Characterizing the human intestinal chondroitin sulfate glycosaminoglycan sulfation signature in inflammatory bowel disease.

The intestinal extracellular matrix (ECM) helps maintain appropriate tissue barrier function and regulate host-microbial interactions. Chondroitin sulfate- and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs) are integral components of the intestinal ECM, and alterations in CS/DS-GAGs have been shown to significantly influence biological functions. Although pathologic ECM remodeling is implicated in inflammatory bowel disease (IBD), it is unknown whether changes in the intestinal CS/DS-GAG composition are also linked to IBD in humans. Our aim was to characterize changes in the intestinal ECM CS/DS-GAG composition in intestinal biopsy samples from patients with IBD using mass spectrometry. We characterized intestinal CS/DS-GAGs in 69 pediatric and young adult patients (n = 13 control, n = 32 active IBD, n = 24 IBD in remission) and 6 adult patients. Here, we report that patients with active IBD exhibit a significant decrease in the relative abundance of CS/DS isomers associated with matrix stability (CS-A and DS) compared to controls, while isomers implicated in matrix instability and inflammation (CS-C and CS-E) were significantly increased. This imbalance of intestinal CS/DS isomers was restored among patients in clinical remission. Moreover, the abundance of pro-stabilizing CS/DS isomers negatively correlated with clinical disease activity scores, whereas both pro-inflammatory CS-C and CS-E content positively correlated with disease activity scores. Thus, pediatric patients with active IBD exhibited increased pro-inflammatory and decreased pro-stabilizing CS/DS isomer composition, and future studies are needed to determine whether changes in the CS/DS-GAG composition play a pathogenic role in IBD.

Reversibility and Low Commitment to Forward Catalysis in the Conjugation of Lipid Alkenals by Glutathione Transferase A4-4.

High concentrations of electrophilic lipid alkenals formed during oxidative stress are implicated in cytotoxicity and disease. However, low concentrations of alkenals are required to induce antioxidative stress responses. An established clearance pathway for lipid alkenals includes conjugation to glutathione (GSH) via Michael addition, which is catalyzed mainly by glutathione transferase isoform A4 (GSTA4-4). Based on the ability of GSTs to catalyze hydrolysis or retro-Michael addition of GSH conjugates, and the antioxidant function of low concentrations of lipid alkenals, we hypothesize that GSTA4-4 contributes a homeostatic role in lipid metabolism. Enzymatic kinetic parameters for retro-Michael addition with trans-2-Nonenal (NE) reveal the chemical competence of GSTA4-4 in this putative role. The forward GSTA4-4-catalyzed Michael addition occurs with the rapid exchange of the C2 proton of NE in D2O as observed by NMR. The isotope exchange was completely dependent on the presence of GSH. The overall commitment to catalysis, or the ratio of first order kcat,f for 'forward' Michael addition to the first order kcat,ex for H/D exchange is remarkably low, approximately 3:1. This behavior is consistent with the possibility that GSTA4-4 is a regulatory enzyme that contributes to steady-state levels of lipid alkenals, rather than a strict 'one way' detoxication enzyme.

Probing the Stability of Proline Cis/Trans Isomers in the Gas Phase with Ultraviolet Photodissociation.

Although most peptide bonds in proteins exist in the trans configuration, when cis peptide bonds do occur, they can have major impact on protein structure and function. The rapid identification of cis peptide bonds is therefore an important task. Peptide bonds containing proline are more likely to adopt the cis configuration because the ring connecting the side chain and backbone in proline flattens the energetic landscape relative to amino acids with free side chains. Examples of cis proline isomers have been identified in both solution and in the gas phase by a variety of structure-probing methods. Mass spectrometry is an attractive potential method for identifying cis proline due to its speed and sensitivity; however, the question remains of whether cis/trans proline isomers originating in solution are preserved during ionization and manipulation within a mass spectrometer. Herein, we investigate the gas-phase stability of isolated solution-phase cis and trans proline isomers using a synthetic peptide sequence with a Tyr-Pro-Pro motif. A variety of dissociation methods were explored to evaluate their potential to distinguish cis/trans configuration, including collision-induced dissociation, radical-directed dissociation, and photodissociation. Only photodissociation employed in conjunction with extremely gentle electrospray and charge solvation by 18-crown-6 ether was able to distinguish cis/trans isomers for our model peptide, suggesting that any thermal activation during transfer or while in the gas phase leads to isomer scrambling. Furthermore, the necessity for 18-crown-6 suggests that intramolecular charge solvation taking place during electrospray ionization can override cis/trans isomer homogeneity. Overall, the results suggest that solution-phase cis/trans proline isomers are fragile and easily lost during electrospray, requiring careful selection of instrument parameters and consideration of charge solvation to prevent cis/trans scrambling.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

A Global Survey of ATPase Activity in Plasmodium falciparum Asexual Blood Stages and Gametocytes.

Effective malaria control and elimination in hyperendemic areas of the world will require treatment of the Plasmodium falciparum (Pf) blood stage that causes disease as well as the gametocyte stage that is required for transmission from humans to the mosquito vector. Most currently used therapies do not kill gametocytes, a highly specialized, non-replicating sexual parasite stage. Further confounding next generation drug development against Pf is the unknown metabolic state of the gametocyte and the lack of known biochemical activity for most parasite gene products in general. Here, we take a systematic activity-based proteomics approach to survey the activity of the large and druggable ATPase family in replicating blood stage asexual parasites and transmissible, non-replicating sexual gametocytes. ATPase activity broadly changes during the transition from asexual schizonts to sexual gametocytes, indicating altered metabolism and regulatory roles of ATPases specific for each lifecycle stage. We further experimentally confirm existing annotation and predict ATPase function for 38 uncharacterized proteins. By mapping the activity of ATPases associated with gametocytogenesis, we assign biochemical activity to a large number of uncharacterized proteins and identify new candidate transmission blocking targets.