What makes a pluripotency reprogramming factor?

Resetting differentiated cells to a pluripotent state is now a widely applied technology and a key step towards personalized cell replacement therapies. Conventionally, combinations of transcription factor proteins are introduced into a differentiated cell to convert gene expression programs and to change cell fates. Yet, the molecular mechanism of nuclear reprogramming is only superficially understood. Specifically, it is unclear what sets pluripotency reprogramming factors (PRFs) molecularly apart from other transcription factor molecules that induce, for example, lineage commitment in embryonic development. Ultimately, PRFs must scan the genome of a differentiated cell, target enhancers of pluripotency factors and initiate gene expression. This requires biochemical properties to selectively recognize DNA sequences, either alone or by cooperating with other PRFs. In this review, we will discuss the molecular make-up of the prominent PRFs Sox2, Oct4, Klf4, Esrrb, Nr5a2 and Nanog and attempt to identify unique features distinguishing them from highly homologous yet functionally contrasting family members. Except for Klf4, the consensus DNA binding motifs are highly conserved for PRFs when compared to non-pluripotency inducing family members, suggesting that the individual DNA sequence preference may not be the distinguishing factor. By contrast, variant composite DNA motifs were found in pluripotency enhancers that lead to a differential assembly of various Sox and Oct family members due selective protein-protein interaction platform. As a consequence, the cooperation of PRFs on distinctly configured DNA motifs may underlie the reprogramming process. Indeed, it has been demonstrated that Sox17 can be rationally engineered into a PRF by modulating its cooperation with Oct4. An in deep understanding of this phenomenon would allow rational engineering and optimization of PRFs. This way, the reprogramming efficiency can be enhanced and fine-tuned to generate optimal synthetic reagents for regenerative medicine.

Lack of human IgE cross-reactivity between mite allergens Blo t 1 and Der p 1.

BACKGROUND: The group 1 mite allergens are the most significant indoor allergens and they belong to the papain-like cysteine protease family. To date there is only one published report on the isolation and characterization of group 1 allergens from Blomia tropicalis mites. The aims of the study are to determine the cross-reactivity between group 1 allergens and to evaluate their clinical importance in allergic patients. METHODS: The full-length Blo t 1 gene was obtained by SMART RACE cDNA amplification method using gene-specific primers. The sequence alignment was performed using LOOK followed by three-dimensional homology modeling. The cDNA was expressed in Pichia pastoris as a secretory protein. Identification of native Blo t 1 in crude mite and spent mite medium extracts was done by Western immunoblot using monoclonal antibody. Allergenicity of recombinant Blo t 1 and native Der p 1 was examined by human IgE ELISA with 80 asthmatic sera. RESULTS: The cDNA sequence consists of 1105 base pairs, including 5'- and 3'-untranslating regions, encoding an open reading frame of 330 amino acid residues. The predicted molecular weight of the deduced protein was approximately 38 kDa. Blo t 1 shared 53 and 34% nucleotide and amino acid, respectively, sequence homology with Der p 1. Native Blo t 1 was detected in both crude mite and spent mite medium extracts, and its estimated molecular weight was about 26 kDa. The recombinant Blo t 1 reacted positively with IgE in 90 and 65% of sera from asthmatic children and adults, respectively, indicating that it is a major allergen. The correlation of human IgE reactivity between Blo t 1 and Der p 1 was low in these sera. CONCLUSION: The full-length cDNA encoding group 1 Blomia tropicalis mite allergen (designated as Blo t 1) has been characterized and expressed from local mites in Singapore. This fecal allergen showed high frequency of human IgE reactivity with asthmatic sera in the tropics and there was a low correlation of IgE reactivity between Blo t 1 and Der p 1.

Nonrandom intragenic variations in patterns of codon bias implicate a sequential interplay between transitional genetic drift and functional amino acid selection.

