Dynactin-dependent cortical dynein and spherical spindle shape correlate temporally with meiotic spindle rotation in Caenorhabditis elegans.

Oocyte meiotic spindles orient with one pole juxtaposed to the cortex to facilitate extrusion of chromosomes into polar bodies. In Caenorhabditis elegans, these acentriolar spindles initially orient parallel to the cortex and then rotate to the perpendicular orientation. To understand the mechanism of spindle rotation, we characterized events that correlated temporally with rotation, including shortening of the spindle in the pole-to pole axis, which resulted in a nearly spherical spindle at rotation. By analyzing large spindles of polyploid C. elegans and a related nematode species, we found that spindle rotation initiated at a defined spherical shape rather than at a defined spindle length. In addition, dynein accumulated on the cortex just before rotation, and microtubules grew from the spindle with plus ends outward during rotation. Dynactin depletion prevented accumulation of dynein on the cortex and prevented spindle rotation independently of effects on spindle shape. These results support a cortical pulling model in which spindle shape might facilitate rotation because a sphere can rotate without deforming the adjacent elastic cytoplasm. We also present evidence that activation of spindle rotation is promoted by dephosphorylation of the basic domain of p150 dynactin.

Free mycolic acid accumulation in the cell wall of the mce1 operon mutant strain of Mycobacterium tuberculosis.

The lipid-rich cell wall of Mycobacterium tuberculosis, the agent of tuberculosis, serves as an effective barrier against many chemotherapeutic agents and toxic host cell effector molecules, and it may contribute to the mechanism of persistence. Mycobacterium tuberculosis strains mutated in a 13-gene operon called mce1, which encodes a putative ABC lipid transporter, induce aberrant granulomatous response in mouse lungs. Because of the postulated role of the mce1 operon in lipid importation, we compared the cell wall lipid composition of wild type and mce1 operon mutant M. tuberculosis H37Rv strains. High resolution mass spectrometric analyses of the mce1 mutant lipid extracts showed unbound mycolic acids to accumulate in the cell wall. Quantitative analysis revealed a 10.7 fold greater amount of free mycolates in the mutant compared to that of the wild type strain. The free mycolates were comprised of alpha, methoxy and keto mycolates in the ratio 1:0.9:0.6, respectively. Since the mce1 operon is regulated in vivo, the free mycolates that accumulate during infection may serve as a barrier for M. tuberculosis against toxic products and contribute to the pathogen's persistence.

The bipolar assembly domain of the mitotic motor kinesin-5.

An outstanding unresolved question is how does the mitotic spindle utilize microtubules and mitotic motors to coordinate accurate chromosome segregation during mitosis? This process depends upon the mitotic motor, kinesin-5, whose unique bipolar architecture, with pairs of motor domains lying at opposite ends of a central rod, allows it to crosslink microtubules within the mitotic spindle and to coordinate their relative sliding during spindle assembly, maintenance and elongation. The structural basis of kinesin-5's bipolarity is, however, unknown, as protein asymmetry has so far precluded its crystallization. Here we use electron microscopy of single molecules of kinesin-5 and its subfragments, combined with hydrodynamic analysis plus mass spectrometry, circular dichroism and site-directed spin label electron paramagnetic resonance spectroscopy, to show how a staggered antiparallel coiled-coil 'BASS' (bipolar assembly) domain directs the assembly of four kinesin-5 polypeptides into bipolar minifilaments.

Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.

Preparation, separation, and conformational analysis of differentially sulfated heparin octasaccharide isomers using ion mobility mass spectrometry.

Heparin is a linear sulfated polysaccharide widely used in medicine because of its anticoagulant properties. The various sulfation and/or acetylation patterns on heparin impart different degrees of conformational change around the glycosidic bonds and subsequently alter its function as an anticoagulant, anticancer, or antiviral drug. Characterization of these structures is important for eventual elucidation of its function but presents itself as an analytical challenge due to the inherent heterogeneity of the carbohydrates. Heparin octasaccharide structural isomers of various sulfation patterns were investigated using ion mobility mass spectrometry (IMMS). In addition to distinguishing the isomers, we report the preparation and tandem mass spectrometry analysis for multiple sulfated or acetylated oligosaccharides. Herein, our data indicate that heparin octasaccharide isomers were separated on the basis of their structural conformations in the ion mobility cell. Subsequent to this separation, isomers were further distinguished using product ions resulting from tandem mass spectrometry. Overall, IMMS analysis was used to successfully characterize and separate individual isomers and subsequently measure their conformations.

