The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80–85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.     

Frustration‐guided motion planning reveals conformational transitions in proteins

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics‐inspired motion planning procedure called dCC‐RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non‐native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC‐RRT to examine how collective, small‐scale motions of four side‐chains in the active site of cyclophilin A propagate through the protein. dCC‐RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non‐canonical capsid binding site 25 Å away, rationalizing NMR and multi‐temperature crystallography experiments. In all, dCC‐RRT can reveal detailed, all‐atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/ .     

Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-<Superscript>13</Superscript>C]Glucose and [1,2-<Superscript>13</Superscript>C]Acetate as Substrates

Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15–90 min with unlabeled glucose in combination with [U-¹³C]glucose or [1,2-¹³C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for ¹³C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography–mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured ¹³C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of ¹³C-labeling observed with [U-¹³C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2-¹³C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using ¹³C-labeling (%) data obtained from mass spectrometry. Based on this approach we suggest that cellular metabolic compartmentation in hippocampus and cerebral cortex is very similar.     

Species richness and evenness respond to diverging land‐use patterns – a cross‐border study of dry tropical woodlands in southern Africa

Cross‐border studies offer unique situations to study the impact of different land‐use regimes on ecosystems. Along the Angolan and Namibian border formed by the Okavango River, the environmental conditions and traditional land‐use practises are the same on either side of the river. However, decades of civil war in Angola led to a stagnant development while political stability in Namibia fostered a recent socio‐economic transformation. We investigated the impact of spatially diffuse land use on plant diversity of the dry tropical woodlands covering the vast, sandy hinterlands of the river. As accessibility is the major factor governing land use, we used distance to road as a proxy for land‐use intensity. Based on 58 vegetation plots sized 20 m × 50 m, we showed that species richness increased with distance to road in Angola while in Namibia it remained constant on a lower level. Evenness showed an inverse pattern to species richness and Shannon diversity index showed no response. Analysing diversity patterns according to life forms revealed that these patterns are primarily driven by woody species. The study showed that spatially diffuse land use has a measurable effect on plant diversity and illustrates that roads act as vectors of change.     

Multi-scale phylogenetic structure in coastal dune plant communities across the globe

Aims Studies integrating phylogenetic history and large-scale community assembly are few, and many questions remain unanswered. Here, we use a global coastal dune plant data set to uncover the important factors in community assembly across scales from the local filtering processes to the global long-term diversification and dispersal dynamics. Coastal dune plant communities occur worldwide under a wide range of climatic and geologic conditions as well as in all biogeographic regions. However, global patterns in the phylogenetic composition of coastal dune plant communities have not previously been studied.Methods The data set comprised vegetation data from 18463 plots in New Zealand, South Africa, South America, North America and Europe. The phylogenetic tree comprised 2241 plant species from 149 families. We calculated phylogenetic clustering (Net Relatedness Index, NRI, and Nearest Taxon Index, NTI) of regional dune floras to estimate the amount of in situ diversification relative to the global dune species pool and evaluated the relative importance of land and climate barriers for these diversification patterns by geographic analyses of phylogenetic similarity. We then tested whether dune plant communities exhibit similar patterns of phylogenetic structure within regions. Finally, we calculated NRI for local communities relative to the regional species pool and tested for an association with functional traits (plant height and seed mass) thought to vary along sea–inland gradients.Important findings Regional species pools were phylogenetically clustered relative to the global pool, indicating regional diversification. NTI showed stronger clustering than NRI pointing to the importance of especially recent diversifications within regions. The species pools grouped phylogenetically into two clusters on either side of the tropics suggesting greater dispersal rates within hemispheres than between hemispheres. Local NRI plot values confirmed that most communities were also phylogenetically clustered within regions. NRI values decreased with increasing plant height and seed mass, indicating greater phylogenetic clustering in communities with short maximum height and good dispersers prone to wind and tidal disturbance as well as salt spray, consistent with environmental filtering along sea–inland gradients. Height and seed mass both showed significant phylogenetic signal, and NRI tended to correlate negatively with both at the plot level. Low NRI plots tended to represent coastal scrub and forest, whereas high NRI plots tended to represent herb-dominated vegetation. We conclude that regional diversification processes play a role in dune plant community assembly, with convergence in local phylogenetic community structure and local variation in community structure probably reflecting consistent coastal-inland gradients. Our study contributes to a better understanding of the globally distributed dynamic coastal ecosystems and the structuring factors working on dune plant communities across spatial scales and regions.     

Mutation-selection models of codon substitution and their use to estimate selective strengths on codon usage

Current models of codon substitution are formulated at the levels of nucleotide substitution and do not explicitly consider the separate effects of mutation and selection. They are thus incapable of inferring whether mutation or selection is responsible for evolution at silent sites. Here we implement a few population genetics models of codon substitution that explicitly consider mutation bias and natural selection at the DNA level. Selection on codon usage is modeled by introducing codon-fitness parameters, which together with mutation-bias parameters, predict optimal codon frequencies for the gene. The selective pressure may be for translational efficiency and accuracy or for fine-tuning translational kinetics to produce correct protein folding. We apply the models to compare mitochondrial and nuclear genes from several mammalian species. Model assumptions concerning codon usage are found to affect the estimation of sequence distances (such as the synonymous rate d(S), the nonsynonymous rate d(N), and the rate at the 4-fold degenerate sites d(4)), as found in previous studies, but the new models produced very similar estimates to some old ones. We also develop a likelihood ratio test to examine the null hypothesis that codon usage is due to mutation bias alone, not influenced by natural selection. Application of the test to the mammalian data led to rejection of the null hypothesis in most genes, suggesting that natural selection may be a driving force in the evolution of synonymous codon usage in mammals. Estimates of selection coefficients nevertheless suggest that selection on codon usage is weak and most mutations are nearly neutral. The sensitivity of the analysis on the assumed mutation model is discussed.     

The 20S Proteasome as an Assembly Platform for the 19S Regulatory Complex

26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base.     

A trypsin-like protease with apparent dual function in early Lepeophtheirus salmonis (Kr?yer) development

Background  

Trypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in Lepeophtheirus salmonis. During these studies a trypsin-like peptidase which differed in a number of traits were identified.