Proteomics and the blood–brain barrier: how recent findings help drug development

The drug discovery and development processes are divided into different stages separated by milestones to indicate that significant progress has been made and that certain criteria for target validation, hits, leads and candidate drugs have been met. Proteomics is a promising approach for the identification of protein targets and biochemical pathways involved in disease process and thus plays an important role in several stages of the drug development. The blood–brain barrier is considered as a major bottleneck when trying to target new compounds to treat neurodegenerative diseases. Based on the survey of recent findings and with a projection on expected improvements, this report attempt to address how proteomics participates in drug development.     

Large bottleneck size in Cauliflower Mosaic Virus populations during host plant colonization

The effective size of populations (Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Despite their high mutation rate and rapid evolution, this parameter is poorly documented experimentally in viruses, particularly plant viruses. All available studies, however, have demonstrated the existence of huge within-host demographic fluctuations, drastically reducing Ne upon systemic invasion of different organs and tissues. Notably, extreme bottlenecks have been detected at the stage of systemic leaf colonization in all plant viral species investigated so far, sustaining the general idea that some unknown obstacle(s) imposes a barrier on the development of all plant viruses. This idea has important implications, as it appoints genetic drift as a constant major force in plant virus evolution. By co-inoculating several genetic variants of Cauliflower mosaic virus into a large number of replicate host plants, and by monitoring their relative frequency within the viral population over the course of the host systemic infection, only minute stochastic variations were detected. This allowed the estimation of the CaMV Ne during colonization of successive leaves at several hundreds of viral genomes, a value about 100-fold higher than that reported for any other plant virus investigated so far, and indicated the very limited role played by genetic drift during plant systemic infection by this virus. These results suggest that the barriers that generate bottlenecks in some plant virus species might well not exist, or can be surmounted by other viruses, implying that severe bottlenecks during host colonization do not necessarily apply to all plant-infecting viruses.     

Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

Background

The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction.

Methodology/Principal Findings

Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death.

Conclusion/Significance

BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.     

Inhibition of platelet aggregation and immunomodulation of NK lymphocytes by administration of ascorbic acid

Platelets aggregation around migrating tumor cells offers protection against the cytotoxic activity of the natural killers cells (NKC). The ascorbic acid in 3 x 10(-3) M concentration completely inhibited platelet aggregation, decreased thromboxane B2 levels, and inhibited the expression of platelet membranic receptor GpIIb/IIIa in non stimulated platelets, and increased the NKC cytotoxicity in an average rate of 105, 61, and 285% in the NKC/targets cells ratios 12.5:1, 25:1 and 50:1 respectively. The results suggest the role of ascorbic acid in increasing the susceptibility of tumor cells to NKC; the ascorbic acid could be used as part of a multidrug therapy to treat diseases which up to now have been treated only through chemotherapy.     

Characterization of unique signature sequences in the divergent maternal protein Bcl2l10

Insertions or deletions (indels) of amino acids residues have been recognized as an important source of genetic and structural divergence between paralogous Bcl-2 family members. However, these signature sequences have not so far been extensively investigated amongst orthologous Bcl-2 family proteins. Bcl2l10 is an antiapoptotic member of the Bcl-2 family that has evolved rapidly throughout the vertebrate lineage and which shows conserved abundant expression in eggs and oocytes. In this paper, we have unraveled two major sites of divergence between human Bcl2l10 and its vertebrate homologs. The first one provides length variation at the N-terminus (before the BH4 domain) and the second one is located between the predicted α5-α6 pore-forming helices, providing an unprecedented case in the superfamily of helix-bundled pore-forming proteins. These two particular indels were studied phylogenetically and through biochemical and cell biological techniques, including truncation and site-directed mutagenesis. While deletion of the N-terminal extension had no significant functional impact in HeLa cells, our results suggest that the human Bcl2l10 protein evolved a calcium-binding motif in its α5-α6 interhelical region by acquiring critical negatively charged residues. Considering the reliance of female eggs on calcium-dependent proteins and calcium-regulated processes and the exceptional longevity of oocytes in the primate lineage, we propose that this microstructural variation may be an adaptive feature associated with high maternal expression of this Bcl-2 family member.     

Impact of internal waves on the spatial distribution of Planktothrix rubescens (cyanobacteria) in an alpine lake

The vertical and horizontal distribution of the cyanobacterium, Planktothrix rubescens, was studied in a deep alpine lake (Lac du Bourget) in a 2-year monitoring program with 11 sampling points, and a 24-h survey at one sampling station. This species is known to proliferate in the metalimnic layer of numerous deep mesotrophic lakes in temperate areas, and also to produce hepatotoxins. When looking at the distribution of P. rubescens at the scale of the entire lake, we found large variations (up to 10 m) in the depth of the biomass peak in the water column. These variations were closely correlated to isotherm displacements. We also found significant variations in the distribution of the cyanobacterial biomass in the northern and southern parts of the lake. We used a physical modeling approach to demonstrate that two internal wave modes can explain these variations. Internal waves are generated by wind events, but can still be detected several days after the end of these events. Finally, our 24-h survey at one sampling point demonstrated that the V1H1 sinusoidal motion could evolve into nonlinear fronts. All these findings show that internal waves have a major impact on the distribution of P. rubescens proliferating in the metalimnic layer of a deep lake, and that this process could influence the growth of this species by a direct impact on light availability.     

RhoD regulates endosome dynamics through Diaphanous-related Formin and Src tyrosine kinase

Early endosomes move bidirectionally between the cell periphery and the interior through a mechanism regulated by the low molecular weight GTPase RhoD. Here, we identify a novel splice variant of human Diaphanous, hDia2C, which specifically binds to RhoD and is recruited onto early endosomes. Expression of RhoD and hDia2C induces a striking alignment of early endosomes along actin filaments and reduces their motility. This activity depends on the membrane recruitment and activation of c-Src kinase, thus uncovering a new role in endosome function. Our results define a novel signal transduction pathway, in which hDia2C and c-Src are sequentially activated by RhoD to regulate the motility of early endosomes through interactions with the actin cytoskeleton.     

Age-dependent changes in body size of Greek boys from 1982 to 2002

Objective: The prevalence of childhood obesity has been rising during the past decades in many parts of the world, including Greece. The dispersion of these trends across age, however, is less clear. The aim of this study was to examine the relationship between age and 20‐year changes in anthropometric characteristics of Greek boys. Research Methods and Procedures: A total of 204 and 106 boys 9 years old, 163 and 274 boys 12 years old, and 161 and 240 boys 15 years old were randomly recruited in 1982 and 2002, respectively, throughout the county of Iraklio, Crete, Greece. Height, weight, and BMI were measured. Results: Contemporary 9 and 12 year olds were taller than their peers in 1982 (+2.9% and +1.2%, respectively; p < 0.005), but this was not the case for 15 year olds (?0.8%; p = 0.083). Body weight and BMI were higher now than in the 1980s, and this held true for all age groups (p < 0.001). Increases in weight also showed a decline with advancing age (+17.4%, +13.9%, and +4.0% for 9, 12, and 15 year olds, respectively), whereas BMI changes were similar for those 9 and 12 years of age (～10.5%), but were almost 2‐fold higher than in 15 year olds (+5.5%). Discussion: Contemporary boys are taller, heavier, and have higher BMI values than their peers in 1982, but the magnitude of these increases gradually declines with advancing age. Rates of increase in BMI, however, seem to have greatly accelerated compared with previous decades.