Equilibrium analysis of allosteric interactions shows zero-order effects

The binding of effector to an allosteric protein exhibits a non-Michaelis-Menten behavior, resulting in either ultrasensitive or subsensitive response. In the present work, a modular approach has been developed to determine the response curve for allosteric systems at higher concentration of allosteric enzyme than that of effector (zero-order sensitivity, as observed in enzyme cascades) by equilibrium analysis. The analysis shows that, in an allosteric system, the zero-order effect can make the response ultrasensitive or subsensitive with respect to the enzyme concentration. The response is dependent on the number of binding sites, cooperativity, and the total effector concentration. The framework was further applied to a well studied allosteric protein, the Escherichia coli aspartate transcarbamoylase. The predictions are found to be consistent with the reported experimental data.     

Topology of factor VIII bound to phosphatidylserine-containing model membranes

Factor VIII (FVIII), a plasma glycoprotein, is an essential cofactor in the blood coagulation cascade. It is a multidomain protein, known to bind to phosphatidylserine (PS)-containing membranes. Based on X-ray and electron crystallography data, binding of FVIII to PS-containing membranes has been proposed to occur only via the C2 domain. Based on these models, the molecular topology of membrane-bound FVIII can be envisioned as one in which only a small fraction of the protein interacts with the membrane, whereas the majority of the molecule is exposed to an aqueous milieu. We have investigated the topology of the membrane-bound FVIII using biophysical and biochemical techniques. Circular dichroism (CD) and fluorescence studies indicate no significant changes in the secondary and tertiary structure of FVIII associated with the membranes. Acrylamide quenching studies show that the protein is predominantly present on the surface of the membrane, exposed to the aqueous milieu. The light scattering and electron microscopy studies indicate the absence of vesicle aggregation and fusion. Binding studies with antibodies directed against specific epitopes in the A1, A2 and C2 domains suggest that FVIII binds to the membrane primarily via C2 domain including the specific phospholipid binding epitope (2303-2332) and may involve subtle conformational changes in this epitope region.     

XdomView: protein domain and exon position visualization

SUMMARY: The relationship between intron distribution in the eukaryotic gene and protein structural elements is essential for understanding the origin and evolution of genes. XdomView is a web-based viewer mapping protein structural domains and intron positions in eukaryotic homologues to its tertiary structure. The association of sequence signals to 3D structure in XdomView provides a valuable visualization environment for eukaryotic gene organization, gene evolution, protein folding and protein structure classification. AVAILABILITY: Freely available from http://surya.bic.nus.edu.sg/xdom.     

Collagen XXVII is developmentally regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens

We have generated an antiserum to the variable domain of mouse collagen XXVII, a recently discovered novel member of the fibrillar collagen family. Collagen XXVII protein was first detectable in the mouse at embryonic day 12.5 (E12.5). By E14.5, the protein localized to cartilage, developing dermis, cornea, the inner limiting membrane of the retina, and major arteries of the heart. However, at E18.5, collagen XXVII protein was no longer apparent in most tissues and appeared restricted mainly to cartilage where expression continued into adulthood. Type XXVII collagen immunolocalized to 10-nm-thick nonstriated fibrils that were distinct from fibrils formed by the classical fibrillar collagens. The transient nature of its expression and unusual fibrillar structure suggest that collagen XXVII plays a developmental role distinct from those of the classical fibrillar collagens.     

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway

The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C₄ traits into C₃ plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C₄ plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C₄ species from C₃ species. To identify genetic factors that specify C₄ leaf anatomy, we generated ethyl methanesulfonate‐ and γ‐ray‐mutagenized populations of the C₄ species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F₂ populations as homozygous recessive alleles. Bulk segregant analysis using next‐generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C₄ and C₃ leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.     

A haplotype map of allohexaploid wheat reveals distinct patterns of selection on homoeologous genomes

BackgroundBread wheat is an allopolyploid species with a large, highly repetitive genome. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed population-scale re-sequencing of a diverse panel of wheat lines.ResultsA sample of 62 diverse lines was re-sequenced using the whole exome capture and genotyping-by-sequencing approaches. We describe the allele frequency, functional significance, and chromosomal distribution of 1.57 million single nucleotide polymorphisms and 161,719 small indels. Our results suggest that duplicated homoeologous genes are under purifying selection. We find contrasting patterns of variation and inter-variant associations among wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. These selected regions are enriched for loci associated with agronomic traits detected in genome-wide association studies.ConclusionsEvidence suggests that directional selection in allopolyploids rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. We hypothesize that allopolyploidy may have increased the likelihood of beneficial allele recovery by broadening the set of possible selection targets.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0606-4) contains supplementary material, which is available to authorized users.     

Nuclear condensation and free radical scavenging: a dual mechanism of bisbenzimidazoles to modulate radiation damage to DNA

The complexing of histones with DNA and the resulting condensation of chromatin protects mammalian cell, from radiation-induced strand breakage. In the present study, benzimidazoles DMA and TBZ showed marked radioprotection through drug-induced compaction of chromatin and direct quenching of free radicals generated by radiation. The mammalian cells were incubated with 100 μM concentration of DMA and TBZ and irradiated at 5 Gy; both the ligands showed nuclei condensation suggesting a probable mechanism to protect DNA from radiation damage. The bisubstituted analogs of Hoechst 33342 are found to be better free radical scavengers and protect DNA against radiation-induced damage at a lower concentration than the parent molecule. Both the ligands also quenched free radicals in isolated free radical system suggesting their dual mode of action against radiation-induced damage to DNA. Molecules binding to the chromatin alter gene expression, whereas in this study both the ligands have not shown any profound effect on the nucleosome assembly and gene expression in vitro and in vivo. Both ligands afford a 2-fold protection by altering DNA structure as well as through direct free radical quenching in bulk solution in comparison to the parent ligand, which acts only through quenching of free radicals. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34⁺ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A⁻ Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55^−/− CD59^−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.