Acylcarnitine Abnormalities Implicate Mitochondrial Dysfunction in Patients With Neovascular Age-Related Macular Degeneration.

Purpose: Abnormalities in lipid metabolism are implicated in age-related macular degeneration (AMD), but the pathways involved remain unclear. We assessed whether acylcarnitine concentrations, a marker of lipid and mitochondrial metabolism, differed between patients with AMD and controls.

Methods: In this cross-sectional case-control study, cases (n = 81) had neovascular AMD and controls (n = 79) had cataract with no other ocular pathology.

An external quality assurance trial to assess mass spectrometry protein testing facilities for identifying multiple human peptides.

The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing.

Structural basis of antigen recognition: crystal structure of duck egg lysozyme.

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported.

A mutually assured destruction mechanism attenuates light signaling in Arabidopsis.

After light-induced nuclear translocation, phytochrome photoreceptors interact with and induce rapid phosphorylation and degradation of basic helix-loop-helix transcription factors, such as PHYTOCHROME-INTERACTING FACTOR 3 (PIF3), to regulate gene expression. Concomitantly, this interaction triggers feedback reduction of phytochrome B (phyB) levels. Light-induced phosphorylation of PIF3 is necessary for the degradation of both proteins.

Integrated community-healthcare diabetes interventions to reduce disparities.

Racial and ethnic minorities suffer disproportionately from diabetes-related morbidity and mortality. With the creation of Accountable Care Organizations (ACOs) under the Affordable Care Act, healthcare organizations may have an increased motivation to implement interventions that collaborate with community resources and organizations. As a result, there will be an increasing need for evidence-based strategies that integrate healthcare and community components to reduce diabetes disparities.

Expanding proteome coverage with orthogonal-specificity α-lytic proteases.

Bottom-up proteomics studies traditionally involve proteome digestion with a single protease, trypsin. However, trypsin alone does not generate peptides that encompass the entire proteome. Alternative proteases have been explored, but most have specificity for charged amino acid side chains.

Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood.

H/ACA small RNA dysfunctions in disease reveal key roles for noncoding RNA modifications in hematopoietic stem cell differentiation.

Noncoding RNAs control critical cellular processes, although their contribution to disease remains largely unexplored. Dyskerin associates with hundreds of H/ACA small RNAs to generate a multitude of functionally distinct ribonucleoproteins (RNPs). The DKC1 gene, encoding dyskerin, is mutated in the multisystem disorder X-linked dyskeratosis congenita (X-DC).

Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity.

Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs.

Global landscape of HIV-human protein complexes.

Human immunodeficiency virus (HIV) has a small genome and therefore relies heavily on the host cellular machinery to replicate. Identifying which host proteins and complexes come into physical contact with the viral proteins is crucial for a comprehensive understanding of how HIV rewires the host's cellular machinery during the course of infection. Here we report the use of affinity tagging and purification mass spectrometry to determine systematically the physical interactions of all 18 HIV-1 proteins and polyproteins with host proteins in two different human cell lines (HEK293 and Jurkat).

Proteomics analysis reveals post-translational mechanisms for cold-induced metabolic changes in Arabidopsis.

Cold-induced changes of gene expression and metabolism are critical for plants to survive freezing. Largely by changing gene expression, exposure to a period of non-freezing low temperatures increases plant tolerance to freezing-a phenomenon known as cold acclimation. Cold also induces rapid metabolic changes, which provide instant protection before temperature drops below freezing point.

Inflammatory stimuli regulate caspase substrate profiles.

The inflammatory caspases, human caspases-1, -4, and -5, proteolytically modulate diverse physiological outcomes in response to proinflammatory signals. Surprisingly, only a few substrates are known for these enzymes, including other caspases and the interleukin-1 family of cytokines. To more comprehensively characterize inflammatory caspase substrates, we combined an enzymatic N-terminal enrichment method with mass spectrometry-based proteomics to identify newly cleaved proteins.

High resolution electron transfer dissociation studies of unfractionated intact histones from murine embryonic stem cells using on-line capillary LC separation: determination of abundant histone isoforms and post-translational modifications.

Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed.

