Thermodynamics of the Interaction between Alzheimer's Disease Related Tau Protein and DNA

Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimer''s disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimer''s pathogenesis.     

The Role of Gcr1p in the Transcriptional Activation of Glycolytic Genes in Yeast Saccharomyces Cerevisiae

To study the interdependence of Gcr1p and Rap1p, we prepared a series of synthetic regulatory sequences that contained various numbers and combinations of CT-boxes (Gcr1p-binding sites) and RPG-boxes (Rap1p-binding sites). The ability of the synthetic oligonucleotides to function as regulatory sequences was tested using an ENO1-lacZ reporter gene. As observed previously, synthetic oligonucleotides containing both CT- and RPG-boxes conferred strong UAS activity. Likewise, a lone CT-box did not show any UAS activity. By contrast, oligonucleotides containing tandem CT-boxes but no RPG-box conferred strong promoter activity. This UAS activity was not dependent on position or orientation of the oligonucleotides in the 5'' noncoding region. However, it was dependent on both GCR1 and GCR2. These results suggest that the ability of Gcr1p to bind Gcr1p-binding sites in vivo is not absolutely dependent on Rap1p. Eleven independent mutants of GCR1 were isolated that conferred weak UAS activity to a single CT-box. Five mutants had single mutations in Gcr1p''s DNA-binding domain and displayed slightly higher affinity for the CT-box. These results support the hypothesis that Gcr1p and Gcr2p play the central role in glycolytic gene expression and that the function of Rap1p is to facilitate the binding of Gcr1p to its target.     

Differential sensitivity to conspecific and allospecific cues in chimpanzees and humans: a comparative eye-tracking study

Previous studies have shown that a variety of animals including humans are sensitive to social cues from others and shift their attention to the same objects attended to by others. However, little is known about how animals process conspecifics'' and another species'' actions, although primates recognize conspecific faces better than those of another species. In this study, using unrestrained eye-tracking techniques, we first demonstrated that conspecific social cues modulated looking behaviours of chimpanzees more than human cues, whereas human observers were equally sensitive to both species. Additionally, first pass gaze duration at the face indicates that chimpanzees looked at the chimpanzees'' face longer than the human face, suggesting that chimpanzees might extract more referential information from a conspecific face. These results also imply that a unique ability for extracting referential information from a variety of social objects has emerged during human evolution.     

The Allosteric Mechanism Induced by Protein Kinase A (PKA) Phosphorylation of Dematin (Band 4.9)

Dematin (band 4.9) is an F-actin binding and bundling protein best known for its role within red blood cells, where it both stabilizes as well as attaches the spectrin/actin cytoskeleton to the erythrocytic membrane. Here, we investigate the structural consequences of phosphorylating serine 381, a covalent modification that turns off F-actin bundling activity. In contrast to the canonical doctrine, in which phosphorylation of an intrinsically disordered region/protein confers affinity for another domain/protein, we found the converse to be true of dematin: phosphorylation of the well folded C-terminal villin-type headpiece confers affinity for its intrinsically disordered N-terminal core domain. We employed analytical ultracentrifugation to demonstrate that dematin is monomeric, in contrast to the prevailing view that it is trimeric. Next, using a series of truncation mutants, we verified that dematin has two F-actin binding sites, one in the core domain and the other in the headpiece domain. Although the phosphorylation-mimicking mutant, S381E, was incapable of bundling microfilaments, it retains the ability to bind F-actin. We found that a phosphorylation-mimicking mutant, S381E, eliminated the ability to bundle, but not bind F-actin filaments. Lastly, we show that the S381E point mutant caused the headpiece domain to associate with the core domain, leading us to the mechanism for cAMP-dependent kinase control of dematin''s F-actin bundling activity: when unphosphorylated, dematin''s two F-actin binding domains move independent of one another permitting them to bind different F-actin filaments. Phosphorylation causes these two domains to associate, forming a compact structure, and sterically eliminating one of these F-actin binding sites.     

Dysferlin Interacts with Tubulin and Microtubules in Mouse Skeletal Muscle

Dysferlin is a type II transmembrane protein implicated in surface membrane repair in muscle. Mutations in dysferlin lead to limb girdle muscular dystrophy 2B, Miyoshi Myopathy and distal anterior compartment myopathy. Dysferlin''s mode of action is not well understood and only a few protein binding partners have thus far been identified. Using affinity purification followed by liquid chromatography/mass spectrometry, we identified alpha-tubulin as a novel binding partner for dysferlin. The association between dysferlin and alpha-tubulin, as well as between dysferlin and microtubules, was confirmed in vitro by glutathione S-transferase pulldown and microtubule binding assays. These interactions were confirmed in vivo by co-immunoprecipitation. Confocal microscopy revealed that dysferlin and alpha-tubulin co-localized in the perinuclear region and in vesicular structures in myoblasts, and along thin longitudinal structures reminiscent of microtubules in myotubes. We mapped dysferlin''s alpha-tubulin-binding region to its C2A and C2B domains. Modulation of calcium levels did not affect dysferlin binding to alpha-tubulin, suggesting that this interaction is calcium-independent. Our studies identified a new binding partner for dysferlin and suggest a role for microtubules in dysferlin trafficking to the sarcolemma.     

