Cysteine Reactivity Profiling to Unveil Redox Regulation in Phytopathogens.

Reactivity-based chemical proteomics is a powerful technology based on the use of tagged chemicals that covalently react with surface-exposed residues on proteins in native proteomes. Reactivity profiling involves the purification, identification, and quantification of labeled peptides by LC-MS/MS. Here, we have detailed a protocol for reactivity profiling of Cys residues using iodoacetamide probes, displaying >1000 reactive Cys residues in the proteome of phytopathogen Pseudomonas syringae pv. tomato DC3000 (PtoDC3000). Comparative reactivity profiling of PtoDC3000 treated with or without hydrogen peroxide (H2O2) identified ~200 H2O2-sensitive Cys residues in antioxidant enzymes, metabolic enzymes, and transcription regulators. Interestingly, half of these H2O2-sensitive Cys residues are more reactive in response to H2O2 and several proteins have multiple Cys residues with opposite reactivities in response to H2O2 exposure.

Quantitative Phosphoproteomics to Study cAMP Signaling.

Cyclic adenosine monophosphate (cAMP) signaling activates multiple downstream cellular targets in response to different stimuli. Specific phosphorylation of key target proteins via activation of the cAMP effector protein kinase A (PKA) is achieved via signal compartmentalization. Termination of the cAMP signal is mediated by phosphodiesterases (PDEs), a diverse group of enzymes comprising several families that localize to distinct cellular compartments. By studying the effects of inhibiting individual PDE families on the phosphorylation of specific targets it is possible to gain information on the subcellular spatial organization of this signaling pathway.We describe a phosphoproteomic approach that can detect PDE family-specific phosphorylation changes in cardiac myocytes against a high phosphorylation background. The method combines dimethyl labeling and titanium dioxide-mediated phosphopeptide enrichment, followed by tandem mass spectrometry.

LRRK2 Is Recruited to Phagosomes and Co-recruits RAB8 and RAB10 in Human Pluripotent Stem Cell-Derived Macrophages.

The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ "maturing" phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.

Triazine Probes Target Ascorbate Peroxidases in Plants.

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), liverwort (Marchantia polymorpha), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in apx1 mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.

Genetic Incorporation of Olefin Cross-Metathesis Reaction Tags for Protein Modification.

Olefin cross-metathesis (CM) is a viable reaction for the modification of alkene-containing proteins. Although allyl sulfide or selenide side-chain motifs in proteins can critically enhance the rate of CM reactions, no efficient method for their site-selective genetic incorporation into proteins has been reported to date. Here, through the systematic evaluation of olefin-bearing unnatural amino acids for their metabolic incorporation, we have discovered S-allylhomocysteine (Ahc) as a genetically encodable Met analogue that is not only processed by translational cellular machinery but also a privileged CM substrate residue in proteins. In this way, Ahc was used for efficient Met codon reassignment in a Met-auxotrophic strain of E. coli (B834 (DE3)) as well as metabolic labeling of protein in human cells and was reactive toward CM in several representative proteins. This expands the use of CM in the toolkit for "tag-and-modify" functionalization of proteins.

Reduction of leucocyte cell surface disulfide bonds during immune activation is dynamic as revealed by a quantitative proteomics workflow (SH-IQ).

Communication through cell surface receptors is crucial for maintaining immune homeostasis, coordinating the immune response and pathogen clearance. This is dependent on the interaction of cell surface receptors with their ligands and requires functionally active conformational states. Thus, immune cell function can be controlled by modulating the structure of either the receptor or the ligand. Reductive cleavage of labile disulfide bonds can mediate such an allosteric change, resulting in modulation of the immune system by a hitherto little studied mechanism. Identifying proteins with labile disulfide bonds and determining the extent of reduction is crucial in elucidating the functional result of reduction. We describe a mass spectrometry-based method-thiol identification and quantitation (SH-IQ)-to identify, quantify and monitor such reduction of labile disulfide bonds in primary cells during immune activation. These results provide the first insight into the extent and dynamics of labile disulfide bond reduction in leucocyte cell surface proteins upon immune activation. We show that this process is thiol oxidoreductase-dependent and mainly affects activatory (e.g. CD132, SLAMF1) and adhesion (CD44, ICAM1) molecules, suggesting a mechanism to prevent over-activation of the immune system and excessive accumulation of leucocytes at sites of inflammation.

