Receptor Autoradiography as a Tool for the Study of the Phylogeny of the Basal Ganglia

Abstract

Recent studies on the neurotransmitter organization of the basal ganglia and forebrain in lower vertebrates suggest that, in contrast to the old concepts of the phylogeny of the brain, there are many similarities between the chemical organization of the brain throughout evolution. By examining neurotransmitter receptors using in vitro autoradiography we have attempted to further our understanding of the evolution of the brain. Receptors enriched in different parts of the basal ganglia in mammals appear to be also enriched in the homologous areas in lower vertebrates. Thus, for example, dopamine and muscarinic receptors, but not serotonin-1A, are enriched in the paleostriatum augmentatum while GABA/benzo-diazepine receptors are enriched in the paleostriatum primitivum corresponding with their localization to the caudate-putamen and globus pallidus respectively. Our results support the concept of a more conservative evolution of the vertebrate brain and demonstrate the usefulness of receptor autoradiography in the understanding of brain evolution.     

The Synthesis of Ribosomes in E. coli: IV. The Synthesis of Ribosomal Protein and the Assembly of Ribosomes

The incorporation of C¹⁴ leucine into the protein moiety of ribosomes has been studied as a sequel to the studies of ribosomal RNA synthesis. In contrast to the latter studies, labeled leucine is incorporated directly into 50S and 30S ribosomes without measurable delay by precursor stages. There is, however, evidence of some transfer of radioactivity from the 43S group of particles to the 50S. The inhibition of protein synthesis by chloramphenicol results in the accumulation of material similar to the eosome—the primary precursor in ribosome synthesis. There is also evidence for the synthesis of some neosome. The results of the studies of ribosomal RNA and protein synthesis are combined into a model of ribosome synthesis. Finally, consideration is made of the significance of these studies of ribosome synthesis for general problems of protein synthesis and information transfer.     

Fraction of Ribosomes Synthesizing Protein as a Function of Specific Growth Rate

The fraction of ribosomes engaged in protein synthesis in Escherichia coli growing at specific growth rates ranging from 0.3 to 1.2 h(-1) was estimated from measurements of nascent protein/ribosome and of polyribosomes. Both measurements showed that the fraction of ribosomes synthesizing protein increased with specific growth rate, from 30 to 70% over the range studied. Polyribosome measurements made at different specific growth rates in four E. coli strains showed no significant differences between strains. The increase in the fraction of polyribosomes had begun within 30 s after a shift-up from glucose-minimal medium, and was complete within 2 to 5 min. These results indicate that the function of ribosomes is regulated.     

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.     

Functional Complementation of a Model Target to Study Vpu Sensitivity

HIV-1 forms infectious particles with Murine Leukemia virus (MLV) Env, but not with the closely related Gibbon ape Leukemia Virus (GaLV) Env. We have determined that the incompatibility between HIV-1 and GaLV Env is primarily caused by the HIV-1 accessory protein Vpu, which prevents GaLV Env from being incorporated into particles. We have characterized the ‘Vpu sensitivity sequence’ in the cytoplasmic tail domain (CTD) of GaLV Env using a chimeric MLV Env with the GaLV Env CTD (MLV/GaLV Env). Vpu sensitivity is dependent on an alpha helix with a positively charged face containing at least one Lysine. In the present study, we utilized functional complementation to address whether all the three helices in the CTD of an Env trimer have to contain the Vpu sensitivity motif for the trimer to be modulated by Vpu. Taking advantage of the functional complementation of the binding defective (D84K) and fusion defective (L493V) MLV and MLV/GaLV Env mutants, we were able to assay the activity of mixed trimers containing both MLV and GaLV CTDs. Mixed trimers containing both MLV and GaLV CTDs were functionally active and remained sensitive to Vpu. However, trimers containing an Env with the GaLV CTD and an Env with no CTD remained functional but were resistant to Vpu. Together these data suggest that the presence of at least one GaLV CTD is sufficient to make an Env trimer sensitive to Vpu, but only if it is part of a trimeric CTD complex.     

Electric Field Encephalography as a Tool for Functional Brain Research: A Modeling Study

We introduce the notion of Electric Field Encephalography (EFEG) based on measuring electric fields of the brain and demonstrate, using computer modeling, that given the appropriate electric field sensors this technique may have significant advantages over the current EEG technique. Unlike EEG, EFEG can be used to measure brain activity in a contactless and reference-free manner at significant distances from the head surface. Principal component analysis using simulated cortical sources demonstrated that electric field sensors positioned 3 cm away from the scalp and characterized by the same signal-to-noise ratio as EEG sensors provided the same number of uncorrelated signals as scalp EEG. When positioned on the scalp, EFEG sensors provided 2–3 times more uncorrelated signals. This significant increase in the number of uncorrelated signals can be used for more accurate assessment of brain states for non-invasive brain-computer interfaces and neurofeedback applications. It also may lead to major improvements in source localization precision. Source localization simulations for the spherical and Boundary Element Method (BEM) head models demonstrated that the localization errors are reduced two-fold when using electric fields instead of electric potentials. We have identified several techniques that could be adapted for the measurement of the electric field vector required for EFEG and anticipate that this study will stimulate new experimental approaches to utilize this new tool for functional brain research.     

