Coexistence of protease sensitive and resistant prion protein in 129VV homozygous sporadic Creutzfeldt-Jakob disease: a case report

Introduction: The coexistence of different molecular types of classical protease-resistant prion protein in the same individual have been described, however, the simultaneous finding of these with the recently described protease-sensitive variant or variably protease-sensitive prionopathy has, to the best of our knowledge, not yet been reported. Case presentation: A 74-year-old Caucasian woman showed a sporadic Creutzfeldt-Jakob disease clinical phenotype with reactive depression, followed by cognitive impairment, akinetic-rigid Parkinsonism with pseudobulbar syndrome and gait impairment with motor apraxia, visuospatial disorientation, and evident frontal dysfunction features such as grasping, palmomental reflex and brisk perioral reflexes. She died at age 77. Neuropathological findings showed: spongiform change in the patient"s cerebral cortex, striatum, thalamus and molecular layer of the cerebellum with proteinase K-sensitive synaptic-like, dot-like or target-like prion protein deposition in the cortex, thalamus and striatum; proteinase K-resistant prion protein in the same regions; and elongated plaque-like proteinase K-resistant prion protein in the molecular layer of the cerebellum. Molecular analysis of prion protein after proteinase K digestion revealed decreased signal intensity in immunoblot, a ladder-like protein pattern, and a 71% reduction of PrPSc signal relative to non-digested material. Her cerebellum showed a 2A prion protein type largely resistant to proteinase K. Genotype of polymorphism at codon 129 was valine homozygous. Conclusion: Molecular typing of prion protein along with clinical and neuropathological data revealed, to the best of our knowledge, the first case of the coexistence of different protease-sensitive prion proteins in the same patient in a rare case that did not fulfill the current clinical diagnostic criteria for either probable or possible sporadic Creutzfeldt-Jakob disease. This highlights the importance of molecular analyses of several brain regions in order to correctly diagnose rare and atypical prionopathies

Conjugation reactions involving maleimides and phosphorothioate oligonucleotides

Phosphorothioate diester oligonucleotides proved to be fully compatible with maleimides in the context of two different conjugation reactions: (a) reaction of 5′diene-[phosphorothioate oligonucleotides] with maleimido-containing compounds to afford the Diels-Alder cycloadduct; (b) conjugation of 5′maleimido-[phosphorothioate oligonucleotides] with thiol-containing compounds. No evidence of reaction between phosphorothioate diesters and maleimides was found in any of these processes. Importantly, in the preparation of 5′maleimido-[phosphorothioate oligonucleotides] from [protected maleimido]-[phosphorothioate oligonucleotides], which requires the maleimide to be deprotected by retro-Diels-Alder reaction (heating for 3-4 h in toluene at 90 °C), no addition of phosphorothioate diester to the maleimide was found either. Finally, maleimide-[phosphorothioate monoester] conjugation was also explored for comparison purposes.

Protein interaction studies point to new functions for Escherichia coli glyceraldehyde-3-phosphate dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is considered a multifunctional protein with defined functions in numerous mammalian cellular processes. GAPDH functional diversity depends on various factors such as covalent modifications, subcellular localization, oligomeric state and intracellular concentration of substrates or ligands, as well as protein-protein interactions. In bacteria, alternative GAPDH functions have been associated with its extracellular location in pathogens or probiotics. In this study, new intracellular functions of E. coli GAPDH were investigated following a proteomic approach aimed at identifying interacting partners using in vivo formaldehyde cross-linking followed by mass spectrometry. The identified proteins were involved in metabolic processes, protein synthesis and folding or DNA repair. Some interacting proteins were also identified in immunopurification experiments in the absence of cross-linking. Pull-down experiments and overlay immunoblotting were performed to further characterize the interaction with phosphoglycolate phosphatase (Gph). This enzyme is involved in the metabolism of 2-phosphoglycolate formed in the DNA repair of 3"-phosphoglycolate ends generated by bleomycin damage. We show that interaction between Gph and GAPDH increases in cells challenged with bleomycin, suggesting involvement of GAPDH in cellular processes linked to DNA repair mechanisms.

A common BACE1 polymorphism is a risk factor for sporadic Creutzfeldt-Jakob disease

The β site APP cleaving enzyme 1 (BACE1) is the rate-limiting β-secretase enzyme in the amyloidogenic processing of APP and Aβ formation, and therefore it has a prominent role in Alzheimer"s disease (AD) pathology. Recent evidence suggests that the prion protein (PrP) interacts directly with BACE1 regulating its β-secretase activity. Moreover, PrP has been proposed as the cellular receptor involved in the impairment of synaptic plasticity and toxicity caused by Aβ oligomers. Provided that common pathophysiologic mechanisms are shared by Alzheimer"s and Creutzfeldt-Jakob (CJD) diseases, we investigated for the first time to the best of our knowledge a possible association of a common synonymous BACE1 polymorphism (rs638405) with sporadic CJD (sCJD). Our results indicate that BACE1 C-allele is associated with an increased risk for developing sCJD, mainly in PRNP M129M homozygous subjects with early onset. These results extend the very short list of genes (other than PRNP) involved in the development of human prion diseases; and support the notion that similar to AD, in sCJD several loci may contribute with modest overall effects to disease risk. These findings underscore the interplay in both pathologies of APP, Aβ oligomers, ApoE, PrP and BACE1, and suggest that aging and perhaps vascular risk factors may modulate disease pathologies in part through these key players

