Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.     

Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.     

Sterile Immunity to Malaria after DNA Prime/Adenovirus Boost Immunization Is Associated with Effector Memory CD8+T Cells Targeting AMA1 Class I Epitopes

Background

Fifteen volunteers were immunized with three doses of plasmid DNA encoding P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1) and boosted with human adenovirus-5 (Ad) expressing the same antigens (DNA/Ad). Four volunteers (27%) demonstrated sterile immunity to controlled human malaria infection and, overall, protection was statistically significantly associated with ELISpot and CD8+ T cell IFN-γ activities to AMA1 but not CSP. DNA priming was required for protection, as 18 additional subjects immunized with Ad alone (AdCA) did not develop sterile protection.

Methodology/Principal Findings

We sought to identify correlates of protection, recognizing that DNA-priming may induce different responses than AdCA alone. Among protected volunteers, two and three had higher ELISpot and CD8+ T cell IFN-γ responses to CSP and AMA1, respectively, than non-protected volunteers. Unexpectedly, non-protected volunteers in the AdCA trial showed ELISpot and CD8+ T cell IFN-γ responses to AMA1 equal to or higher than the protected volunteers. T cell functionality assessed by intracellular cytokine staining for IFN-γ, TNF-α and IL-2 likewise did not distinguish protected from non-protected volunteers across both trials. However, three of the four protected volunteers showed higher effector to central memory CD8+ T cell ratios to AMA1, and one of these to CSP, than non-protected volunteers for both antigens. These responses were focused on discrete regions of CSP and AMA1. Class I epitopes restricted by A*03 or B*58 supertypes within these regions of AMA1 strongly recalled responses in three of four protected volunteers. We hypothesize that vaccine-induced effector memory CD8+ T cells recognizing a single class I epitope can confer sterile immunity to P. falciparum in humans.

Conclusions/Significance

We suggest that better understanding of which epitopes within malaria antigens can confer sterile immunity and design of vaccine approaches that elicit responses to these epitopes will increase the potency of next generation gene-based vaccines.     

Open Source Tracking and Analysis of Adult Drosophila Locomotion in Buridan's Paradigm with and without Visual Targets

Background

Insects have been among the most widely used model systems for studying the control of locomotion by nervous systems. In Drosophila, we implemented a simple test for locomotion: in Buridan''s paradigm, flies walk back and forth between two inaccessible visual targets [1]. Until today, the lack of easily accessible tools for tracking the fly position and analyzing its trajectory has probably contributed to the slow acceptance of Buridan''s paradigm.

Methodology/Principal Findings

We present here a package of open source software designed to track a single animal walking in a homogenous environment (Buritrack) and to analyze its trajectory. The Centroid Trajectory Analysis (CeTrAn) software is coded in the open source statistics project R. It extracts eleven metrics and includes correlation analyses and a Principal Components Analysis (PCA). It was designed to be easily customized to personal requirements. In combination with inexpensive hardware, these tools can readily be used for teaching and research purposes. We demonstrate the capabilities of our package by measuring the locomotor behavior of adult Drosophila melanogaster (whose wings were clipped), either in the presence or in the absence of visual targets, and comparing the latter to different computer-generated data. The analysis of the trajectories confirms that flies are centrophobic and shows that inaccessible visual targets can alter the orientation of the flies without changing their overall patterns of activity.

Conclusions/Significance

Using computer generated data, the analysis software was tested, and chance values for some metrics (as well as chance value for their correlation) were set. Our results prompt the hypothesis that fixation behavior is observed only if negative phototaxis can overcome the propensity of the flies to avoid the center of the platform. Together with our companion paper, we provide new tools to promote Open Science as well as the collection and analysis of digital behavioral data.     

Lymphocytic choriomeningitis virus infection yields overlapping CD4+ and CD8+ T-cell responses

Activation of CD4⁺ T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4⁺ T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2^d. This is quite disparate to the H-2^b setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2^d or whether additional CD4⁺ T-cell epitopes could be identified in the setting of the H-2^b background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4⁺ T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4⁺ epitopes, four of them also stimulate CD8⁺ T cells in a statistically significant manner. Furthermore, we assessed these CD4⁺ T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4⁺ and CD8⁺ T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.     

Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance

The primary CD8(+) T cell response of C57BL/6J mice against the 28 known epitopes of lymphocytic choriomeningitis virus (LCMV) is associated with a clear immunodominance hierarchy whose mechanism has yet to be defined. To evaluate the role of epitope competition in immunodominance, we manipulated the number of CD8(+) T cell epitopes that could be recognized during LCMV infection. Decreasing epitope numbers, using a viral variant lacking dominant epitopes or C57BL/6J mice lacking H-2K(b), resulted in minor response increases for the remaining epitopes and no new epitopes being recognized. Increasing epitope numbers by using F(1) hybrid mice, delivery by recombinant vaccinia virus, or epitope delivery as a pool in IFA maintained the overall response pattern; however, changes in the hierarchy did become apparent. MHC binding affinity of these epitopes was measured and was found to not strictly predict the hierarchy since in several cases similarly high binding affinities were associated with differences in immunodominance. In these instances the naive CD8(+) T cell precursor frequency, directly measured by tetramer staining, correlated with the response hierarchy seen after LCMV infection. Finally, we investigated an escape mutant of the dominant GP33-41 epitope that elicited a weak response following LCMV variant virus infection. Strikingly, dominance loss likely reflects a substantial reduction in frequencies of naive precursors specific for this epitope. Thus, our results indicate that an intrinsic property of the epitope (MHC binding affinity) and an intrinsic property of the host (naive precursor frequency) jointly dictate the immunodominance hierarchy of CD8(+) T cell responses.     

The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.     

A comparison of random forest and its Gini importance with standard chemometric methods for the feature selection and classification of spectral data

Background  

Regularized regression methods such as principal component or partial least squares regression perform well in learning tasks on high dimensional spectral data, but cannot explicitly eliminate irrelevant features. The random forest classifier with its associated Gini feature importance, on the other hand, allows for an explicit feature elimination, but may not be optimally adapted to spectral data due to the topology of its constituent classification trees which are based on orthogonal splits in feature space.     