Emetine Binding to Ribosomes of Entamoeba histolytica—Inhibition of Protein Synthesis and Amebicidal Action*

After demonstration that emetine is amebicidal by inhibiting protein synthesis, the question arose whether active protein synthesis is required for emetine's amebicidal effect. The answer appears to be “no,” as derived from experiments on intact amebae. Responses were compared for log- and stationary-growth phase amebae. In the latter, protein synthesis is significantly slower, and sensitivity to emetine, i.e. degree of inhibition of protein synthesis, was maintained independently of rate of protein synthesis. Both stages equally bound tritiated emetine to their ribcsomes. Binding of [³H]emetine was not affected by certain drugs that interfere with energy metabolism, protein synthesis, and/or ribosomal function, e.g. dinitrophenol, puromycin, chloroquine, and acriflavin. High concentrations of EDTA combined with puromycin (which disaggregates ribosomes into their subunits) lowered binding by 50%. In chase experiments the ribosomes of intact amebae were prelabeled with [³H]emetine or [³H]isoemetine, then exposed to relatively high concentrations of unlabeled emetine. Labeled isoemetine was displaced almost completely, whereas no displacement of [³H]emetine occurred; evidently, the high stability of the emetine-ribosome binding is due in part to a hydrogen-bonding reaction of the C-1' atom of the emetine molecule with the chain-elongation site. Finally, evidence was obtained that capacity to bind emetine is an index of drug resistance.     

Structure-Based Phylogeny as a Diagnostic for Functional Characterization of Proteins with a Cupin Fold

Background

The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary associations and the nature of bound metal ion, despite having a conserved β-barrel structural scaffold. Here, an attempt has been made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which the structures are available through world-wide structural genomics initiatives but characterized as “hypothetical”. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function.

Methodology/Principal Findings

A 3-D structure-based phylogenetic approach was undertaken. Interestingly, a dendrogram generated solely on the basis of structural dissimilarity measure at the level of domain folds was found to cluster functionally similar members. This clustering also reflects an independent evolution of the two domains in bicupins. Close examination of structural superposition of members across various functional clusters reveals structural variations in regions that not only form the active site pocket but are also involved in interaction with another domain in the same polypeptide or in the oligomer.

Conclusions/Significance

Structure-based phylogeny of cupins can influence identification of functions of proteins of yet unknown function with cupin fold. This approach can be extended to other proteins with a common fold that show high evolutionary divergence. This approach is expected to have an influence on the function annotation in structural genomics initiatives.     

The Mode of Action of Pederin,a Drug Inhibiting Protein Synthesis in Eucaryotic Organisms

Abstract

Pederin, a toxic substance isolated from the insect Paederus fuscipes, inhibits growth of Saccharomyces cerevisiae and EUE cells but not of Bacillus subtilis. Protein synthesis in vitro appears to be inhibited by the drug in preparations obtained from organisms containing 80 S ribosomes (yeast, EUE cells and rat liver) but not in those from organisms endowed with 70 S ribosomes (E. coli and B. subtilis). Pederin inhibits protein synthesis at a stage subsequent to the formation of the ternary complex between ribosomes, aminoacyl-tRNA and messenger RNA. Resistance or susceptibility to the drug appears to be a characteristic of ribosomes.     

Fluorescence Quenching as a Tool to Investigate Quinolone Antibiotic Interactions with Bacterial Protein OmpF

The outer membrane porin OmpF is an important protein for the uptake of antibiotics through the outer membrane of gram-negative bacteria; however, the possible binding sites involved in this uptake are still not recognized. Determination, at the molecular level, of the possible sites of antibiotic interaction is very important, not only to understand their mechanism of action but also to unravel bacterial resistance. Due to the intrinsic OmpF fluorescence, attributed mainly to its tryptophans (Trp²¹⁴, Trp⁶¹), quenching experiments were used to assess the site(s) of interaction of some quinolone antibiotics. OmpF was reconstituted in different organized structures, and the fluorescence quenching results, in the presence of two quenching agents, acrylamide and iodide, certified that acrylamide quenches Trp⁶¹ and iodide Trp²¹⁴. Similar data, obtained in presence of the quinolones, revealed distinct behaviors for these antibiotics, with nalidixic acid interacting near Trp²¹⁴ and moxifloxacin near Trp⁶¹. These studies, based on straightforward and quick procedures, show the existence of conformational changes in the protein in order to adapt to the different organized structures and to interact with the quinolones. The extent of reorganization of the protein in the presence of the different quinolones allowed an estimate on the sites of protein/quinolone interaction.     

