Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.     

Mitochondrial DNA in metazoa: degree of freedom in a frozen event

The mitochondrial genome (mtDNA), due to its peculiar features such as exclusive presence of orthologous genes, uniparental inheritance, lack of recombination, small size and constant gene content, certainly represents a major model system in studies on evolutionary genomics in metazoan. In 800 million years of evolution the gene content of metazoan mitochondrial genomes has remained practically frozen but several evolutionary processes have taken place. These processes, reviewed here, include rearrangements of gene order, changes in base composition and arising of compositional asymmetry between the two strands, variations in the genetic code and evolution of codon usage, lineage-specific nucleotide substitution rates and evolutionary patterns of mtDNA control regions.     

A high-throughput method for liquid chromatography–tandem mass spectrometry determination of plasma alkylresorcinols,biomarkers of whole grain wheat and rye intake

Plasma alkylresorcinols are increasingly analyzed in cohort studies to improve estimates of whole grain intake and their relationship with disease incidence. Current methods require large volumes of solvent (>10 ml/sample) and have relatively low daily sample throughput. We tested five different supported extraction methods for extracting alkylresorcinols from plasma and improved a normal-phase liquid chromatography coupled to a tandem mass spectrometer method to reduce sample analysis time. The method was validated and compared with gas chromatography–mass spectrometry analysis. Sample preparation with HybridSPE supported extraction was most effective for alkylresorcinol extraction, with recoveries of 77–82% from 100 μl of plasma. The use of 96-well plates allowed extraction of 160 samples per day. Using a 5-cm NH₂ column and heptane reduced run times to 3 min. The new method had a limit of detection and limit of quantification equivalent to 1.1–1.8 nmol/L and 3.5–6.1 nmol/L plasma, respectively, for the different alkylresorcinol homologues. Accuracy was 93–105%, and intra- and inter-batch precision values were 4–18% across different plasma concentrations. This method makes it possible to quantify plasma alkylresorcinols in 100 μl of plasma at a rate of at least 160 samples per day without the need for large volumes of organic solvents.     

Cell surface orifices of caveolae and localization of caveolin to the necks of caveolae in adipocytes

Caveolae are noncoated invaginations of the plasma membrane that form in the presence of the protein caveolin. Caveolae are found in most cells, but are especially abundant in adipocytes. By high-resolution electron microscopy of plasma membrane sheets the detailed structure of individual caveolae of primary rat adipocytes was examined. Caveolin-1 and -2 binding was restricted to the membrane proximal region, such as the ducts or necks attaching the caveolar bulb to the membrane. This was confirmed by transfection with myc-tagged caveolin-1 and -2. Essentially the same results were obtained with human fibroblasts. Hence caveolin does not form the caveolar bulb in these cells, but rather the neck and may thus act to retain the caveolar constituents, indicating how caveolin participates in the formation of caveolae. Caveolae, randomly distributed over the plasma membrane, were very heterogeneous, varying in size between 25 and 150 nm. There was about one million caveolae in an adipocyte, which increased the surface area of the plasma membrane by 50%. Half of the caveolae, those larger than 50 nm, had access to the outside of the cell via ducts and 20-nm orifices at the cell surface. The rest of the caveolae, those smaller than 50 nm, were not open to the cell exterior. Cholesterol depletion destroyed both caveolae and the cell surface orifices.     

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.     

Repeated labilization-reconsolidation processes strengthen declarative memory in humans

The idea that memories are immutable after consolidation has been challenged. Several reports have shown that after the presentation of a specific reminder, reactivated old memories become labile and again susceptible to amnesic agents. Such vulnerability diminishes with the progress of time and implies a re-stabilization phase, usually referred to as reconsolidation. To date, the main findings describe the mechanisms associated with the labilization-reconsolidation process, but little is known about its functionality from a biological standpoint. Indeed, two functions have been proposed. One suggests that destabilization of the original memory after the reminder allows the integration of new information into the background of the original memory (memory updating), and the other suggests that the labilization-reconsolidation process strengthens the original memory (memory strengthening). We have previously reported the reconsolidation of human declarative memories, demonstrating memory updating in the framework of reconsolidation. Here we deal with the strengthening function attributed to the reconsolidation process. We triggered labilization-reconsolidation processes successively by repeated presentations of the proper reminder. Participants learned an association between five cue-syllables and their respective response-syllables. Twenty-four hours later, the paired-associate verbal memory was labilized by exposing the subjects to one, two or four reminders. The List-memory was evaluated on Day 3 showing that the memory was improved when at least a second reminder was presented in the time window of the first labilization-reconsolidation process prompted by the earlier reminder. However, the improvement effect was revealed on Day 3, only when at least two reminders were presented on Day 2 and not as a consequence of only retrieval. Therefore, we propose central concepts for the reconsolidation process, emphasizing its biological role and the parametrical constrains for this function to be operative.     

Proteome profile of human urine with two-dimensional liquid phase fractionation

Two-dimensional liquid chromatography separation (2-DL), based on chromatofocusing for first dimension and hydrophobicity for second, can be used as a complementary method to two-dimensional gel electrophoresis (2-DE). A platform now available, ProteomeLab PF 2D provided by Beckman Coulter, (Fullerton, CA, USA), assembles these methods in automation. This system was applied to resolve large numbers of urine proteins. Reproducibility and sensitivity in protein resolution were evaluated in this study using urines collected from male blood donors. About 1000 peaks were detected at a pH range of 4.0-8.5 by applying 1 mg of proteins. Furthermore, the same fractions showing peaks with high absorbance intensities in second dimension were collected and subjected to matrix-assisted laser desorption/ionization-time of flight/mass spectrometry analysis for identification. The results showed that the 2-DL provides high reproducibility of two-dimensional protein map, and lends fractions to subsequent mass spectrometry analysis without the further need for extraction or solubilization of samples as required for spots excised from 2-DE gels. In addition, this system also allows to separate particularly proteins with 40-9 kDa molecular weight.     

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.