Although most codon third bases appear to be functionless, the synonymous codons so defined exhibit a strikingly nonrandom distribution (codon bias) within human and other genes. To examine this phenomenon further, we generated a database of DNA sequences encoding human transmembrane cell-surface receptor proteins. Using this database we show here that the guanine and cytosine content of codon third bases (GC3) varies intragenically with the nature of the specified receptor domains (transmembrane > extracellular > intracellular domains; p < 0.001), the phenotype of the encoded amino acids (hydrophobic > hydrophilic > neutral amino acids; p < 0.001), and the receptor affiliation of the transmembrane (G-protein-coupled receptors > receptor tyrosine kinases; p < 0.001). Within gene regions specifying transmembrane domains, GC3 declines as domain functionality becomes redundant with increasing hydrophobicity (p < 0.001). Codons containing the second-base cytosine (XCZ, which encodes neutral amino acids) are selectively depleted of third-base adenine content (A3: XCA codons) when encoding transmembrane domain residues, consistent with positive selection for transitional mutation of XCG to XTG (which encodes hydrophobic amino acids) rather than to the synonymous XCA. Supporting this XCG --> XTG mechanism of codon bias, the G3:A3 ratio of codons specifying the transmembrane amino acid glycine (GGZ) is intermediate between that of its functional homolog alanine (GCZ) and that of hydrophobic valine (GTZ), even though the C3:T3 ratios are similar. Conversely, nearest-neighbor analysis of third bases 5' to codons specifying valine and leucine (CTZ) confirms a significant difference in C3:T3 but not G3:A3 ratios (i.e., C3/G1 --> T3/G1 > C3/A1; p < 0.001), consistent with the functionally advantageous retention of hydrophobic residues. These data raise the possibility that patterns of intragenic codon bias reflect a balance between negative and positive selection, suggesting in turn that analysis of codon third-base usage may help to predict the functional significance of encoded products.

Androgen receptor mutations causing human androgen insensitivity syndromes show a key role of residue M807 in Helix 8-Helix 10 interactions and in receptor ligand-binding domain stability.

Transactivation activity of the androgen receptor (AR) is induced by the binding of an androgen to its ligand-binding domain (LBD). The tertiary architecture of the AR LBD, in common with other steroid/nuclear receptors, is a sandwich of 12 alpha-helices (H). We have encountered a missense substitution, M807T, which was associated with partially defective androgen binding in a 46,XY infant with ambiguous genitalia. In contrast, two other substitutions in the same residue 807 to valine and arginine, resulted in almost total abrogation of androgen-binding and complete androgen insensitivity syndrome in two unrelated individuals. We recreated these substitutions in residue 807 and observed that disruption of ligand-binding and transactivation activities was total for M807R and partial for M807V, while the least-affected was M807T. Modelling of the AR LBD indicate that van der Waal interactions between residue 807 (H8) to H9 and H10 were severely disrupted for the arginine mutant, but relatively preserved for the threonine and valine mutants. However, there was a subtle difference between these two variants in that M807T, but not M807V, improved van der Waal contacts with another residue L859 in H10, suggesting the importance of interactions between M807 and L859 for LBD stability. Atomic distances of M807 (H8) to L859 (H10) in corresponding residues of the distantly related ER alpha, RXR alpha, PPAR gamma and VDR LBD are highly conserved and almost invariant, suggesting that H8/H10 interactions are critical for LBD stability in other members of the steroid/nuclear receptor superfamily.

Towards the MHC-peptide combinatorics.

The exponentially increased sequence information on major histocompatibility complex (MHC) alleles points to the existence of a high degree of polymorphism within them. To understand the functional consequences of MHC alleles, 36 nonredundant MHC-peptide complexes in the protein data bank (PDB) were examined. Induced fit molecular recognition patterns such as those in MHC-peptide complexes are governed by numerous rules. The 36 complexes were clustered into 19 subgroups based on allele specificity and peptide length. The subgroups were further analyzed for identifying common features in MHC-peptide binding pattern. The four major observations made during the investigation were: (1) the positional preference of peptide residues defined by percentage burial upon complex formation is shown for all the 19 subgroups and the burial profiles within entries in a given subgroup are found to be similar; (2) in class I specific 8- and 9-mer peptides, the fourth residue is consistently solvent exposed, however this observation is not consistent in class I specific 10-mer peptides; (3) an anchor-shift in positional preference is observed towards the C terminal as the peptide length increases in class II specific peptides; and (4) peptide backbone atoms are proportionately dominant at the MHC-peptide interface.

