Rational Design of Temperature-Sensitive Alleles Using Computational Structure Prediction

Temperature-sensitive (ts) mutations are mutations that exhibit a mutant phenotype at high or low temperatures and a wild-type phenotype at normal temperature. Temperature-sensitive mutants are valuable tools for geneticists, particularly in the study of essential genes. However, finding ts mutations typically relies on generating and screening many thousands of mutations, which is an expensive and labor-intensive process. Here we describe an in silico method that uses Rosetta and machine learning techniques to predict a highly accurate “top 5” list of ts mutations given the structure of a protein of interest. Rosetta is a protein structure prediction and design code, used here to model and score how proteins accommodate point mutations with side-chain and backbone movements. We show that integrating Rosetta relax-derived features with sequence-based features results in accurate temperature-sensitive mutation predictions

Three-dimensional structure of β-cell-specific zinc transporter, ZnT-8, predicted from the type 2 diabetes-associated gene variant SLC30A8 R325W

<p>Abstract</p> <p>Background</p> <p>We examined the effects of the R325W mutation on the three-dimensional (3D) structure of the β-cell-specific Zn²⁺(zinc) transporter ZnT-8.</p> <p>Methods</p> <p>A model of the C-terminal domain of the human ZnT-8 protein was generated by homology modeling based on the known crystal structure of the <it>Escherichia coli </it>(<it>E. coli</it>) zinc transporter YiiP at 3.8 Å resolution.</p> <p>Results</p> <p>The homodimer ZnT-8 protein structure exists as a Y-shaped architecture with Arg325 located at the ultimate bottom of this motif at approximately 13.5 Å from the transmembrane domain juncture. The C-terminal domain sequences of the human ZnT-8 protein and the <it>E. coli </it>zinc transporter YiiP share 12.3% identical and 39.5% homologous residues resulting in an overall homology of 51.8%. Validation statistics of the homology model showed a reasonable quality of the model. The C-terminal domain exhibited an αββαβ fold with Arg325 as the penultimate N-terminal residue of the α2-helix. The side chains of both Arg325 and Trp325 point away from the interface with the other monomer, whereas the ε-NH₃⁺group of Arg325 is predicted to form an ionic interaction with the β-COO^-group of Asp326 as well as Asp295. An amino acid alignment of the β2-α2 C-terminal loop domain revealed a variety of neutral amino acids at position 325 of different ZnT-8 proteins.</p> <p>Conclusions</p> <p>Our validated homology models predict that both Arg325 and Trp325, amino acids with a helix-forming behavior, and penultimate N-terminal residues in the α2-helix of the C-terminal domain, are shielded by the planar surface of the three cytoplasmic β-strands and hence unable to affect the sensing capacity of the C-terminal domain. Moreover, the amino acid residue at position 325 is too far removed from the docking and transporter parts of ZnT-8 to affect their local protein conformations. These data indicate that the inherited R325W abnormality in SLC30A8 may be tolerated and results in adequate zinc transfer to the correct sites in the pancreatic islet cells and are consistent with the observation that the <it>SLC30A8 </it>gene variant R325W has a low predicted value for future type 2 diabetes at population-based level.</p

Global report on preterm birth and stillbirth (2 of 7): discovery science

<p>Abstract</p> <p>Background</p> <p>Normal and abnormal processes of pregnancy and childbirth are poorly understood. This second article in a global report explains what is known about the etiologies of preterm births and stillbirths and identifies critical gaps in knowledge. Two important concepts emerge: the continuum of pregnancy, beginning at implantation and ending with uterine involution following birth; and the multifactorial etiologies of preterm birth and stillbirth. Improved tools and data will enable discovery scientists to identify causal pathways and cost-effective interventions.</p> <p>Pregnancy and parturition continuum</p> <p>The biological process of pregnancy and childbirth begins with implantation and, after birth, ends with the return of the uterus to its previous state. The majority of pregnancy is characterized by rapid uterine and fetal growth without contractions. Yet most research has addressed only uterine stimulation (labor) that accounts for <0.5% of pregnancy.</p> <p>Etiologies</p> <p>The etiologies of preterm birth and stillbirth differ by gestational age, genetics, and environmental factors. Approximately 30% of all preterm births are indicated for either maternal or fetal complications, such as maternal illness or fetal growth restriction. Commonly recognized pathways leading to preterm birth occur most often during the gestational ages indicated: (1) inflammation caused by infection (22-32 weeks); (2) decidual hemorrhage caused by uteroplacental thrombosis (early or late preterm birth); (3) stress (32-36 weeks); and (4) uterine overdistention, often caused by multiple fetuses (32-36 weeks). Other contributors include cervical insufficiency, smoking, and systemic infections. Many stillbirths have similar causes and mechanisms. About two-thirds of late fetal deaths occur during the antepartum period; the other third occur during childbirth. Intrapartum asphyxia is a leading cause of stillbirths in low- and middle-income countries.</p> <p>Recommendations</p> <p>Utilizing new systems biology tools, opportunities now exist for researchers to investigate various pathways important to normal and abnormal pregnancies. Improved access to quality data and biological specimens are critical to advancing discovery science. Phenotypes, standardized definitions, and uniform criteria for assessing preterm birth and stillbirth outcomes are other immediate research needs.</p> <p>Conclusion</p> <p>Preterm birth and stillbirth have multifactorial etiologies. More resources must be directed toward accelerating our understanding of these complex processes, and identifying upstream and cost-effective solutions that will improve these pregnancy outcomes.</p