Nebulin: A Study of Protein Repeat Evolution

Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains.Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates.In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination.Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein.Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance.     

Population Waves as a Condition for Increasing Ecological Stability of Organisms in the Process of Vertical Evolution

Ecological stability of an organism, which determines the possibility of its existence under changing environmental conditions, can be estimated as the probability of the participation of each viable offspring in reproduction. In developing species, the periodic rises and falls in the population size (Chetverikov's waves of life) can lead to changes in ecological stability, which is of macroevolutionary importance. Under conditions of isolation such changes generally result in specialization of intraspecific races but they could then lead to an increase in ecological stability of hybrid forms. Ecological stability of prosperous species increases during macroevolution due to combinative recombination between specialized intraspecific races or closely related species.     

Fluorescence Spectroscopy as a Tool to Unravel the Dynamics of Protein Nanoparticle Formation by Liquid Antisolvent Precipitation

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.     

A Case Study of the De Novo Evolution of a Complex Odometric Behavior in Digital Organisms

Investigating the evolution of animal behavior is difficult. The fossil record leaves few clues that would allow us to recapitulate the path that evolution took to build a complex behavior, and the large population sizes and long time scales required prevent us from re-evolving such behaviors in a laboratory setting. We present results of a study in which digital organisms–self-replicating computer programs that are subject to mutations and selection–evolved in different environments that required information about past experience for fitness-enhancing behavioral decisions. One population evolved a mechanism for step-counting, a surprisingly complex odometric behavior that was only indirectly related to enhancing fitness. We examine in detail the operation of the evolved mechanism and the evolutionary transitions that produced this striking example of a complex behavior.     

On the Use of Variance per Genotype as a Tool to Identify Quantitative Trait Interaction Effects: A Report from the Women's Genome Health Study

Testing for genetic effects on mean values of a quantitative trait has been a very successful strategy. However, most studies to date have not explored genetic effects on the variance of quantitative traits as a relevant consequence of genetic variation. In this report, we demonstrate that, under plausible scenarios of genetic interaction, the variance of a quantitative trait is expected to differ among the three possible genotypes of a biallelic SNP. Leveraging this observation with Levene''s test of equality of variance, we propose a novel method to prioritize SNPs for subsequent gene–gene and gene–environment testing. This method has the advantageous characteristic that the interacting covariate need not be known or measured for a SNP to be prioritized. Using simulations, we show that this method has increased power over exhaustive search under certain conditions. We further investigate the utility of variance per genotype by examining data from the Women''s Genome Health Study. Using this dataset, we identify new interactions between the LEPR SNP rs12753193 and body mass index in the prediction of C-reactive protein levels, between the ICAM1 SNP rs1799969 and smoking in the prediction of soluble ICAM-1 levels, and between the PNPLA3 SNP rs738409 and body mass index in the prediction of soluble ICAM-1 levels. These results demonstrate the utility of our approach and provide novel genetic insight into the relationship among obesity, smoking, and inflammation.     

The Stress of Protein Misfolding: From Single Cells to Multicellular Organisms

Organisms survive changes in the environment by altering their rates of metabolism, growth, and reproduction. At the same time, the system must ensure the stability and functionality of its macromolecules. Fluctuations in the environment are sensed by highly conserved stress responses and homeostatic mechanisms, and of these, the heat shock response (HSR) represents an essential response to acute and chronic proteotoxic damage. However, unlike the strategies employed to maintain the integrity of the genome, protection of the proteome must be tailored to accommodate the normal flux of nonnative proteins and the differences in protein composition between cells, and among individuals. Moreover, adult cells are likely to have significant differences in the rates of synthesis and clearance that are influenced by intrinsic errors in protein expression, genetic polymorphisms, and fluctuations in physiological and environmental conditions. Here, we will address how protein homeostasis (proteostasis) is achieved at the level of the cell and organism, and how the threshold of the stress response is set to detect and combat protein misfolding. For metazoans, the requirement for coordinated function and growth imposes additional constraints on the detection, signaling, and response to misfolding, and requires that the HSR is integrated into various aspects of organismal physiology, such as lifespan. This is achieved by hierarchical regulation of heat shock factor 1 (HSF1) by the metabolic state of the cell and centralized neuronal control that could allow optimal resource allocation between cells and tissues. We will examine how protein folding quality control mechanisms in individual cells may be integrated into a multicellular level of control, and further, even custom-designed to support individual variability and impose additional constraints on evolutionary adaptation.