The atomic resolution structure of bucandin, a novel toxin isolated from the Malayan krait, determined by direct methods.

Bucandin is a novel presynaptic neurotoxin isolated from Bungarus candidus (Malayan krait). It has the unique property of enhancing presynaptic acetylcholine release and represents a family of three-finger toxins with an additional disulfide in the first loop. There are no existing structures from this sub-category of three-finger toxins. The X-ray crystal structure of bucandin has been determined by the Shake-and-Bake direct-methods procedure. The resulting electron-density maps were of outstanding quality and allowed the automated tracing of 61 of the 63 amino-acid residues, including their side chains, and the placement of 48 solvent molecules. The 0.97 A resolution full-matrix least-squares refinement converged to a crystallographic R factor of 12.4% and the final model contains 118 solvent molecules. This is the highest resolution structure of any member of the three-finger toxin family and thus it can serve as the best model for other members of the family. Furthermore, the structure of this novel toxin will help in understanding its unique ability to enhance acetylcholine release. The unique structure resulting from the fifth disulfide bond residing in the first loop improves the understanding of other toxins with a similar arrangement of disulfide bonds.

Knowledge-based grouping of modeled HLA peptide complexes.

Human leukocyte antigens are the most polymorphic of human genes and multiple sequence alignment shows that such polymorphisms are clustered in the functional peptide binding domains. Because of such polymorphism among the peptide binding residues, the prediction of peptides that bind to specific HLA molecules is very difficult. In recent years two different types of computer based prediction methods have been developed and both the methods have their own advantages and disadvantages. The nonavailability of allele specific binding data restricts the use of knowledge-based prediction methods for a wide range of HLA alleles. Alternatively, the modeling scheme appears to be a promising predictive tool for the selection of peptides that bind to specific HLA molecules. The scoring of the modeled HLA-peptide complexes is a major concern. The use of knowledge based rules (van der Waals clashes and solvent exposed hydrophobic residues) to distinguish binders from nonbinders is applied in the present study. The rules based on (1) number of observed atomic clashes between the modeled peptide and the HLA structure, and (2) number of solvent exposed hydrophobic residues on the modeled peptide effectively discriminate experimentally known binders from poor/nonbinders. Solved crystal complexes show no vdW Clash (vdWC) in 95% cases and no solvent exposed hydrophobic peptide residues (SEHPR) were seen in 86% cases. In our attempt to compare experimental binding data with the predicted scores by this scoring scheme, 77% of the peptides are correctly grouped as good binders with a sensitivity of 71%.

IE-Kb: intron exon knowledge base.

SUMMARY: IE-Kb (Intron Exon-Knowledge base) illustrates the intron-exon dynamics in eukaryotic genes. We have developed three different knowledge sets, namely 'Non-redundant ExInt', 'Non-redundant Pfam-ExInt complement' and 'Non-redundant GenBank eukaryotic subdivisional sets' to understand this phenomenon. Statistical analysis is performed on each knowledge set and the results are made available online. The entries in knowledge sets are ranked based on their intron length, exon length and protein length with relational hyper-links to the corresponding intron phase, intron position, intron sequence, gene definition and parent GenBank entry.

Crystallization and preliminary crystallographic study of stonustoxin, a protein lethal factor isolated from the stonefish (Synanceja horrida) venom.

Crystals of stonustoxin have been obtained and diffract to 3.4 A resolution. Stonustoxin is a protein lethal factor isolated from the venom of the stonefish, Synanceja horrida. The crystals belong to the tetragonal space group P422, with unit cell constants a = b = 109.0 A, c = 245.7 A. A native stonustoxin molecule has two subunits, designated alpha and beta, respectively, and there is one stonustoxin molecule per asymmetric unit.

