Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.     

A Systematic Approach for the Genetic Dissection of Protein Complexes in Living Cells

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αB-Crystallin Interacts with Cytoplasmic Intermediate Filament Bundles during Mitosis

The small heat-shock protein αB-crystallin interacts with intermediate filament proteins. Using cosedimentation assay, we showed previously that in vitro binding of αB-crystallin to peripherin and vimentin was temperature-dependent. Furthermore, when NIH 3T3 cells were submitted to different stress conditions a dynamic reorganization of the intermediate filament network was observed concomitantly with the recruitment of αB-crystallins on the intermediate filament proteins. Thus, the intracellular state of αB-crystallin correlated directly with the remodeling of the intermediate filament network in response to stress. Here, we show data suggesting that αB-crystallin is implicated in remodeling of intermediate filaments during cell division. We investigated the intracellular distribution of αB-crystallin in naturally occurring mitotic NIH 3T3 cells and in neuroblastoma N2a and N1E115 cells. In NIH 3T3 cells, αB-crystallin remained diffused throughout the cell cycle. Subcellular fractionation of αB-crystallin showed that αB-crystallin remained in the cytosolic compartment during mitosis. Furthermore, αB-crystallin accumulated in mitotically arrested NIH 3T3 cells. This increased level of αB-crystallin protein was due to an increased level of αB-crystallin mRNA in mitotic NIH 3T3 cells. In the neuroblastoma cells, the intermediate filaments were rearranged into thick cable-like structures and αB-crystallin was recruited onto them. In neuroblastoma N2a cells the level of expression did not change during the cell cycle. However, a small fraction of αB-crystallin switched onto the insoluble fraction in mitotically arrested N2a cells. Our results suggested that depending on the state of rearrangement of the intermediate filament network during mitosis αB-crystallin was either recruited onto the intermediate filaments or upregulated in the cytosolic compartment.     

Biochemical properties of alligator (Alligator mississippiensis) bone collagen

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) isolated and purified from alligator (Alligator mississippiensis) bone were studied for molecular size, amino acid profile, secondary structure, and denaturation temperature by SDS-PAGE, HPLC, circular dichroism, and viscometry. Two collagen subunits, alpha1 and alpha2 were identified by SDS-PAGE. The molecular masses for alpha1 and alpha2 chains of ASC were 124 kDa and 111 kDa, respectively. The molecular masses were 123 kDa for alpha1 and 110 kDa for alpha2 chains of the PSC preparation. The molecular masses for ([alpha1](2) alpha2) of ASC and PSC were 359 kDa and 356 kDa, respectively. The major composition of alligator bone ASC and PSC was found to be typical type I collagen. The amino acid profiles of alligator ASC and PSC were similar to amino acid profile of subtropical fish black drum (Pogonias cromis, Sciaenidae) bone. Comparison of amino acid profiles with shark cartilage PSC, showed differences in alanine, hydroxylysine, lysine, and histidine contents. The denaturation temperatures (T(d)) of alligator ASC and PSC collagen measured by viscometry were 38.1 and 38.2 degrees C, respectively. Thermal denaturation temperatures, measured by melting point using circular dichroism, were 37.6 and 37.9 degrees C, respectively. Taken together, these results suggest that alligator bone collagen may find a wide range of applications in biological research, functional foods and nutraceuticals, and biomedical and pharmaceutical research.     

Exploring large-scale brain networks in functional MRI.

Increasing emphasis has been recently put on large-scale network processing of brain functions. To explore these networks, many approaches have been proposed in functional magnetic resonance imaging (fMRI). Their objective is to answer the following two questions: (1) what brain regions are involved in the functional process under investigation? and (2) how do these regions interact? We review some of the key concepts and corresponding methods to cope with both issues.     

Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain

We have recently described the final steps leading to the crystallization of a mammalian membrane protein, the rabbit sarcoplasmic reticulum Ca2+-ATPase, after heterologous expression. Here, we detail the initial steps leading to this new purification method. A biotin acceptor domain was fused at the C-terminal part of Ca2+-ATPase and a thrombin site was inserted between both coding regions. The recombinant protein was expressed under the control of a galactose-inducible promoter in the yeast Saccharomyces cerevisiae. The biotinylation reaction of the protein was performed directly in vivo in yeast. After solubilization of the yeast light membrane fraction, the biotinylated protein was retained specifically using the strong biotin-avidin interaction. Finally, digestion by the protease thrombin allowed the separation of the Ca2+-ATPase from the biotinylated domain. At this step, Ca2+-ATPase is in a relatively purified form (about 40%). After a size-exclusion HPLC step, the purity of the protein is about 70%, and evaluation of the conformational changes during the catalytic cycle by monitoring the intrinsic fluorescence is demonstrated. The major advantage of this avidin procedure is the particularly good specific ATPase activity as compared with that of a purified His-tagged Ca2+-ATPase.     

Hemispheric dissociation in access to the human semantic system

Patient studies suggest that speech and environmental sounds are differentially processed by the left and right hemispheres. Here, using functional imaging in normal subjects, we compared semantic processing of spoken words to equivalent processing of environmental sounds, after controlling for low-level perceptual differences. Words enhanced activation in left anterior and posterior superior temporal regions, while environmental sounds enhanced activation in a right posterior superior temporal region. This left/right dissociation was unchanged by different attentional/working memory contexts, but it was specific to tasks requiring semantic analysis. While semantic processing involves widely distributed networks in both hemispheres, our results support the hypothesis of a dual access route specific for verbal and nonverbal material, respectively.     

An example of molecular co-evolution: reactive oxygen species (ROS) and ROS scavenger levels in Schistosoma mansoni/Biomphalaria glabrata interactions

The co-evolution between hosts and parasites involves huge reciprocal selective pressures on both protagonists. However, relatively few reports have evaluated the impact of these reciprocal pressures on the molecular determinants at the core of the relevant interaction, such as the factors influencing parasitic virulence and host resistance. Here, we address this question in a host-parasite model that allows co-evolution to be monitored in the field: the interaction between the mollusc, Biomphalaria glabrata, and its trematode parasite, Schistosoma mansoni. Reactive oxygen species (ROS) produced by the haemocytes of B. glabrata are known to play a crucial role in killing S. mansoni. Therefore, the parasite must defend itself against oxidative damage caused by ROS using ROS scavengers in order to survive. In this context, ROS and ROS scavengers are involved in a co-evolutionary arms race, and their respective production levels by sympatric host and parasite could be expected to be closely related. Here, we test this hypothesis by comparing host oxidant and parasite antioxidant capabilities between two S. mansoni/B. glabrata populations that have co-evolved independently. As expected, our findings show a clear link between the oxidant and antioxidant levels, presumably resulting from sympatric co-evolution. We believe this work provides the first supporting evidence of the Red Queen Hypothesis of reciprocal evolution for functional traits at the field-level in a model involving a host and a eukaryotic parasite.     

Toward rational design of bacterial genomes

The advent of genetic engineering-the ability to edit and insert DNA into living organisms-in the latter half of the 20th century created visions of a new era of synthetic biology, where novel biological functions could be designed and implemented for useful purposes. We are witnessing an exciting revolution of scale, wherein technical progresses allow for the manipulation of genetic material at the whole genome level. This will enable the manufacture of increasingly complex genetic designs to solve pressing challenges in health, energy and the environment-if and when such designs can be specified. We argue that the organized development of key common application organisms, engineered for engineerability, and attendant libraries of parts, pathways and standardized manufacturing are necessary for this genome-scale technology to realize its promise.