On the origin of the selectivity of plasmidic H-NS towards horizontally acquired DNA: Linking H-NS oligomerization and cooperative DNA binding

The nucleoid-associated protein H-NS is a global modulator of the expression of genes associated with adaptation to environmental changes. A variant of H-NS expressed in the R27 plasmid was previously shown to selectively modulate the expression of horizontally acquired genes, with minimal effects on core genes that are repressed by the chromosomal form of H-NS. Both H-NS proteins are formed by an oligomerization domain and a DNA-binding domain, which are connected by a linker that is highly flexible in the absence of DNA. We studied DNA binding by means of oligomer-forming chimeric proteins in which domains of the chromosomal and plasmidic variants are exchanged, as well as in monomeric truncated forms containing the DNA-binding domain and variable portions of the linker. Point mutations in the linker were also examined in full-length and truncated H-NS constructs. These experiments show that the linker region contributes to DNA binding affinity and that it is a main component of the distinct DNA binding properties of chromosomal and plasmidic H-NS. We propose that interactions between the linker and DNA limit the flexibility of the connection between H- NS oligomerization and DNA binding and provide an allosteric indirect readout mechanism to detect long- range distortions of DNA, thus enabling discrimination between core and horizontally acquired DNA.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins

Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Computational toolbox towards evolutionary domain mapping of membrane proteins

Membrane proteins account for about 20% to 30% of all proteins encoded in a typical genome. They play central roles in multiple cellular processes mediating the interaction of the cell with its surrounding. Over 60% of all drug targets contain a membrane domain. The experimental difficulties of obtaining a crystal structural severely limits our ability or understanding of membrane protein function. Computational evolutionary studies of proteins are crucial for the prediction of 3D structures. In this project, we construct a tool able to quantify the evolutionary positive selective pressure on each residue of membrane proteins through maximum likelihood phylogeny reconstruction. The conservation plot combined with a structural homology model is also a potent tool to predict those residues that have essentials roles in the structure and function of a membrane protein and can be very useful in the design of validation experiments.

Characterization of an antennal carboxylesterase from the pest moth Spodoptera littoralis degrading a host plant odorant.

Background: Carboxyl/cholinesterases (CCEs) are highly diversified in insects. These enzymes have a broad range of proposed functions, in neuro/developmental processes, dietary detoxification, insecticide resistance or hormone/pheromone degradation. As few functional data are available on purified or recombinant CCEs, the physiological role of most of these enzymes is unknown. Concerning their role in olfaction, only two CCEs able to metabolize sex pheromones have been functionally characterized in insects. These enzymes are only expressed in the male antennae, and secreted into the lumen of the pheromone-sensitive sensilla. CCEs able to hydrolyze other odorants than sex pheromones, such as plant volatiles, have not been identified. Methodology: In Spodoptera littoralis, a major crop pest, a diversity of antennal CCEs has been previously identified. We have employed here a combination of molecular biology, biochemistry and electrophysiology approaches to functionally characterize an intracellular CCE, SlCXE10, whose predominant expression in the olfactory sensilla suggested a role in olfaction. A recombinant protein was produced using the baculovirus system and we tested its catabolic properties towards a plant volatile and the sex pheromone components. Conclusion: We showed that SlCXE10 could efficiently hydrolyze a green leaf volatile and to a lesser extent the sex pheromone components. The transcript level in male antennae was also strongly induced by exposure to this plant odorant. In antennae, SlCXE10 expression was associated with sensilla responding to the sex pheromones and to plant odours. These results suggest that a CCE-based intracellular metabolism of odorants could occur in insect antennae, in addition to the extracellular metabolism occurring within the sensillar lumen. This is the first functional characterization of an Odorant- Degrading Enzyme active towards a host plant volatile.

On the role of G protein-coupled receptors oligomerization

The existence of a supramolecular organization of the G protein-coupled receptor (GPCR) is now being widely accepted by the scientific community. Indeed, GPCR oligomers may enhance the diversity and performance by which extracellular signals are transferred to the G proteins in the process of receptor transduction, although the mechanism that underlies this phenomenon still remains unsolved. Recently, it has been proposed that a trans-conformational switching model could be the mechanism allowing direct inhibition/activation of receptor activation/inhibition, respectively. Thus, heterotropic receptor-receptor allosteric regulations are behind the GPCR oligomeric function. In this paper we want to revise how GPCR oligomerization impinges on several important receptor functions like biosynthesis, plasma membrane diffusion or velocity, pharmacology and signaling. In particular, the rationale of receptor oligomerization might lie in the need of sensing complex whole cell extracellular signals and translating them into a simple computational model.

Ligand binding to heparan sulfate proteoglycans induces their aggregation and distribution along actin cytoskeleton

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.

