Modulation of the Chaperone DnaK Allosterism by the Nucleotide Exchange Factor GrpE

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70_NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70_SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaK_NBD, with ATP inducing the open, substrate-receptive DnaK_SBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaK_NBD. DnaK_SBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaK_NBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaK_SBD near DnaK_NBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaK_SBD, whereas the DnaK_NBD-DnaK_SBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.     

Structural and functional studies of LRP6 ectodomain reveal a platform for Wnt signaling

LDL-receptor-related protein 6 (LRP6), alongside Frizzled receptors, transduces Wnt signaling across the plasma membrane. The LRP6 ectodomain comprises four tandem β-propeller-EGF-like domain (PE) pairs that harbor binding sites for Wnt morphogens and their antagonists including Dickkopf 1 (Dkk1). To understand how these multiple interactions are integrated, we combined crystallographic analysis of the third and fourth PE pairs with electron microscopy (EM) to determine the complete ectodomain structure. An extensive inter-pair interface, conserved for the first-to-second and third-to-fourth PE interactions, contributes to a compact platform-like architecture, which is disrupted by mutations implicated in developmental diseases. EM reconstruction of the LRP6 platform bound to chaperone Mesd exemplifies a binding mode spanning PE pairs. Cellular and binding assays identify overlapping Wnt3a- and Dkk1-binding surfaces on the third PE pair, consistent with steric competition, but also suggest a model in which the platform structure supports an interplay of ligands through multiple interaction sites.     

Evidence for a remodelling of DNA-PK upon autophosphorylation from electron microscopy studies

The multi-subunit DNA-dependent protein kinase (DNA-PK), a crucial player in DNA repair by non-homologous end-joining in higher eukaryotes, consists of a catalytic subunit (DNA-PKcs) and the Ku heterodimer. Ku recruits DNA-PKcs to double-strand breaks, where DNA-PK assembles prior to DNA repair. The interaction of DNA-PK with DNA is regulated via autophosphorylation. Recent SAXS data addressed the conformational changes occurring in the purified catalytic subunit upon autophosphorylation. Here, we present the first structural analysis of the effects of autophosphorylation on the trimeric DNA-PK enzyme, performed by electron microscopy and single particle analysis. We observe a considerable degree of heterogeneity in the autophosphorylated material, which we resolved into subpopulations of intact complex, and separate DNA-PKcs and Ku, by using multivariate statistical analysis and multi-reference alignment on a partitioned particle image data set. The proportion of dimeric oligomers was reduced compared to non-phosphorylated complex, and those dimers remaining showed a substantial variation in mutual monomer orientation. Together, our data indicate a substantial remodelling of DNA-PK holo-enzyme upon autophosphorylation, which is crucial to the release of protein factors from a repaired DNA double-strand break.     

Association study of MIA3 rs17465637 polymorphism with cardiovascular disease in rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a complex polygenic inflammatory disease associated with accelerated atherosclerosis. Melanoma inhibitor protein 3 (MIA3) is required for the export of collagen VlI (COL7A1) from the endoplasmic reticulum and it appears to be a tumor suppressor of malignant melanoma. Genome-wide association studies have described an association between MIA3 rs17465637 A/C polymorphisms and coronary artery disease and myocardial infarction. Because of that, we assessed the MIA3 rs17465637 polymorphism in 1505 RA Spanish patients stratified according to the presence/absence of cardiovascular (CV) disease. Also, a subgroup of patients without CV events was assessed for the presence of subclinical atherosclerosis using carotid ultrasound to establish carotid intima-media wall thickness and carotid plaques and brachial ultrasonography to determine the presence of endothelial dysfunction by flow-mediated endothelium-dependent and independent vasodilatation. MIA3 rs17465637 allele A showed a trend for association with the presence of carotid plaques (odds ratio 1.56, 95% confidence interval [0.96-2.51]; p=0.07). However, apart from an association of the MIA3 rs17465637 A allele with the risk of CV events in RA patients with dyslipidemia (p=0.018), no other significant associations were found between the presence of MIA3 rs17465637 A allele and the risk of suffering CV events or other surrogate markers of atherosclerosis. In conclusion, our results suggest a potential association of the MIA3 rs17465637 with CV disease in dyslipidemic patients with RA. However, additional studies are required to better establish the role of the MIA3 gene in mechanisms leading to the accelerated atherogenesis observed in RA.     

Blood-soluble Fas levels are increased in type 2 diabetic patients with peripheral vascular disease.

AIM: To investigate the possible utility of plasma sFas (soluble Fas) levels as a marker of peripheral vascular disease (PVD) in type 2 diabetic patients, and the relationship between classical cardiovascular risk factors and sFas levels in these patients. MATERIALS AND METHODS: sFas levels were measured in 57 type 2 diabetic patients with and 60 without PVD matched for age and sex. Diagnosis of PVD was established in presence of at least one of the following criteria: leg or foot amputation of vascular cause, lower-extremity arterial angioplasty or surgical by-pass, or ankle-braquial index (ABI) less than 1 in at least one side of the body. ELISA was used to measure sFas levels. RESULTS: None of the risk factors assessed total cholesterol, HDL cholesterol, triglycerides, CRP, ACE, fibrinogen, Lp(a) and homocysteine was significantly different between both groups of patients. However, patients with PVD had higher plasma sFas levels than the group without PVD (10.25+/-3.7 ng/ml VS. 8.86+/-2.6 ng/ml; p=0.02). Levels of sFas were 1.45 ng/ml (95% CI: 0.32-2.58; p=0.013) higher in PVD patients when adjusting by age, total, HDL and LDL cholesterol, triglycerides, homocysteine, CRP, ACE, arterial hypertension and tobacco smoking. Using multiple logistic regression sFas is a predictor of PVD, although not potent. CONCLUSION: Plasma sFas may be an independent marker of PVD in type 2 diabetes mellitus patients.     

Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex

Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that regulates gene expression and mRNA quality. A complex network of macromolecular interactions regulates NMD initiation, which is only partially understood. According to prevailing models, NMD begins by the assembly of the SURF (SMG1–UPF1–eRF1–eRF3) complex at the ribosome, followed by UPF1 activation by additional factors such as UPF2 and UPF3. Elucidating the interactions between NMD factors is essential to comprehend NMD, and here we demonstrate biochemically and structurally the interaction between human UPF2 and eukaryotic release factor 3 (eRF3). In addition, we find that UPF2 associates with SURF and ribosomes in cells, in an UPF3-independent manner. Binding assays using a collection of UPF2 truncated variants reveal that eRF3 binds to the C-terminal part of UPF2. This region of UPF2 is partially coincident with the UPF3-binding site as revealed by electron microscopy of the UPF2–eRF3 complex. Accordingly, we find that the interaction of UPF2 with UPF3b interferes with the assembly of the UPF2–eRF3 complex, and that UPF2 binds UPF3b more strongly than eRF3. Together, our results highlight the role of UPF2 as a platform for the transient interactions of several NMD factors, including several components of SURF.     

Bushmeat Hunting Alters Recruitment of Large-seeded Plant Species in Central Africa

We investigated the effect of local hunting on plant recruitment at the community level in the Ngotto Forest, Central African Republic. It is the first study of this kind in the afrotropics. To compare our results with previous studies conducted in the neotropics, we used the protocol proposed byNuñez-Iturri and Howe. We compared animal relative abundances and seed length, density and diversity of seedlings at two sites with contrasting anthropogenic pressures: one with low hunting pressure (LH) and one with high hunting pressure (HH). Furthermore, we investigated how density and diversity of recruitment vary with seed length in the two sites. Both sites exhibit similar soil, climate and tree species composition, and have never been logged. Large mammals (frugivores and herbivores) were extirpated from HH and relative abundance of medium-sized frugivores drastically decreased in HH compared with LH. In HH, diversity of seedlings was reduced compared with LH, especially for large-seeded plant species dispersed by large game mammals. The approach used in this study shows promising perspectives to investigate the effects of human disturbances on the regeneration of tropical forests. Furthermore, in the afrotropical context of a lack of data on plant–animal ecological relationships, seed length appears to be a good predictor of the strength of the deficit of plant recruitment due to large mammal extirpation because of hunting. Thus, more efforts are needed to study the ecological roles of large-seeded plants in order to better understand the effects of their disappearance on tropical forest resilience.     

Human posterior parietal cortex flexibly determines reference frames for reaching based on sensory context

Current models of sensorimotor transformations emphasize the dominant role of gaze-centered representations for reach planning in the posterior parietal cortex (PPC). Here we exploit fMRI repetition suppression to test whether the sensory modality of a target determines the reference frame used to define the motor goal in the PPC and premotor cortex. We show that when targets are defined visually, the anterior precuneus selectively encodes the motor goal in gaze-centered coordinates, whereas the parieto-occipital junction, Brodman Area 5 (BA 5), and PMd use a mixed gaze- and body-centered representation. In contrast, when targets are defined by unseen proprioceptive cues, activity in these areas switches to represent the motor goal predominantly in body-centered coordinates. These results support computational models arguing for flexibility in reference frames for action according to sensory context. Critically, they provide neuroanatomical evidence that flexibility is achieved by exploiting a multiplicity of reference frames that can be expressed within individual areas.     

Membrane fluidity determines sensitivity of filamentous fungi to chitosan

The antifungal mode of action of chitosan has been studied for the last 30 years, but is still little understood. We have found that the plasma membrane forms a barrier to chitosan in chitosan‐resistant but not chitosan‐sensitive fungi. The plasma membranes of chitosan‐sensitive fungi were shown to have more polyunsaturated fatty acids than chitosan‐resistant fungi, suggesting that their permeabilization by chitosan may be dependent on membrane fluidity. A fatty acid desaturase mutant of Neurospora crassa with reduced plasma membrane fluidity exhibited increased resistance to chitosan. Steady‐state fluorescence anisotropy measurements on artificial membranes showed that chitosan binds to negatively charged phospholipids that alter plasma membrane fluidity and induces membrane permeabilization, which was greatest in membranes containing more polyunsaturated lipids. Phylogenetic analysis of fungi with known sensitivity to chitosan suggests that chitosan resistance may have evolved in nematophagous and entomopathogenic fungi, which naturally encounter chitosan during infection of arthropods and nematodes. Our findings provide a method to predict the sensitivity of a fungus to chitosan based on its plasma membrane composition, and suggests a new strategy for antifungal therapy, which involves treatments that increase plasma membrane fluidity to make fungi more sensitive to fungicides such as chitosan.