Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding proteins

Background

Myelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress.

Results

We performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2.

Conclusions

Overall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.

     

Microtubule defects in mesenchymal stromal cells distinguish patients with Progressive Supranuclear Palsy

Progressive Supranuclear Palsy (PSP) is a rare neurodegenerative disease whose etiopathogenesis remains elusive. The intraneuronal accumulation of hyperphosphorylated Tau, a pivotal protein in regulating microtubules (MT), leads to include PSP into tauopathies. Pathological hallmarks are well known in neural cells but no word yet if PSP‐linked dysfunctions occur also in other cell types. We focused on bone marrow mesenchymal stromal cells (MSCs) that have recently gained attention for therapeutic interventions due to their anti‐inflammatory, antiapoptotic and trophic properties. Here, we aimed to investigate MSCs biology and to disclose if any disease‐linked defect occurs in this non‐neuronal compartment. First, we found that cells obtained from patients showed altered morphology and growth. Next, Western blotting analysis unravelled the imbalance in α‐tubulin post‐translational modifications and in MT stability. Interestingly, MT mass is significantly decreased in patient cells at baseline and differently changes overtime compared to controls, suggesting their inability to efficiently remodel MT cytoskeleton during ageing in culture. Thus, our results provide the first evidence that defects in MT regulation and stability occur and are detectable in a non‐neuronal compartment in patients with PSP. We suggest that MSCs could be a novel model system for unravelling cellular processes implicated in this neurodegenerative disorder.     

Linkage disequilibrium clustering‐based approach for association mapping with tightly linked genomewide data

Genomewide association studies (GWAS) aim to identify genetic markers strongly associated with quantitative traits by utilizing linkage disequilibrium (LD) between candidate genes and markers. However, because of LD between nearby genetic markers, the standard GWAS approaches typically detect a number of correlated SNPs covering long genomic regions, making corrections for multiple testing overly conservative. Additionally, the high dimensionality of modern GWAS data poses considerable challenges for GWAS procedures such as permutation tests, which are computationally intensive. We propose a cluster‐based GWAS approach that first divides the genome into many large nonoverlapping windows and uses linkage disequilibrium network analysis in combination with principal component (PC) analysis as dimensional reduction tools to summarize the SNP data to independent PCs within clusters of loci connected by high LD. We then introduce single‐ and multilocus models that can efficiently conduct the association tests on such high‐dimensional data. The methods can be adapted to different model structures and used to analyse samples collected from the wild or from biparental F₂ populations, which are commonly used in ecological genetics mapping studies. We demonstrate the performance of our approaches with two publicly available data sets from a plant (Arabidopsis thaliana) and a fish (Pungitius pungitius), as well as with simulated data.     

Inference of genetic architecture from chromosome partitioning analyses is sensitive to genome variation,sample size,heritability and effect size distribution

Genomewide association studies have contributed immensely to our understanding of the genetic basis of complex traits. One major conclusion arising from these studies is that most traits are controlled by many loci of small effect, confirming the infinitesimal model of quantitative genetics. A popular approach to test for polygenic architecture involves so‐called “chromosome partitioning” where phenotypic variance explained by each chromosome is regressed on the size of the chromosome. First developed for humans, this has now been repeatedly used in other species, but there has been no evaluation of the suitability of this method in species that can differ in their genome characteristics such as number and size of chromosomes. Nor has the influence of sample size, heritability of the trait, effect size distribution of loci controlling the trait or the physical distribution of the causal loci in the genome been examined. Using simulated data, we show that these characteristics have major influence on the inferences of the genetic architecture of traits we can infer using chromosome partitioning analyses. In particular, small variation in chromosome size, small sample size, low heritability, a skewed effect size distribution and clustering of loci can lead to a loss of power and consequently altered inference from chromosome partitioning analyses. Future studies employing this approach need to consider and derive an appropriate null model for their study system, taking these parameters into consideration. Our simulation results can provide some guidelines on these matters, but further studies examining a broader parameter space are needed.     

Alphavirus Replicase Protein NSP1 Induces Filopodia and Rearrangement of Actin Filaments

Expression of the NSP1 protein of Semliki Forest virus and Sindbis virus in cultured cells induced filopodia-like extensions containing NSP1 but not F actin. The actin stress fibers disappeared, whereas vimentin, keratin, and tubulin networks remained intact. The effects of NSP1 were dependent on its palmitoylation but not on its enzymatic activities and were also observed in virus-infected cells.     

Intracellular Distribution of Rubella Virus Nonstructural Protein P150

Antiserum prepared against an amino-terminal fragment of rubella virus (RUB) nonstructural polyprotein was used to study RUB-infected Vero cells. Replicase protein P150 was associated with vesicles and vacuoles of endolysosomal origin and later with large, convoluted, tubular membrane structures. Newly incorporated bromouridine was associated with the same structures and specifically with small membrane invaginations, spherules, indicating that these structures may be the sites of viral RNA synthesis.