Nicotinic agonist binding site mapped by methionine- and tyrosine-scanning coupled with azidochloropyridinyl photoaffinity labeling.

Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tyrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.

Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: characterization of oxoammonium cation-induced modifications.

Spin scavenging combined with chromatographic and mass spectrometric procedures can, in principle, be employed to detect and identify protein-based radicals within complex biological matrices. This approach is based on the well-known ability of stable synthetic nitroxide radicals to scavenge carbon-centered radicals, forming stable diamagnetic addition products. Hence, characterization of these addition products would allow for the identification of specific free radicals.

Promiscuous aggregate-based inhibitors promote enzyme unfolding.

One of the leading sources of false positives in early drug discovery is the formation of organic small molecule aggregates, which inhibit enzymes nonspecifically at micromolar concentrations in aqueous solution. The molecular basis for this widespread problem remains hazy. To investigate the mechanism of inhibition at a molecular level, we determined changes in solvent accessibility that occur when an enzyme binds to an aggregate using hydrogen-deuterium exchange mass spectrometry.

Histone acetylation and chromatin remodeling are required for UV-B-dependent transcriptional activation of regulated genes in maize.

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B-tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B-sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors.

Comprehensive mechanistic analysis of hits from high-throughput and docking screens against beta-lactamase.

High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against beta-lactamase using quantitative HTS (qHTS).

Atypical nicotinic agonist bound conformations conferring subtype selectivity.

The nicotinic acetylcholine (ACh) receptor (nAChR) plays a crucial role in excitatory neurotransmission and is an important target for drugs and insecticides. Diverse nAChR subtypes with various subunit combinations confer differential selectivity for nicotinic drugs. We investigated the subtype selectivity of nAChR agonists by comparing two ACh-binding proteins (AChBPs) as structural surrogates with distinct pharmacological profiles [i.

Impaired dexamethasone-mediated induction of tryptophan 2,3-dioxygenase in heme-deficient rat hepatocytes: translational control by a hepatic eIF2alpha kinase, the heme-regulated inhibitor.

Tryptophan 2,3-dioxygenase (TDO), a liver-specific cytosolic hemoprotein, is the rate-limiting enzyme in L-tryptophan catabolism and thus a key serotonergic determinant. Glucocorticoids transcriptionally activate the TDO gene with marked enzyme induction. TDO is also regulated by heme, its prosthetic moiety, as its expression and function are significantly reduced after acute hepatic heme depletion.

Sulfopeptide fragmentation in electron-capture and electron-transfer dissociation.

Sulfopeptides can be misassigned as phosphopeptides because of the isobaric nature of the sulfo- and the phosphomoieties. Instruments having the ability to measure mass with high accuracy may be employed to distinguish these moieties based on their mass defect (the sulfo-group is 9 mmu lighter than the phosphomoiety). However, the assignment of the exact site(s) of post-translational modification is required to probe biological function.

Defining nicotinic agonist binding surfaces through photoaffinity labeling.

Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein.

Mapping the elusive neonicotinoid binding site.

Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid and thiacloprid, are nonprotonated and selective for insects and their nicotinic receptors, whereas nicotinoids such as nicotine and epibatidine are cationic and selective for mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), as a structural surrogate for the extracellular ligand-binding domain of the nicotinic receptor, is similarly sensitive to neonicotinoids and nicotinoids.

Dendritic iodinated contrast agents with PEG-cores for CT imaging: synthesis and preliminary characterization.

The purpose of this study was to design, synthesize, and initially characterize a representative set of novel constructs for large-molecular radiographic/computed tomography (CT) contrast agents, intended for a primarily intravascular distribution. A new assembly of well-known and biocompatible components consists of paired, symmetrical dendritic polylysines initiated from both ends of a poly(ethylene glycol) (PEG) core, yielding an array of multiple free amino groups to which were conjugated highly soluble and stable triiodophthalamide ("triiodo") moieties. An array of six dendritic contrast agents was synthesized originally, using three different PEG cores (3, 6, 12 kDa) with t-Boc lysine-generated dendrimer "amplifiers" (from three to five generations) containing 16 to 64 amino groups for conjugation with reactive triiodo moieties.