Biologically Relevant Metal-Cation Binding Induces Conformational Changes in Heparin Oligosaccharides as Measured by Ion Mobility Mass Spectrometry.

Heparin interacts with many proteins and is involved in biological processes such as anticoagulation, angiogenesis, and antitumorigenic activities. These heparin-protein interactions can be influenced by the binding of various metal ions to these complexes. In particular, physiologically relevant metal cations influence heparin-protein conformations through electronic interactions inherent to this polyanion. In this study, we employed ion mobility mass spectrometry (IMMS) to observe conformational changes that occur in fully-sulfated heparin octasaccharides after the successive addition of metal ions. Our results indicate that binding of positive counter ions causes a decrease in collision cross section (CCS) measurements, thus promoting a more compact octasaccharide structure.

Identification of novel protein kinase A phosphorylation sites in the M-domain of human and murine cardiac myosin binding protein-C using mass spectrometry analysis.

Cardiac myosin binding protein-C (cMyBP-C) is a large multidomain accessory protein bound to myosin thick filaments in striated muscle sarcomeres. It plays an important role in the regulation of muscle contraction, and mutations in the gene encoding cMyBP-C are a common cause of familial hypertrophic cardiomyopathy, the leading cause of sudden cardiac death in young people. (1) The N-terminal domains including the C0, C1, cMyBP-C motif, and C2 domains play a crucial role in maintaining and modulating actomyosin interactions (keeping normal cardiac function) in a phosphorylation-dependent manner. The cMyBP-C motif or "M-domain" is a highly conserved linker domain in the N-terminus of cMyBP-C that contains three to five protein kinase A (PKA) phosphorylation sites, depending on species. For the human isoform, three PKA sites were previously identified (Ser(275), Ser(284), and Ser(304)), while three homologous sites exist in the murine isoform (Ser(273), Ser(282), and Ser(302)). The murine cMyBP-C isoform contains an additional conserved consensus site, Ser(307) that is not present in the human isoform. In this study, we investigated sites of PKA phosphorylation of murine and human cMyBP-C by treating the recombinant protein C0C2 ( approximately 50 KDa, which contains the N-terminal C0, C1, M, and C2 domains) and C1C2 (approximately 35 KDa, contains C1, M, and C2 domains) with PKA and assessing the phosphorylation states using SDS-PAGE with ProQ Diamond staining, and powerful hybrid mass spectrometric analyses. Both high-accuracy bottom-up and measurements of intact proteins mass spectrometric approaches were used to determine the phosphorylation states of C0C2 and C1C2 proteins with or without PKA treatment. Herein, we report for the first time that there are four PKA phosphorylation sites in both murine and human M-domains; both murine Ser(307) and a novel human Ser(311) can be phosphorylated in vitro by PKA. Future studies are needed to investigate the phosphorylation state of murine and human cMyBP-C in vivo.

Subunit architecture of multiprotein assemblies determined using restraints from gas-phase measurements.

Protein interaction networks are becoming an increasingly important area of research within structural genomics. Here we present an ion mobility-mass spectrometry approach capable of distinguishing the overall subunit architecture of protein complexes. The approach relies on the simultaneous measurement in the gas phase of the mass and size of intact assemblies and subcomplexes. These data are then used as restraints to generate topological models of protein complexes. To test and develop our method, we have chosen two well-characterized homo-dodecameric protein complexes: ornithine carbamoyl transferase and glutamine synthetase. By forming subcomplexes related to the comparative strength of the subunit interfaces, acquiring ion mobility data, and subsequent modeling, we show that these "building blocks" retain their native interactions and do not undergo major rearrangement in either solution or gas phases. We apply this approach to study two subcomplexes of the human eukaryotic initiation factor 3, for which there is no high-resolution structure.

NASA Radiation Biomarker Workshop, September 27-28, 2007.