Taxol-stabilized microtubules promote the formation of filaments from unmodified full-length Tau in vitro

Tau is a neuronal protein that stabilizes the microtubule (MT) network, but it also forms filaments associated with Alzheimer''s disease. Understanding Tau–MT and Tau–Tau interactions would help to establish Tau function in health and disease. For many years, literature reports on Tau–MT binding behavior and affinity have remained surprisingly contradictory (e.g., 10-fold variation in Tau–MT affinity). Tau–Tau interactions have also been investigated, but whether MTs might affect Tau filament formation is unknown. We have addressed these issues through binding assays and microscopy. We assessed Tau–MT interactions via cosedimentation and found that the measured affinity of Tau varies greatly, depending on the experimental design and the protein concentrations used. To investigate this dependence, we used fluorescence microscopy to examine Tau–MT binding. Strikingly, we found that Taxol-stabilized MTs promote Tau filament formation without characterized Tau-filament inducers. We propose that these novel Tau filaments account for the incongruence in Tau–MT affinity measurements. Moreover, electron microscopy reveals that these filaments appear similar to the heparin-induced Alzheimer''s model. These observations suggest that the MT-induced Tau filaments provide a new model for Alzheimer''s studies and that MTs might play a role in the formation of Alzheimer''s-associated neurofibrillary tangles.     

Lesions to right posterior parietal cortex impair visual depth perception from disparity but not motion cues

The posterior parietal cortex (PPC) is understood to be active when observers perceive three-dimensional (3D) structure. However, it is not clear how central this activity is in the construction of 3D spatial representations. Here, we examine whether PPC is essential for two aspects of visual depth perception by testing patients with lesions affecting this region. First, we measured subjects'' ability to discriminate depth structure in various 3D surfaces and objects using binocular disparity. Patients with lesions to right PPC (N = 3) exhibited marked perceptual deficits on these tasks, whereas those with left hemisphere lesions (N = 2) were able to reliably discriminate depth as accurately as control subjects. Second, we presented an ambiguous 3D stimulus defined by structure from motion to determine whether PPC lesions influence the rate of bistable perceptual alternations. Patients'' percept durations for the 3D stimulus were generally within a normal range, although the two patients with bilateral PPC lesions showed the fastest perceptual alternation rates in our sample. Intermittent stimulus presentation reduced the reversal rate similarly across subjects. Together, the results suggest that PPC plays a causal role in both inferring and maintaining the perception of 3D structure with stereopsis supported primarily by the right hemisphere, but do not lend support to the view that PPC is a critical contributor to bistable perceptual alternations.This article is part of the themed issue ‘Vision in our three-dimensional world’.     

Mirror agnosia.

Normal people rarely confuse the mirror image of an object with a real object so long as they realize they are looking into a mirror. We report a new neurological sign, ''mirror agnosia'', following right parietal lesions in which this ability is severely compromised. We studied four right hemisphere stroke patients who had left visual field ''neglect''. i.e. they were indifferent to objects in their left visual field even though they were not blind. We then placed a vertical parasagittal mirror on each patients'' right so that they could clearly see the reflection of objects placed in the (neglected) visual field. When shown a candy or pen on their left, the patients kept banging their hand into the mirror or groped behind it attempting to grab the reflection; they did not reach for the real object on the left, even though they were mentally quite lucid and knew they were looking into a mirror. Remarkably, all four patients kept complaining that the object was ''in the mirror'', ''outside my reach'' or ''behind the mirror''. Thus, even the patients'' ability to make simple logical inferences about mirrors has been selectively warped to accommodate the strange new sensory world that they now inhabit. The finding may have implications for understanding how the brain creates representations of mirror reflections.     

Pleiotropic Functions of Tumor Suppressor WWOX in Normal and Cancer Cells

WW domain-containing oxidoreductase (WWOX), originally marked as a likely tumor suppressor gene, has over the years become recognized for its role in a much wider range of cellular activities. Phenotypic effects displayed in animal studies, along with resolution of WWOX''s architecture, fold, and binding partners, point to the protein''s multifaceted biological functions. Results from a series of complementary experiments seem to indicate WWOX''s involvement in metabolic regulation. More recently, clinical studies involving cases of severe encephalopathy suggest that WWOX also plays a part in controlling CNS development, further expanding our understanding of the breadth and complexity of WWOX behavior. Here we present a short overview of the various approaches taken to study this dynamic gene, emphasizing the most recent findings regarding WWOX''s metabolic- and CNS-associated functions and their underlying molecular basis.     

