Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Expression and membrane integration of SARS-CoV M protein

SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46–68 and 78–100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14–36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Predicting the success of an invader: Niche shift versus niche conservatism

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.     

A functional variant in SLC1A3 influences ADHD risk by disrupting a hsa‐miR‐3171 binding site: A two‐stage association study

Attention‐deficit hyperactivity disorder (ADHD) is one of the most common neuropsychiatric disorders in children and adolescents with high heritability. Evidence is accumulating that SLC1A3 may play a role in ADHD etiology. Therefore, a two‐stage case‐control study was conducted on 752 cases and 774 controls to explore the role of SLC1A3 in ADHD. Bioinformatic annotations and functional experiments were applied to reveal the potential biological mechanisms. Finally, SLC1A3 rs1049522 showed significant association with ADHD risk in two stages with CA genotype vs AA genotype, odds ratio (OR) = 0.694 (95% confidence interval, CI = 0.570‐0.844) and dominant model, OR = 0.749 (95% CI = 0.621‐0.904) in the combined stage. Besides, rs1049522 was found to be related to ADHD hyperactive/impulsive symptom, and rs1049522‐C showed increased SLC1A3 mRNA expression in the cerebellar cortex. Dual‐luciferase reporter assay further indicated that rs1049522‐C allele enhanced SLC1A3 expression by disrupting the hsa‐miR‐3171 binding site. In conclusion, SLC1A3 variant rs1049522 was implicated in ADHD susceptibility in a Chinese Han population probably by enhancing the SLC1A3 expression in a miRNA‐mediated manner.     

Putting the genes into community genetics

As part of the long‐term fusion of evolutionary biology and ecology (Ford, 1964), the field of community genetics has made tremendous progress in describing the impacts of plant genetic variation on community and ecosystem processes. In the “genes‐to‐ecosystems” framework (Whitham et al., 2003), genetically based traits of plant species have ecological consequences, but previous studies have not identified specific plant genes responsible for community phenotypes. The study by Barker et al. (2019) in this issue of Molecular Ecology uses an impressive common garden experiment of trembling aspen (Figure 1) to test for the genetic basis of tree traits that shape the insect community composition. Using a Genome‐Wide Association Study (GWAS), they found that genomic regions associated with phytochemical traits best explain variation in herbivore community composition, and identified specific genes associated with different types of leaf‐modifying herbivores and ants. This is one of the first studies to identify candidate genes underlying the heritable plant traits that explain patterns of insect biodiversity.     

Epistatic partners of neurogenic genes modulate Drosophila olfactory behavior

The extent to which epistasis affects the genetic architecture of complex traits is difficult to quantify, and identifying variants in natural populations with epistatic interactions is challenging. Previous studies in Drosophila implicated extensive epistasis between variants in genes that affect neural connectivity and contribute to natural variation in olfactory response to benzaldehyde. In this study, we implemented a powerful screen to quantify the extent of epistasis as well as identify candidate interacting variants using 203 inbred wild‐derived lines with sequenced genomes of the Drosophila melanogaster Genetic Reference Panel (DGRP). We crossed the DGRP lines to P[GT1]‐element insertion mutants in Sema‐5c and neuralized (neur), two neurodevelopmental loci which affect olfactory behavior, and to their coisogenic wild‐type control. We observed significant variation in olfactory responses to benzaldehyde among F₁ genotypes and for the DGRP line by mutant genotype interactions for both loci, showing extensive nonadditive genetic variation. We performed genome‐wide association analyses to identify the candidate modifier loci. None of these polymorphisms were in or near the focal genes; therefore, epistasis is the cause of the nonadditive genetic variance. Candidate genes could be placed in interaction networks. Several candidate modifiers are associated with neural development. Analyses of mutants of candidate epistatic partners with neur (merry‐go‐round (mgr), prospero (pros), CG10098, Alhambra (Alh) and CG12535) and Sema‐5c (CG42540 and bruchpilot (brp)) showed aberrant olfactory responses compared with coisogenic controls. Thus, integrating genome‐wide analyses of natural variants with mutations at defined genomic locations in a common coisogenic background can unmask specific epistatic modifiers of behavioral phenotypes.