A summary is provided of presentations and discussions at the NASA Radiation Biomarker Workshop held September 27-28, 2007 at NASA Ames Research Center in Mountain View, CA. Invited speakers were distinguished scientists representing key sectors of the radiation research community. Speakers addressed recent developments in the biomarker and biotechnology fields that may provide new opportunities for health-related assessment of radiation-exposed individuals, including those exposed during long-duration space travel. Topics discussed included the space radiation environment, biomarkers of radiation sensitivity and individual susceptibility, molecular signatures of low-dose responses, multivariate analysis of gene expression, biomarkers in biodefense, biomarkers in radiation oncology, biomarkers and triage after large-scale radiological incidents, integrated and multiple biomarker approaches, advances in whole-genome tiling arrays, advances in mass spectrometry proteomics, radiation biodosimetry for estimation of cancer risk in a rat skin model, and confounding factors. A summary of conclusions is provided at the end of the report.

Mass spectrometric kinetic analysis of human tyrosylprotein sulfotransferase-1 and -2.

Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, tyrosylprotein sulfotransferase-1 and -2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified tyrosylprotein sulfotransferase (TPST)-1 and -2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono- and disulfated products, and our results show that the K(m,app) for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.

Determination of the sites of tyrosine O-sulfation in peptides and proteins.

Tyrosine O-sulfation is a key post-translational modification that regulates protein-protein interactions in extracellular space. We describe a subtractive strategy to determine the sites of tyrosine O-sulfation in proteins. Hydroxyl groups on unsulfated tyrosines are blocked by stoichiometric acetylation in a one-step reaction using sulfosuccinimidyl acetate (S-NHSAc) in the presence of imidazole at pH 7.0. The presence of sulfotyrosine is indicated by the detection of free tyrosine after tandem mass spectrometry (MS/MS) analysis under conditions in which the sulfuryl group of sulfotyrosine is labile. Since phosphorylation and sulfation of tyrosine are isobaric, we used alkaline phosphatase treatment to distinguish these two modifications. Using this methodology we identified the sites and the order of sulfation of several peptides mediated by purified human tyrosylprotein sulfotransferases (TPSTs), and unambiguously determined the tyrosine sulfation sites in mouse lumican and human vitronectin.

Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5'-phosphosulfate reductase.

APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.

Effects of sulfate position on heparin octasaccharide binding to CCL2 examined by tandem mass spectrometry.

Chemokine-glycosaminoglycan (GAG) interactions have been shown to be essential for in vivo chemokine signaling, which functions in such diverse processes as inflammation, development, and cancer metastasis. Despite the importance of these interactions, the saccharide sequence dependency of chemokine-GAG interactions is poorly understood. In a recent study, FT-ICR mass spectrometry was used to show that the chemokine CCL2 (monocyte chemoattractant protein 1) binds only to the 11- and 12-sulfated components of a heparin octasaccharide library. Although the exact structure of the fully sulfated, 12-sulfated octasaccharide is known, the 11-sulfated species could have a number of sulfated disaccharide sequences. In the current study, the composition of the 11-sulfated heparin octasaccharides, as well as the composition of CCL2 affinity purified 11-sulfated heparin octasaccharides, were examined by tandem MS. Of the three possible singly desulfated disaccharides, one species, III-S, is enriched by CCL2 affinity purification, indicating that the 11-sulfated heparin octasaccharides containing this disaccharide are preferentially bound to CCL2. These data suggest that 2-O and N sulfation of heparin may be of greater importance to CCL2-heparin binding than 6-O sulfation.

Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: implications for substrate binding and catalysis.

The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.

A conserved mechanism for sulfonucleotide reduction.

Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor.

Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry.

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0-PAPS and Stf0-trehalose binary complexes, and a Stf0-3'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase.

An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach.

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay.

A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.

Stereoselective beta-Hydrogen Elimination from Nickel(II)-N-Glycoside Complexes.

Octahedral nickel(II)-N-glycoside complexes of glucose, galactose, mannose, and talose were synthesized and analyzed by electrospray ionization (ESI). A resulting tricoordinate species generated from the octahedral complex was subjected to collision-induced dissociation. A highly stereoselective dissociation pathway involving beta-hydrogen elimination and cross-ring cleavages was observed in complexes possessing equatorial C-2 substituents. (2)H- and (13)C-labeling experiments indicate that the hydrogen on C-2 and a labile proton are involved in the beta-hydrogen elimination. Additionally, C-4, C-5, and C-6 are shown to be lost from the monosaccharide as a result of the cross-ring cleavages. A mechanism is postulated to explain the stereoselectivity of this dissociation.