Detection of two distinct forms of apoC-I in great apes

ApoC-I, the smallest of the soluble apolipoproteins, associates with both TG-rich lipoproteins and HDL. Mass spectral analyses of human apoC-I previously had demonstrated that in the circulation there are two forms, either a 57 amino acid protein or a 55 amino acid protein, due to the loss of two amino acids from the N-terminus. In our analyses of the apolipoproteins of the other great apes by mass spectrometry, four forms of apoC-I were detected. Two of these showed a high degree of identity to the mature and truncated forms of human apoC-I. The other two were homologous to the virtual protein and its truncated form that are encoded by a human pseudogene. In humans, the genes for apoC-I and its pseudogene are located on chromosome 19, the pseudogene being 2.5 kb downstream from the apoC-I gene. Based on the similarity between the apoC-I gene and the pseudogene, it has been concluded that the latter arose from the former as a result of gene duplication approximately 35 Mya. Interestingly, the virtual protein encoded by the pseudogene is acidic, not basic like apoC-I. In the chimpanzee, there also are two genes for apoC-I, the one upstream encodes a basic protein and the downstream gene, rather than being a pseudogene, encodes an acidic protein (P86336). In addition to reporting on the molecular masses of great ape apoC-I, we were able to clearly demonstrate by “Top-down” sequencing that the acidic form arose from a separate gene. In our analyses, we have measured the molecular masses of apoC-I associated with the HDL of the following great apes: bonobo (Pan paniscus), chimpanzee (Pan troglodytes), and the Sumatran orangutan (Pongo abelii). Genomic variations in chromosome 19 among great apes, baboons and macaques as they relate to both genes for apoC-I and the pseudogene are compared and discussed.     

Deconvolution and Database Search of Complex Tandem Mass Spectra of Intact Proteins: A COMBINATORIAL APPROACH*