Deciphering the Preference and Predicting the Viability of Circular Permutations in Proteins

Circular permutation (CP) refers to situations in which the termini of a protein are relocated to other positions in the structure. CP occurs naturally and has been artificially created to study protein function, stability and folding. Recently CP is increasingly applied to engineer enzyme structure and function, and to create bifunctional fusion proteins unachievable by tandem fusion. CP is a complicated and expensive technique. An intrinsic difficulty in its application lies in the fact that not every position in a protein is amenable for creating a viable permutant. To examine the preferences of CP and develop CP viability prediction methods, we carried out comprehensive analyses of the sequence, structural, and dynamical properties of known CP sites using a variety of statistics and simulation methods, such as the bootstrap aggregating, permutation test and molecular dynamics simulations. CP particularly favors Gly, Pro, Asp and Asn. Positions preferred by CP lie within coils, loops, turns, and at residues that are exposed to solvent, weakly hydrogen-bonded, environmentally unpacked, or flexible. Disfavored positions include Cys, bulky hydrophobic residues, and residues located within helices or near the protein's core. These results fostered the development of an effective viable CP site prediction system, which combined four machine learning methods, e.g., artificial neural networks, the support vector machine, a random forest, and a hierarchical feature integration procedure developed in this work. As assessed by using the hydrofolate reductase dataset as the independent evaluation dataset, this prediction system achieved an AUC of 0.9. Large-scale predictions have been performed for nine thousand representative protein structures; several new potential applications of CP were thus identified. Many unreported preferences of CP are revealed in this study. The developed system is the best CP viability prediction method currently available. This work will facilitate the application of CP in research and biotechnology

Breeding on the leading edge of a northward range expansion: differences in morphology and the stress response in the arctic Gambel's white-crowned sparrow

Individuals at the forefront of a range shift are likely to exhibit phenotypic traits that distinguish them from the population breeding within the historic range. Recent studies have examined morphological, physiological and behavioral phenotypes of individuals at the edge of their range. Several studies have found differences in the hypothalamic-pituitary-adrenal (HPA) axis activity in response to acute restraint stress in individuals at the range limits. HPA axis activation leads to elevations in glucocorticoids that regulate physiology and behavior. Here we compare the hormonal profiles and morphometrics from Gambel's white-crowned sparrows (Zonotrichia leucophrys gambelii) breeding at the northern limit of the population's range to those birds breeding within the historic population range. Birds breeding at the northern limit experienced a harsher environment with colder temperatures; however, we found no differences in arthropod prey biomass between the northern limit and more southern (historic) sites. Males at the northern limit had higher body condition scores (mass corrected for body size) compared to individuals within the historic range, but no differences were found in beak and tarsus lengths, wing chord, muscle profile or fat stores. In males during the pre-parental stage, before breeding commenced, HPA axis activity was elevated in birds at the northern limit of the range, but no differences were found during the parental or molt stages. Females showed no differences in HPA axis activity during the parental stage. This study suggests that "pioneering" individuals at the limits of their breeding range exhibit physiology and morphology that are distinct from individuals within the historic range

A new Groucho TLE4 protein may regulate the repressive activity of Pax5 in human B lymphocytes

During mouse B-cell development, Pax5 is an essential transcription factor that acts as an activator of B-cell-specific genes and as a repressor of alternative lineage fates. The repressive function is mediated by the recruitment of members of the Groucho co-repressor family. Using an RNA display approach, we have isolated a transcript, called QD, specifically expressed in human pro-B and pre-B cells, which is derived from the human Groucho TLE4 gene. The QD transcript contains the first four TLE4 exons and an intronic sequence 3′ of exon 4, demonstrating that QD is a splice variant of TLE4. The putative resulting protein of 94 amino acids corresponds to approximately half of an N-terminal tetramerization domain. We also show specific expression of TLE4 transcripts in human B cells and of TLE4 proteins in B-cell nuclei. Moreover, we demonstrate that recombinant QD protein binds to the TLE4 Q domain and is able to abolish the TLE4/Pax5 interaction. Thus, QD could act as a negative regulator of TLE4 function, in early B-cell differentiation