Elastase Digests: New Ammunition for Shotgun Membrane Proteomics

Despite many advances in membrane proteomics during the last decade the fundamental problem of accessing the transmembrane regions itself has only been addressed to some extent. The present study establishes a method for the nano-LC-based analysis of complex membrane proteomes on the basis of a methanolic porcine pancreatic elastase digest to increase transmembrane coverage. Halobacterium salinarium purple and Corynebacterium glutamicum membranes were successfully analyzed by using the new protocol. We demonstrated that elastase digests yield a large proportion of transmembrane peptides, facilitating membrane protein identification. The potential for characterization of a membrane protein through full sequence coverage using elastase is there but is restricted to the higher abundance protein components. Compatibility of the work flow with the two most common mass spectrometric ionization techniques, ESI and MALDI, was shown. Currently better results are obtained using ESI mainly because of the low response of MALDI for strictly neutral peptides. New findings concerning elastase specificity in complex protein mixtures reveal a new prospect beyond the application in shotgun experiments. Furthermore peptide mass fingerprinting with less specific enzymes might be done in the near future but requires an adaptation of current search algorithms to the new proteases.Upon the introduction of modern mass spectrometric ionization techniques, such as MALDI (1) and ESI (2), extremely powerful and valuable tools were given to researchers for the identification and characterization of proteins. Nevertheless the intricate analysis of membrane subproteomes still represents one of the major challenges despite the amount of “success” reported in the literature. Until now, there was no general protocol available to address membrane proteomes as a whole, demonstrating their degree of difficulty and complexity.During the past years, two different strategies in proteome analysis have evolved: the more widespread bottom-up and top-down proteomics. The latter approach has been shown to provide access to the transmembranal regions of membrane proteins and has the power to characterize complete protein primary structures including labile covalent modifications (3, 4). Nevertheless there are limitations due to sample complexity, emphasizing the need for a liquid chromatographic separation on the protein level, which is comparable to LC peptide separation. Therefore, the bottom-up variant is the most commonly used method for the proteomics analysis of complex membrane samples. Improvements in the bottom-up work flow were achieved by adapting sample cleanup and prefractionation processes (5–10) and subsequently by the development of modified and optimized separation techniques. The two main work flows, the gel-based approach mainly carried out with 2D¹ SDS-PAGE (11, 12) and the shotgun identification of proteolytically digested protein mixtures and their multidimensional separation via liquid chromatography (13), had to be adapted. The separation of proteins via classical 2D SDS-PAGE is only possible up to a GRAVY score of ∼0.4 (14, 15). Despite lacking the separation power of the IEF-SDS-PAGE system, derived techniques like the doubled SDS- (16) and 16-benzyldimethyl-n-hexadecylammonium chloride/SDS-PAGE (17) represent an improvement for hydrophobic proteins. Advances in the nLC separation of hydrophobic peptides, e.g. the separation at elevated temperatures (18) and the use of LC-compatible detergents (19), yielded significant success. When combining both prominent separation techniques, the one-dimensional SDS-LC analysis proved to be more effective than 2D PAGE as well (20, 21).One of the remaining steps still offering room for improvements is the proteolytic procedure. The original multidimensional protein identification technique using trypsin has been successfully improved by the application of proteinase K under high pH conditions, yielding an increased number of membrane protein identifications (22). Further modification of this method by recleaving the isolated membranous parts with cyanogen bromide increased the accessibility to membrane-spanning peptides (18).Despite the suitability of proteinase K for the shotgun analysis of membrane proteomes, trypsin is the prevalent enzyme choice in most current proteomics approaches because of its very specific cleavage behavior. Calculations have implied that alternative proteases are preferentially suited for the analysis of membrane proteomes (23). Proteases other than trypsin have been utilized only to a small degree and mostly for the targeted analysis of protein complexes. Pepsin, for example, was used for the characterization of an aquaporin (24), and elastase and subtilisin were used as proteases in a triple digest approach of protein complexes and lens tissue (25). Further improvements in the accessibility of the membrane proteome have been shown for tryptic (26, 27) and tryptic/chymotryptic (28) digests by performing the proteolytic treatment in the presence of methanol.During membrane proteomics method development, purple membranes from Halobacterium sp. NRC-1, consisting to a large extent of the H⁺-ATPase bacteriorhodopsin, have widely been used as reference in a variety of cases. Full BR sequence coverage has been reported by several publications using different techniques (27, 29). Such exorbitant sequence coverage amounts are only achievable for the most abundant proteins within a complex mixture. The identification of less expressed proteins, which certainly are more in the researcher''s interest, occurs via lower peptide numbers (27).Generally the basic tryptic cleavage sites are predominantly located in the loops facing the cytoplasm and to a lesser extent in the extracellular or periplasmic loops but are very scarce within the transmembrane helix stretches. This mostly limits the potential tryptic fragments to loop components and larger peptides containing at least one TM helix. The use of another protease, preferentially cleaving after neutral aliphatic residues, should not succumb to this lack of cleavage sites.One of the previously mentioned proteases, porcine pancreatic elastase, was reported to possess potential cleavage specificity at the carboxyl-terminal side of small neutral amino acids (30). It has been used previously for the examination of single protein phosphorylations (31) but was described not to be suited for the cleavage of complex membrane-containing samples because of a supposedly limited activity when applied to them (22). Despite this finding, elastase has been successfully utilized in a mass spectrometric analysis as early as 1974 when it was regarded as an ideal protease for future mass spectrometric studies (32). At the same time, most of the experiments to characterize elastase by analyzing its S₁ pocket (for nomenclature, see Ref. 33) binding capabilities (34) as well as cleavage preferences of ester and protein substrates (35–39) were performed.Based on previous research concerning elastase, we set up an nLC-based membrane proteomics analysis. This large data set was used to characterize the protease behavior of elastase and the physicochemical properties of the detected peptides. The PM model was used to establish a method to analyze more complex Corynebacterium glutamicum membranes, which have been analyzed previously using different 2D techniques (7, 28). We demonstrated that a combination of elastase and methanol is particularly suitable for an nLC-based membrane proteome analysis and results in a significantly increased number of TM peptides. Additionally the promising PMF application of elastase digests is discussed.