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks. This raises both pattern recognition and combinatorial challenges for spectral deconvolution. We present MS-Deconv, a combinatorial algorithm for spectral deconvolution. The algorithm first generates a large set of candidate isotopomer envelopes for a spectrum, then represents the spectrum as a graph, and finally selects its highest scoring subset of envelopes as a heaviest path in the graph. In contrast with other approaches, the algorithm scores sets of envelopes rather than individual envelopes. We demonstrate that MS-Deconv improves on Thrash and Xtract in the number of correctly recovered monoisotopic masses and speed. We applied MS-Deconv to a large set of top-down spectra from Yersinia rohdei (with a still unsequenced genome) and further matched them against the protein database of related and sequenced bacterium Yersinia enterocolitica. MS-Deconv is available at http://proteomics.ucsd.edu/Software.html.Top-down proteomics is a mass spectrometry-based approach for identification of proteins and their post-translational modifications (PTMs)1 (1–14). Unlike the “bottom-up” approach where proteins are first digested into peptides and then a peptide mixture is analyzed by mass spectrometry, the top-down approach analyzes intact proteins. Thus, it has advantages in detecting and localizing PTMs as well as identifying multiple protein species (e.g. proteolytically processed protein species). Despite its advantages, top-down proteomics presents many challenges. These include requirement of high sample quantity, sophisticated instrumentation, protein separation, and robust computational analysis tools. For this reason, top-down proteomics has rarely been used for analyzing complex mixtures (12–18), and it is typically used to study single purified proteins. However, this situation is quickly changing with recent top-down studies of complex protein mixtures (14, 19).Because of the existence of natural isotopes, fragment ions of the same chemical formula and charge state are usually represented by a collection of spectral peaks in tandem mass spectra called an isotopomer envelope. The monoisotopic mass of a chemical formula is the sum of the masses of the atoms using the principal (most abundant) isotope for each element. Spectral deconvolution focuses on grouping spectral peaks into isotopomer envelopes. By doing so, the charge state and monoisotopic mass of each envelope are effectively determined. A complex multi-isotopic peak list in the m/z space is translated into a simple monoisotopic mass list that is easier to analyze.Given the monoisotopic mass and charge state of a fragment ion, its theoretical isotopic distribution can be predicted by assuming the fragment ion has an average elemental composition with respect to its mass (20) or using its precise elemental composition if the protein is known. Exploiting this, many deconvolution methods use theoretical isotopic distributions to detect and evaluate candidate isotopomer envelopes, which is the envelope detection problem (Fig. 1). To evaluate the fit of a candidate envelope to its theoretical isotopic distribution, many metrics have been proposed (20–32).Open in a separate windowFig. 1.Envelope detection. a, a theoretical isotopic distribution is predicted with the monoisotopic mass and charge state of a fragment ion. b, an observed envelope is detected by mapping peaks in the theoretical distribution to the spectrum. c, match between the theoretical isotopic distribution and the observed envelope. d, the theoretical isotopic distribution is scaled (the intensities of the peaks are multiplied by a constant) to have the best fit with the intensities of peaks in the observed envelope. Finally, a score for the observed envelope can be computed by comparing it with the intensity-scaled theoretical isotopic distribution.The candidate envelopes often overlap and share peaks, leading to a combinatorial problem of selecting the list of envelopes that best explains the spectrum (Fig. 2). In contrast to the well studied envelope detection problem, the envelope selection problem remains poorly explored. Most deconvolution algorithms follow a simple greedy approach to selecting the set of envelopes where the highest scoring envelopes are iteratively selected and removed from the spectrum. Although this approach often generates reasonable sets of envelopes for simple spectra, its performance deteriorates in cases of complex spectra.Open in a separate windowFig. 2.Envelope selection problem. Overlapping envelopes lead to a difficult combinatorial problem of selecting an optimal set of envelopes. We illustrate two cases where a deconvolution method that follows a greedy envelope selection outputs the envelope E₂, whereas the optimal solution consists of the envelopes E₁ and E₃. Example a illustrates the case where envelopes do not share peaks, and example b illustrates the case where envelopes share a spectral peak (E₁ and E₃).In particular, the greedy approach performs well when the envelopes are distributed sparsely along the m/z axis. Large proteins have many fragments that appear in multiple charge states. The high number of envelopes/peaks and the small m/z spread of the fragments with high charge states result in narrow m/z regions with high peak density. In these peak-dense regions, envelopes may overlap and share peaks, and the greedy approach and even manual interpretation often fail to find the optimal combination of envelopes (supplemental Fig. 1).Several methods have been proposed to explore the envelope selection problem. McIlwain et al. (33) presented a dynamic programming algorithm for selecting a set of envelopes such that the m/z ranges of the envelopes do not overlap. This non-overlapping condition becomes too restrictive for complex spectra of intact proteins. Samuelsson et al. (34) proposed a method that follows a non-negative sparse regression scheme. Du and Angeletti (35) and Renard et al. (36) addressed the envelope selection problem as a statistical problem of variable selection and used LASSO to solve it.Here, we present MS-Deconv, a combinatorial algorithm for spectral deconvolution. MS-Deconv (i) generates a large set of candidate envelopes, (ii) constructs an envelope graph encoding all envelopes and relationships between them, and (iii) finds a heaviest path in the envelope graph. Although the envelope graph of a complex spectrum is large (exceeding a million nodes in some cases), the heaviest path algorithm can efficiently find an optimal set of envelopes. MS-Deconv explicitly scores combinations of candidate envelopes rather than individual envelopes as in previous approaches.We tested MS-Deconv on a data set of top-down spectra from known proteins and evaluated the monoisotopic masses recovered by MS-Deconv. A mass was classified as a true positive if it was matched to the monoisotopic mass of a theoretical fragment ion of the protein within a specific parts per million (ppm) tolerance. We compared the performance of MS-Deconv with the widely used Thrash (20) and Xtract (37) and demonstrated that, with a few exceptions, MS-Deconv recovers more true positive masses. For example, for the collisionally activated dissociation (CAD) spectrum of bacteriorhodopsin (BR) with charge 10, the percentage of true positive masses among the top 150 masses is above 70% for MS-Deconv and less than 50% for Thrash. Additionally, MS-Deconv is ∼33 times faster than Thrash and 4 times faster than Xtract. Furthermore, MS-Deconv implements some user-friendly features: (i) outputs the set of peptide sequence tags, (ii) provides protein and spectral annotations, and (iii) allows one to inspect the recovered envelopes. We also tested MS-Deconv on a large LC-MS/MS data set from Yersinia rohdei (with a still unsequenced genome) (19). Y. rohdei is a non-pathogenic bacterium that is often used as a simulant for the potential bioterrorism agent Yersinia pestis, the causative agent of plague. We applied MS-Deconv to extract monoisotopic mass lists from top-down spectra and compared the mass lists with those reported by Thrash. We used ProSightPC (38) and the spectral alignment algorithm (39) to identify related proteins from a protein database of Yersinia enterocolitica (with a closely related and sequenced genome). The results demonstrated that MS-Deconv reported more matched fragments than Thrash for most proteins. Additionally, using spectral alignment, we identified eight proteins in Y. rohdei that were not reported in the ProSightPC-based searches (19) of the Y. enterocolitica protein database.     

Characteristics and programme-defined treatment outcomes among childhood tuberculosis (TB) patients under the national TB programme in Delhi

Background

Childhood tuberculosis (TB) patients under India''s Revised National TB Control Programme (RNTCP) are managed using diagnostic algorithms and directly observed treatment with intermittent thrice-weekly short-course treatment regimens for 6–8 months. The assignment into pre-treatment weight bands leads to drug doses (milligram per kilogram) that are lower than current World Health Organization (WHO) guidelines for some patients.

Objectives

The main aim of our study was to describe the baseline characteristics and treatment outcomes reported under RNTCP for registered childhood (age <15 years) TB patients in Delhi. Additionally, we compared the reported programmatic treatment completion rates between children treated as per WHO recommended anti-TB drug doses with those children treated with anti-TB drug doses below that recommended in WHO guidelines.

