The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation

Amphipathic helices target perilipins 1-3 to lipid droplets

Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs inSaccharomyces cerevisiae,demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targetingin vivoandin vitro Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment.This work was supported by grants from The Wellcome Trust (091551 and 107064 to DBS), the U.K. NIHR Cambridge Biomedical Research Centre, the Medical Research Council (G0701446 to SS and a Doctoral training grant awarded to the University of Cambridge for ERR), core facilities at the MRC Metabolic Diseases Unit (MC_UU_12012/5) and by the Innovative Medicines Initiative Joint Undertaking, EMIF-Metabolism award.This is the final version of the article. It first appeared from ASBMB via https://doi.org/10.1074/jbc.M115.69104

Inter-domain Communication Mechanisms in an ABC Importer: A Molecular Dynamics Study of the MalFGK2E Complex

ATP-Binding Cassette transporters are ubiquitous membrane proteins that convert the energy from ATP-binding and hydrolysis into conformational changes of the transmembrane region to allow the translocation of substrates against their concentration gradient. Despite the large amount of structural and biochemical data available for this family, it is still not clear how the energy obtained from ATP hydrolysis in the ATPase domains is “transmitted” to the transmembrane domains. In this work, we focus our attention on the consequences of hydrolysis and inorganic phosphate exit in the maltose uptake system (MalFGK2E) from Escherichia coli. The prime goal is to identify and map the structural changes occurring during an ATP-hydrolytic cycle. For that, we use extensive molecular dynamics simulations to study three potential intermediate states (with 10 replicates each): an ATP-bound, an ADP plus inorganic phosphate-bound and an ADP-bound state. Our results show that the residues presenting major rearrangements are located in the A-loop, in the helical sub-domain, and in the “EAA motif” (especially in the “coupling helices” region). Additionally, in one of the simulations with ADP we were able to observe the opening of the NBD dimer accompanied by the dissociation of ADP from the ABC signature motif, but not from its corresponding P-loop motif. This work, together with several other MD studies, suggests a common communication mechanism both for importers and exporters, in which ATP-hydrolysis induces conformational changes in the helical sub-domain region, in turn transferred to the transmembrane domains via the “coupling helices”

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one perilipin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific perilipins present on its LDs. Despite their early discovery, exactly how perilipins target LDs and why they displace each other in a ‘hierarchical’ manner remains unclear. They all share an amino-terminal 11-mer repeat amphipathic region suggested to be involved in LD targeting. Here, we show that in vivo this domain functions as a primary highly reversible LD targeting motif in perilipins1-3 and, in vitro, we document reversible and competitive binding between a wildtype purified perilipin1 11-mer repeat peptide and a mutant with reduced binding affinity to both ‘naked’ and phospholipid coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in perilipin1 more effectively than in perilipin2, whereas in perilipin3 it weakens binding. These findings suggest that dual amphipathic helical regions mediat

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a "hierarchical" manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1-3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both "naked" and phospholipid-coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1-3 to LDs

Comparative performances of machine learning methods for classifying Crohn Disease patients using genome-wide genotyping data

Abstract: Crohn Disease (CD) is a complex genetic disorder for which more than 140 genes have been identified using genome wide association studies (GWAS). However, the genetic architecture of the trait remains largely unknown. The recent development of machine learning (ML) approaches incited us to apply them to classify healthy and diseased people according to their genomic information. The Immunochip dataset containing 18,227 CD patients and 34,050 healthy controls enrolled and genotyped by the international Inflammatory Bowel Disease genetic consortium (IIBDGC) has been re-analyzed using a set of ML methods: penalized logistic regression (LR), gradient boosted trees (GBT) and artificial neural networks (NN). The main score used to compare the methods was the Area Under the ROC Curve (AUC) statistics. The impact of quality control (QC), imputing and coding methods on LR results showed that QC methods and imputation of missing genotypes may artificially increase the scores. At the opposite, neither the patient/control ratio nor marker preselection or coding strategies significantly affected the results. LR methods, including Lasso, Ridge and ElasticNet provided similar results with a maximum AUC of 0.80. GBT methods like XGBoost, LightGBM and CatBoost, together with dense NN with one or more hidden layers, provided similar AUC values, suggesting limited epistatic effects in the genetic architecture of the trait. ML methods detected near all the genetic variants previously identified by GWAS among the best predictors plus additional predictors with lower effects. The robustness and complementarity of the different methods are also studied. Compared to LR, non-linear models such as GBT or NN may provide robust complementary approaches to identify and classify genetic markers