Purification and Proteomics of Influenza Virions.

This chapter describes a basic workflow for analyzing the protein composition of influenza virions. In order to obtain suitable material, the chapter describes how to concentrate influenza virions from the growth media of infected cells and to purify them by ultracentrifugation through a density gradient. This approach is also suitable for purifying influenza virions from the allantoic fluid of embryonated chicken eggs. As a small quantity of microvesicles are co-purified with virions, optional steps are included to increase the stringency of purification by enriching material with viral receptor binding and cleaving activity. Material purified in this way can be used for a variety of downstream applications, including proteomics. As a detailed example of this, the chapter also describes a standard workflow for analyzing the protein composition of concentrated virions by liquid chromatography and tandem mass spectrometry.

Ligand binding to a G protein-coupled receptor captured in a mass spectrometer.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors belong to the largest family of membrane-embedded cell surface proteins and are involved in a diverse array of physiological processes. Despite progress in the mass spectrometry of membrane protein complexes, G protein-coupled receptors have remained intractable because of their low yield and instability after extraction from cell membranes. We established conditions in the mass spectrometer that preserve noncovalent ligand binding to the human purinergic receptor P2Y1. Results established differing affinities for nucleotides and the drug MRS2500 and link antagonist binding with the absence of receptor phosphorylation. Overall, therefore, our results are consistent with drug binding, preventing the conformational changes that facilitate downstream signaling. More generally, we highlight opportunities for mass spectrometry to probe effects of ligand binding on G protein-coupled receptors.

CD44 Binding to Hyaluronic Acid Is Redox Regulated by a Labile Disulfide Bond in the Hyaluronic Acid Binding Site.

CD44 is the primary leukocyte cell surface receptor for hyaluronic acid (HA), a component of the extracellular matrix. Enzymatic post translational cleavage of labile disulfide bonds is a mechanism by which proteins are structurally regulated by imparting an allosteric change and altering activity. We have identified one such disulfide bond in CD44 formed by Cys77 and Cys97 that stabilises the HA binding groove. This bond is labile on the surface of leukocytes treated with chemical and enzymatic reducing agents. Analysis of CD44 crystal structures reveal the disulfide bond to be solvent accessible and in the-LH hook configuration characteristic of labile disulfide bonds. Kinetic trapping and binding experiments on CD44-Fc chimeric proteins show the bond is preferentially reduced over the other disulfide bonds in CD44 and reduction inhibits the CD44-HA interaction. Furthermore cells transfected with CD44 no longer adhere to HA coated surfaces after pre-treatment with reducing agents. The implications of CD44 redox regulation are discussed in the context of immune function, disease and therapeutic strategies.

Immunoregulation through membrane proteins modified by reducing conditions induced by immune reactions.

Selected disulfide bonds in membrane proteins are labile and are thus susceptible to changes in redox potential and/or the presence of thiol isomerase enzymes. Modification of these disulfide bonds can lead to conformational changes of the protein that in turn may alter protein activity and function. This occurs in the entry of several enveloped viruses into their host cells, e.g. HIV, hepatitis C virus and Newcastle disease virus. Labile disulfide bonds are also important in platelet activation, cytokine signalling and in a variety of diseases including cancer and arthritis. In this review we will concentrate on recent advances in understanding the conditions that lead to disulfide bond reduction in membrane proteins and their effects in regulating immune function.

FixK2, a key regulator in Bradyrhizobium japonicum, is a substrate for the protease ClpAP in vitro.

FixK2 is a CRP-like transcription factor that controls the endosymbiotic lifestyle of Bradyrhizobium japonicum. The reason for its noticeable protease sensitivity was explored here. The repertoire of Clp chaperone-proteases in B. japonicum was examined, and specifically ClpAP1 and ClpXP1 were purified and tested. FixK2 was found to be degraded by ClpAP1 but not by ClpXP1. Degradation was inhibited by the ClpS1 adaptor protein, indicating that FixK2 is a direct substrate for ClpAP1. The last 12 amino acids of FixK2 appeared to be recognized by ClpA. The results suggest that the ClpAP system is involved in the cellular turnover of FixK2.