Methods

For this cross-sectional retrospective study, we reviewed programme records of all 1089 TB patients aged <15 years registered for TB treatment from January to June, 2008 in 6 randomly selected districts of Delhi. WHO disease classification and treatment outcome definitions are used by RNTCP, and these were extracted as reported in programme records.

Results and Conclusions

Among 1074 patients with records available, 651 (61%) were females, 122 (11%) were <5 years of age, 1000 (93%) were new cases, and 680 (63%) had extra-pulmonary TB (EP-TB)—most commonly peripheral lymph node disease [310 (46%)]. Among 394 pulmonary TB (PTB) cases, 165 (42%) were sputum smear-positive. The overall reported treatment completion rate was 95%. Similar reported treatment completion rates were found in all subgroups assessed, including those patients whose drug dosages were lower than that currently recommended by WHO. Further studies are needed to assess the reasons for the low proportion of under-5 years of age TB case notifications, address challenges in reaching all childhood TB patients by RNTCP, the accuracy of diagnosis, and the clinical validity of reported programme defined treatment completion.     

Cpd-1 Null Mice Display a Subtle Neurological Phenotype

Background

CPD1 (also known as ANP32-E) belongs to a family of evolutionarily conserved acidic proteins with leucine rich repeats implicated in a variety of cellular processes regulating gene expression, vesicular trafficking, intracellular signaling and apoptosis. Because of its spatiotemporal expression pattern, CPD1 has been proposed to play an important role in brain morphogenesis and synaptic development.

Methodology/Principal Findings

We have generated CPD1 knock-out mice that we have subsequently characterized. These mice are viable and fertile. However, they display a subtle neurological clasping phenotype and mild motor deficits.

Conclusions/Significance

CPD1 is not essential for normal development; however, it appears to play a role in the regulation of fine motor functions. The minimal phenotype suggests compensatory biological mechanisms.     

Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus

The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin.     

Nuclear localization and in situ DNA damage by Mycobacterium tuberculosis nucleoside-diphosphate kinase

Nucleoside-diphosphate kinase of Mycobacterium tuberculosis (mNdK) is a secretory protein, but the rationale behind secreting an enzyme involved in the maintenance of cellular pool of nucleoside triphosphates is not clearly understood. To elucidate the biological significance of mNdK secretion, we expressed mNdK fused to green fluorescent protein in HeLa and COS-1 cells. Interestingly, mNdK was detected in the nuclei of HeLa and COS-1 cells. Incubation of mNdK with nuclei isolated from HeLa and COS-1 cells led to in situ damage of chromosomal DNA. Surface plasmon resonance studies demonstrated that mNdK binds supercoiled plasmid DNA lacking apurinic/apyrimidinic sites with a dissociation constant of 30 +/- 3.2 mum. Plasmid cleavage by mNdK was found to be dependent on the specific divalent metal ion and inhibited by a metal ion chelator. Moreover, the metal ion-dependent DNA cleavage by mNdK was mediated by superoxide radicals as detected by electron paramagnetic resonance. The cleavage reaction was inhibited under nitrogen atmosphere confirming the necessity of molecular oxygen for DNA cleavage. In view of the findings that mNdK is secreted by intracellular mycobacteria and damages the nuclear DNA, it can be postulated that mNdK may cause cell death that could help in the dissemination of the pathogen.     

Characterization of a cytotoxic pilin subunit of Xenorhabdus nematophila

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.     

Free radical scavenging potential of Picrorhiza kurrooa Royle ex Benth

For assessing free radical scavenging potential of P. kurrooa, the antioxidant activity of P. kurrooa extract was studied by lipid peroxidation assay using rat liver homogenate. The extract (1 mg/ml) showed marked protection (up to 66.68%) against peroxidation of liver phospholipids. Besides, reduced glutathione showed very encouraging activity. The extract also exhibited significant scavenging activity. Thus augmenting the wide use of plant in the indigenous system of medicine, which may partly be due to antioxidant and free radical scavening activity of the extract.     

The role of glycosphingolipids in HIV signaling, entry and pathogenesis

Although HIV uses CD4 and coreceptors (CCR5 and CXCR4) for productive infection of T cells, glycosphingolipids (GSL) may play ancillary roles in lymphoid and non-lymphoid cells. Interactions of the HIV Envelope Glycoprotein (Env) with GSL may help HIV in various steps of its pathogenesis. Physical-chemical aspects of the interactions between HIV Env and GSL leading to CD4-dependent entry into lymphocytes, the role of GSL in HIV transcytosis, and CD4-independent entry into non-lymphoid cells are reviewed. An overview of signaling properties of HIV receptors is provided with some speculation on how GSL may play a role in these events by virtue of being in membrane rafts. Finally, we summarize how interactions between HIV and coreceptors leading to signaling and/or fusion can be analyzed by the use of various tyrosine kinase and cytoskeletal inhibitors. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.