Decoding protein modifications using top-down mass spectrometry

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.     

Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide

Phosphinothricin tripeptide (PTT, phosphinothricylalanylalanine) is a natural-product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 (ref. 1) and Streptomyces viridochromogenes DSM 40736 (ref. 2). PTT has attracted widespread interest because of its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see ref. 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of catalytic steps that to our knowledge has not been described so far, including a highly unusual reaction for carbon bond cleavage. In sum, we define a pathway for early PTT biosynthesis that is more complex than previously appreciated.     

ProSight Annotator: Complete control and customization of protein entries in UniProt XML files

The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu .     

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.     

Agonist-independent, high constitutive activity of the human melanocortin 1 receptor

The melanocortins (alpha-melanocyte-stimulating hormone and adrenocorticotropin) act on epidermal melanocytes to increase melanogenesis, the eumelanin/pheomelanin ratio and dendricity. These actions are mediated by the heptahelical melanocortin 1 receptor (MC1R), positively coupled to adenylyl cyclase. Gain-of-function mouse Mc1r alleles are associated with a dark, eumelanic coat. Conversely, loss-of-function variants, or overexpression of agouti, a natural melanocortin antagonist, yield yellow, pheomelanic furs. In humans, loss-of-function MC1R variants are associated with fair skin, poor tanning, propensity to freckle and increased skin cancer risk. Therefore, MC1R is a key regulator of mammalian pigmentation. Several observations such as induction of constitutive pigmentation in amelanotic mouse melanoma cells following expression of MC1R indicate that the receptor might display agonist-independent activity. We report a systematic and comparative study of MC1R and Mc1r constitutive activity. We show that expression of MC1R in heterologous systems leads to an agonist-independent increase in cyclic adenosine monophophate (cAMP). Basal signalling is a function of receptor expression and is two to fourfold higher for MC1R than for Mc1r. Moreover, it is observed in human melanoma cells over-expressing the MC1R. Constitutive signalling is abolished or reduced by point mutations of MC1R impairing the response to agonists, and is only doubled by the Lys94Glu mutation, mimicking the constitutively active mouse E(so-3J) allele. Stable or transient expression of wild-type MC1R, but not of loss-of-function mutants, potently stimulates forskolin activation of adenylyl cyclase, a common feature of constitutively active Gs-coupled receptors. Therefore, human MC1R displays a strong agonist-independent constitutive activity.     

Comparison of effects of exercise and diuretic on left ventricular geometry, mass, and insulin resistance in older hypertensive adults

To compare the effects of exercise training and hydrochlorothiazide on left ventricular (LV) geometry and mass, blood pressure (BP), and hyperinsulinemia in older hypertensive adults, we studied 28 patients randomized either to a group (age 66.4 +/- 1.3 yr; n = 16) that exercised or to a group (age 65.3 +/- 1.2 yr; n = 12) that received hydrochlorothiazide for 6 mo. Endurance exercise training induced a 15% increase in peak aerobic power. The reduction in systolic BP was twofold greater with thiazide than with exercise (26.6 +/- 12.2 vs. 11.5 +/- 10.9 mmHg). Exercise and thiazide reduced LV wall thickness, LV mass index (14% in each group), and the LV wall thickness-to-radius ratio (h/r) similarly (exercise: before 0.48 +/- 0.2, after 0.42 +/- 0.01; thiazide: before 0.47 +/- 0.04, after 0.40 +/- 0.04; P = 0.017). The reductions in systolic BP and h/r were correlated in the exercise group (r = 0.70, P = 0.005) but not in the thiazide group. Exercise training reduced glucose-stimulated hyperinsulinemia (before: 13.65 +/- 2.6 vs. 9.84 +/- 1.5 mU.ml(-1).min; P = 0.04) and insulin resistance. Thiazide did not affect plasma insulin levels. The results suggest that although exercise is less effective in reducing systolic BP than thiazide, it can induce regression of LV hypertrophy similar in magnitude to thiazide. Unlike hydrochlorothiazide, exercise training can improve insulin resistance and aerobic capacity in older hypertensive people.     

Antioxidant mechanisms of polyphenolic caffeic acid oligomers, constituents of Salvia officinalis

Caffeic acid, rosmarinic acid and oligomers of caffeic acid with multiple catechol groups are all constituents of Salvia officinalis. Their antioxidant potential was investigated with regard to their radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic studies revealed very high rate constants with hydroxyl radicals. Evidence from kinetic modeling calculations suggested an unusual complex behavior due to the presence of both O4- and O3-semiquinones and formation and decay of a hydroxyl radical adduct at the vinyl side chain. The radical structures observed by EPR spectroscopy after autoxidation in slightly alkaline solutions were only partially identified due to their instability and generally represented dissociated O4-semiquinones. Hybrid density-functional calculations of the potential radical structures showed distinct differences between the resonance stabilization of the O4- and O3-semiquinones of caffeic and dihydrocaffeic acids, reflected also in the considerably faster decay of the O3-semiquinone observed by pulse radiolysis. No evidence was found for dimerization reactions via Cbeta radicals typical for lignin biosynthesis.