Antibodies binding diverse pertactin epitopes protect mice from Bordetella pertussis infection

Infection by the bacterium Bordetella pertussis continues to cause considerable morbidity and mortality worldwide. Many current acellular pertussis vaccines include the antigen pertactin, which has presumptive adhesive and immunomodulatory activities, but is rapidly lost from clinical isolates after the introduction of these vaccines. To better understand the contributions of pertactin antibodies to protection and pertactin''s role in pathogenesis, we isolated and characterized recombinant antibodies binding four distinct epitopes on pertactin. We demonstrate that four of these antibodies bind epitopes that are conserved across all three classical Bordetella strains, and competition assays further showed that antibodies binding these epitopes are also elicited by B. pertussis infection of baboons. Surprisingly, we found that representative antibodies binding each epitope protected mice against experimental B. pertussis infection. A cocktail of antibodies from each epitope group protected mice against a subsequent lethal dose of B. pertussis and greatly reduced lung colonization levels after sublethal challenge. Each antibody reduced B. pertussis lung colonization levels up to 100-fold when administered individually, which was significantly reduced when antibody effector functions were impaired, with no antibody mediating antibody-dependent complement-induced lysis. These data suggest that antibodies binding multiple pertactin epitopes protect primarily by the same bactericidal mechanism, which overshadows contributions from blockade of other pertactin functions. These antibodies expand the available tools to further dissect pertactin''s role in infection and understand the impact of antipertactin antibodies on bacterial fitness.     

The evolutionary dynamics of receptor binding avidity in influenza A: a mathematical model for a new antigenic drift hypothesis

The most salient feature of influenza evolution in humans is its antigenic drift. This process is characterized by structural changes in the virus''s B-cell epitopes and ultimately results in the ability of the virus to evade immune recognition and thereby reinfect previously infected hosts. Until recently, amino acid substitutions in epitope regions of the viral haemagglutinin were thought to be positively selected for their ability to reduce antibody binding and therefore were thought to be responsible for driving antigenic drift. However, a recent hypothesis put forward by Hensley and co-workers posits that cellular receptor binding avidity is the dominant phenotype under selection, with antigenic drift being a side effect of these binding avidity changes. Here, we present a mathematical formulation of this new antigenic drift model and use it to show how rates of antigenic drift depend on epidemiological parameters. We further use the model to evaluate how two different vaccination strategies can impact antigenic drift rates and ultimately disease incidence levels. Finally, we discuss the assumptions present in the model formulation, predictions of the model, and future work that needs to be done to determine the consistency of this hypothesis with known patterns of influenza''s genetic and antigenic evolution.     

Indirect Readout of DNA Sequence by P22 Repressor: Roles of DNA and Protein Functional Groups in Modulating DNA Conformation

The repressor of bacteriophage P22 (P22R) discriminates between its various DNA binding sites by sensing the identity of non-contacted base pairs at the center of its binding site. The “indirect readout” of these non-contacted bases is apparently based on DNA's sequence-dependent conformational preferences. The structures of P22R–DNA complexes indicate that the non-contacted base pairs at the center of the binding site are in the B′ state. This finding suggests that indirect readout and therefore binding site discrimination depend on P22R's ability to either sense and/or impose the B′ state on the non-contacted bases of its binding sites. We show here that the affinity of binding sites for P22R depends on the tendency of the central bases to assume the B′-DNA state. Furthermore, we identify functional groups in the minor groove of the non-contacted bases as the essential modulators of indirect readout by P22R. In P22R–DNA complexes, the negatively charged E44 and E48 residues are provocatively positioned near the negatively charged DNA phosphates of the non-contacted nucleotides. The close proximity of the negatively charged groups on protein and DNA suggests that electrostatics may play a key role in the indirect readout process. Changing either of two negatively charged residues to uncharged residues eliminates the ability of P22R to impose structural changes on DNA and to recognize non-contacted base sequence. These findings suggest that these negatively charged amino acids function to force the P22R-bound DNA into the B′ state and therefore play a key role in indirect readout by P22R.     

MEG Correlates of Learning Novel Objects Properties in Children

Learning the functional properties of objects is a core mechanism in the development of conceptual, cognitive and linguistic knowledge in children. The cerebral processes underlying these learning mechanisms remain unclear in adults and unexplored in children. Here, we investigated the neurophysiological patterns underpinning the learning of functions for novel objects in 10-year-old healthy children. Event-related fields (ERFs) were recorded using magnetoencephalography (MEG) during a picture-definition task. Two MEG sessions were administered, separated by a behavioral verbal learning session during which children learned short definitions about the “magical” function of 50 unknown non-objects. Additionally, 50 familiar real objects and 50 other unknown non-objects for which no functions were taught were presented at both MEG sessions. Children learned at least 75% of the 50 proposed definitions in less than one hour, illustrating children''s powerful ability to rapidly map new functional meanings to novel objects. Pre- and post-learning ERFs differences were analyzed first in sensor then in source space. Results in sensor space disclosed a learning-dependent modulation of ERFs for newly learned non-objects, developing 500–800 msec after stimulus onset. Analyses in the source space windowed over this late temporal component of interest disclosed underlying activity in right parietal, bilateral orbito-frontal and right temporal regions. Altogether, our results suggest that learning-related evolution in late ERF components over those regions may support the challenging task of rapidly creating new semantic representations supporting the processing of the meaning and functions of novel objects in children.