Dual role of LldR in regulation of the lldPRD operon, involved in l-Lactate metabolism in Escherichia coli.

The lldPRD operon of Escherichia coli, involved in L-lactate metabolism, is induced by growth in this compound. We experimentally identified that this system is transcribed from a single promoter with an initiation site located 110 nucleotides upstream of the ATG start codon. On the basis of computational data, it had been proposed that LldR and its homologue PdhR act as regulators of the lldPRD operon. Nevertheless, no experimental data on the function of these regulators have been reported so far. Here we show that induction of an lldP-lacZ fusion by L-lactate is lost in an lldR mutant, indicating the role of LldR in this induction. Expression analysis of this construct in a pdhR mutant ruled out the participation of PdhR in the control of lldPRD. Gel shift experiments showed that LldR binds to two operator sites, O1 (positions 105 to 89) and O2 (positions 22 to 38), with O1 being filled at a lower concentration of LldR. L-Lactate induced a conformational change in LldR that did not modify its DNA binding activity. Mutations in O1 and O2 enhanced the basal transcriptional level. However, only mutations in O1 abolished induction by L-lactate. Mutants with a change in helical phasing between O1 and O2 behaved like O2 mutants. These results were consistent with the hypothesis that LldR has a dual role, acting as a repressor or an activator of lldPRD. We propose that in the absence of L-lactate, LldR binds to both O1 and O2, probably leading to DNA looping and the repression of transcription. Binding of L-lactate to LldR promotes a conformational change that may disrupt the DNA loop, allowing the formation of the transcription open complex.

Comparative genomics of Hedgehog loci in chordates and the origin of Shh regulatory novelties

The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications.

Divergent evolution and purifying selection of the flaA gene sequences in Aeromonas

BACKGROUND: The bacterial flagellum is the most important organelle of motility in bacteria and plays a key role in many bacterial lifestyles, including virulence. The flagellum also provides a paradigm of how hierarchical gene regulation, intricate protein-protein interactions and controlled protein secretion can result in the assembly of a complex multi-protein structure tightly orchestrated in time and space. As if to stress its importance, plants and animals produce receptors specifically dedicated to the recognition of flagella. Aside from motility, the flagellum also moonlights as an adhesion and has been adapted by humans as a tool for peptide display. Flagellar sequence variation constitutes a marker with widespread potential uses for studies of population genetics and phylogeny of bacterial species. RESULTS: We sequenced the complete flagellin gene (flaA) in 18 different species and subspecies of Aeromonas. Sequences ranged in size from 870 (A. allosaccharophila) to 921 nucleotides (A. popoffii). The multiple alignment displayed 924 sites, 66 of which presented alignment gaps. The phylogenetic tree revealed the existence of two groups of species exhibiting different FlaA flagellins (FlaA1 and FlaA2). Maximum likelihood models of codon substitution were used to analyze flaA sequences. Likelihood ratio tests suggested a low variation in selective pressure among lineages, with an omega ratio of less than 1 indicating the presence of purifying selection in almost all cases. Only one site under potential diversifying selection was identified (isoleucine in position 179). However, 17 amino acid positions were inferred as sites that are likely to be under positive selection using the branch-site model. Ancestral reconstruction revealed that these 17 amino acids were among the amino acid changes detected in the ancestral sequence. CONCLUSION: The models applied to our set of sequences allowed us to determine the possible evolutionary pathway followed by the flaA gene in Aeromonas, suggesting that this gene have probably been evolving independently in the two groups of Aeromonas species since the divergence of a distant common ancestor after one or several episodes of positive selection. REVIEWERS: This article was reviewed by Alexey Kondrashov, John Logsdon and Olivier Tenaillon (nominated by Laurence D Hurst).

BioSuper: A web tool for the superimposition of biomolecules and assemblies with rotational symmetry

Background Most of the proteins in the Protein Data Bank (PDB) are oligomeric complexes consisting of two or more subunits that associate by rotational or helical symmetries. Despite the myriad of superimposition tools in the literature, we could not find any able to account for rotational symmetry and display the graphical results in the web browser. Results BioSuper is a free web server that superimposes and calculates the root mean square deviation (RMSD) of protein complexes displaying rotational symmetry. To the best of our knowledge, BioSuper is the first tool of its kind that provides immediate interactive visualization of the graphical results in the browser, biomolecule generator capabilities, different levels of atom selection, sequence-dependent and structure-based superimposition types, and is the only web tool that takes into account the equivalence of atoms in side chains displaying symmetry ambiguity. BioSuper uses ICM program functionality as a core for the superimpositions and displays the results as text, HTML tables and 3D interactive molecular objects that can be visualized in the browser or in Android and iOS platforms with a free plugin. Conclusions BioSuper is a fast and functional tool that allows for pairwise superimposition of proteins and assemblies displaying rotational symmetry. The web server was created after our own frustration when attempting to superimpose flexible oligomers. We strongly believe that its user-friendly and functional design will be of great interest for structural and computational biologists who need to superimpose oligomeric proteins (or any protein). BioSuper web server is freely available to all users at http://ablab.ucsd.edu/BioSuper webcite.

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription.