-refined structures is well-defined, with an average rmsd value to the mean structure of 0.72 +/- 0.12 A for backbone and 1.32 +/- 0.11 A for heavy atoms for amino acids 3-47. Attractin contains two antiparallel helices, made up of residues Ile9-Gln16 and I30-S36. The NMR distance constraints are consistent with the three disulfide bonds determined by mass spectroscopy (C4-C41, C13-C33, and C20-C26), where the first two could be directly determined from NOESY cross-peaks between CH beta protons of the corresponding cysteines. The second helix contains the (L/I)(29)IEECKTS(36) sequence conserved in attractins from five species of Aplysia that could interact with the receptor. The sequence and structure of this region are similar to those of the recognition helix of the Er-11 pheromone of the unicellular ciliate Euplotes raikovi, suggesting a possible common pathway for intercellular communication of these two distinct pheromone families.

Amino Acid Sequence↗
PubMed2003

Detecting potential IgE-reactive sites on food proteins using a sequence and structure database, SDAP-food.

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.

Allergens↗
PubMed2003

Identifying property based sequence motifs in protein families and superfamilies: application to DNase-1 related endonucleases.

MOTIVATION: Identification of short conserved sequence motifs common to a protein family or superfamily can be more useful than overall sequence similarity in suggesting the function of novel gene products. Locating motifs still requires expert knowledge, as automated methods using stringent criteria may not differentiate subtle similarities from statistical noise. RESULTS: We have developed a novel automatic method, based on patterns of conservation of 237 physical-chemical properties of amino acids in aligned protein sequences, to find related motifs in proteins with little or no overall sequence similarity. As an application, our web-server MASIA identified 12 property-based motifs in the apurinic/apyrimidinic endonuclease (APE) family of DNA-repair enzymes of the DNase-I superfamily. Searching with these motifs located distantly related representatives of the DNase-I superfamily, such as Inositol 5'-polyphosphate phosphatases in the ASTRAL40 database, using a Bayesian scoring function. Other proteins containing APE motifs had no overall sequence or structural similarity. However, all were phosphatases and/or had a metal ion binding active site. Thus our automated method can identify discrete elements in distantly related proteins that define local structure and aspects of function. We anticipate that our method will complement existing ones to functionally annotate novel protein sequences from genomic projects. AVAILABILITY: MASIA WEB site: http://www.scsb.utmb.edu/masia/masia.html SUPPLEMENTARY INFORMATION: The dendrogram of 42 APE sequences used to derive motifs is available on http://www.scsb.utmb.edu/comp_biol.html/DNA_repair/publication.html

Algorithms↗