Structural studies of two rhinovirus serotypes complexed with fragments of their cellular receptor.

Two human rhinovirus serotypes complexed with two- and five-domain soluble fragments of the cellular receptor, intercellular adhesion molecule-1, have been investigated by X-ray crystallographic analyses of the individual components and by cryo-electron microscopy of the complexes. The three-dimensional image reconstructions provide a molecular envelope within which the crystal structures of the viruses and the receptor fragments can be positioned with accuracy. The N-terminal domain of the receptor binds to the rhinovirus 'canyon' surrounding the icosahedral 5-fold axes. Fitting of molecular models into the image reconstruction density identified the residues on the virus that interact with those on the receptor surface, demonstrating complementarity of the electrostatic patterns for the tip of the N-terminal receptor domain and the floor of the canyon. The complexes seen in the image reconstructions probably represent the first stage of a multistep binding process. A mechanism is proposed for the subsequent viral uncoating process.

The structure of the two amino-terminal domains of human intercellular adhesion molecule-1 suggests how it functions as a rhinovirus receptor.

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also utilized as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2 A resolution and fitted into a cryo-electron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE and FG loops of the amino-terminal immunoglobulin-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1 which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the I-(insertion) domain on the alpha chain of LFA-1 and the carboxy group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Interconversion of the kinetic identities of the tandem catalytic domains of receptor-like protein-tyrosine phosphatase PTPalpha by two point mutations is synergistic and substrate-dependent.

The two tandem homologous catalytic domains of PTPalpha possess different kinetic properties, with the membrane proximal domain (D1) exhibiting much higher activity than the membrane distal (D2) domain. Sequence alignment of PTPalpha-D1 and -D2 with the D1 domains of other receptor-like PTPs, and modeling of the PTPalpha-D1 and -D2 structures, identified two non-conserved amino acids in PTPalpha-D2 that may account for its low activity. Mutation of each residue (Val-536 or Glu-671) to conform to its invariant counterpart in PTPalpha-D1 positively affected the catalytic efficiency of PTPalpha-D2 toward the in vitro substrates para-nitrophenylphosphate and the phosphotyrosyl-peptide RR-src. Together, they synergistically transformed PTPalpha-D2 into a phosphatase with catalytic efficiency for para-nitrophenylphosphate equal to PTPalpha-D1 but not approaching that of PTPalpha-D1 for the more complex substrate RR-src. In vivo, no gain in D2 activity toward p59(fyn) was effected by the double mutation. Alteration of the two corresponding invariant residues in PTPalpha-D1 to those in D2 conferred D2-like kinetics toward all substrates. Thus, these two amino acids are critical for interaction with phosphotyrosine but not sufficient to supply PTPalpha-D2 with a D1-like substrate specificity for elements of the phosphotyrosine microenvironment present in RR-src and p59(fyn). Whether the structural features of D2 can uniquely accommodate a specific phosphoprotein substrate or whether D2 has an alternate function in PTPalpha remains an open question.

Development of software tools at BioInformatics Centre (BIC) at the National University of Singapore (NUS).

There is burgeoning volume of information and data arising from the rapid research and unprecedented progress in molecular biology. This has been particularly affected by the Human Genome Project which is trying to completely sequence three billion nucleotides of the human genome (1),(1a). Other genome sequencing projects are also contributing substantially to this exponential growth in the number of DNA nucleotides and proteins sequenced. The number of journals, reports and research papers and tools required for the analysis of these sequences has also increased. For this the life sciences today needs tools in information technology and computation to prevent degeneration of this data into an inchoate accretion of unconnected facts and figures. The recently formed BioInformatics Centre (BIC) at the National University of Singapore (NUS) provides access to various commonly used computational tools available over the World Wide Web (WWW)--using a uniform interface and easy access. We have also come up with a new database tool. BioKleisli, which allows you to interact with various geographically scattered, heterogeneous, structurally complex and constantly evolving data sources. This paper summarises the importance of network access and database integration to biomedical research and gives a glimpse of current research conducted at BIC.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand.

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-A resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus-ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the alpha chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

Integration of bioInformatics tools at the National University of Singapore (NUS).

In the past decade "Big Science" such as the Genome Project has generated an enormous amount of data in the life sciences. Concurrently, the synergy of this project with existing research has quickened the pace of biological discovery. But the major drawback that is beginning to be felt worldwide is the primitive level of organisation in the data accumulated. Without a proper framework or knowledge scaffold to hang and interconnect the various bits of data and information, the national knowledge-to-data ratio is declining rapidly. We are trying to serve a solution to this enigma by providing a World Wide Web (WWW) interface to Biosoftware and at the same time have come up with a database integration tool that can query heterogeneous, geographically scattered and disparate databases simultaneously. In this report we will talk about BioInformatics in general with specific reference to BioInformatics Centre (BIC) at the National University of Singapore.

Structural analysis of Urechis caupo hemoglobin.

The structure of Urechis caupo hemoglobin in the cyanomet state has been refined to R = 0.148 at 2.5 A resolution. Although the tertiary structure is similar to that of other vertebrate and invertebrate hemoglobins the quaternary structures of this tetramer is unique as suggested by the earlier determination of the 5.0 A resolution structure. The G and H helices of the hemoglobin are on the outside of the tetramer facing the solvent in contrast to human hemoglobin where the G and H helices form inter-subunit contacts. A substantial number of tightly bound water molecules help mediate interactions between subunits. The unusual arrangement of subunits is consistent with the general lack of co-operativity of oxygen uptake for Urechis caupo hemoglobin.

Crystallographic and cryo EM analysis of virion-receptor interactions.

Cryoelectron microscopy has been used to determine the first structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 (HRV16) complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule-1 (ICAM-1) shows that ICAM-1 binds into the 12 A deep "canyon" on the surface of the virus. This is consistent with the prediction that the viral receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of HRV14 and CD4, homologous to HRV16 and ICAM-1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.

The hemoglobins of the bullfrog, Rana catesbeiana. The cDNA-derived amino acid sequences of the alpha chains of adult hemoglobins B and C: their roles in deoxygenation-induced aggregation.

The adult bullfrog (Rana catesbeiana) has two major tetrameric hemoglobins, B and C, which share a common beta chain but have different alpha chains. Components B and C associate upon deoxygenation to form a complex of the form BC2, a trimer of tetramers that depends on contacts between the alpha B and alpha C chains. Nucleotide sequences of cDNA transcripts for these chains have been determined. Transcripts were identified by analysis of the amino acid compositions of the tryptic peptides of the components and by partial amino acid sequencing. These results, together with the amino acid sequence of the beta chain (Tam, L.-T., Gray, G. P., and Riggs, A. F. (1986) J. Biol. Chem. 261, 8290-8294), permit an analysis of the structures of the alpha 2 beta 2 tetramers of hemoglobins B and C. Molecular modeling suggests possible residues at the alpha B-alpha C interfaces in the BC2 trimer and additional alpha C-alpha C contacts that would form a closed ring of six alpha chain subunits that would further stabilize the BC2 trimer. Phylogenetic analysis of the alpha B sequence suggests that it may be a "tadpole" chain, the temporal expression of which has shifted from larva to adult.

Structure of a human rhinovirus complexed with its receptor molecule.

Cryoelectron microscopy has been used to determine the structure of a virus when complexed with its glycoprotein cellular receptor. Human rhinovirus 16 complexed with the two amino-terminal, immunoglobulin-like domains of the intercellular adhesion molecule 1 shows that the intercellular adhesion molecule 1 binds into the 12-A deep "canyon" on the viral surface. This result confirms the prediction that the viral-receptor attachment site lies in a cavity inaccessible to the host's antibodies. The atomic structures of human rhinovirus 14 and CD4, homologous to human rhinovirus 16 and intercellular adhesion molecule 1, showed excellent correspondence with observed density, thus establishing the virus-receptor interactions.