Tyrosine Sulfation Research Articles

TPST2-mediated receptor tyrosine sulfation enhances leukocidin cytotoxicity and S. aureus infection.

An essential fact underlying the severity of Staphylococcus aureus (S. aureus) infection is the bicomponent leukocidins released by the pathogen to target and lyse host phagocytes through specific binding cell membrane receptors. However, little is known about the impact of post-transcriptional modification of receptors on the leukocidin binding. In this study, we used small interfering RNA library (Horizon/Dharmacon) to screen potential genes that affect leukocidin binding on receptors. The cell permeability was investigated through flow cytometry measuring the internalization of 4',6-diamidino-2-phenylindole. Expression of C5a anaphylatoxin chemotactic receptor 1 (C5aR1), sulfated C5aR1 in, and binding of 6x-His-tagged Hemolysin C (HlgC) and Panton-Valentine leukocidin (PVL) slow-component to THP-1 cell lines was detected and analyzed via flow cytometry. Bacterial burden and Survival analysis experiment was conducted in WT and myeloid TPST-cko C57BL/6N mice. After short hairpin RNA (shRNA) knockdown of TPST2 gene in THP-1, HL-60, and RAW264.7, the cytotoxicity of HlgAB, HlgCB, and Panton-Valentine leukocidin on THP-1 or HL-60 cells was decreased significantly, and the cytotoxicity of HlgAB on RAW264.7 cells was also decreased significantly. Knockdown of TPST2 did not affect the C5aR1 expression but downregulated cell surface C5aR1 tyrosine sulfation on THP-1. In addition, we found that the binding of HlgC and LukS-PV on cell surface receptor C5aR1 was impaired in C5aR1+TPST2- and C5aR1-TPST2- cells. Phagocyte knockout of TPST2 protects mice from S. aureus infection and improves the survival of mice infected with S. aureus. These results indicate that phagocyte TPST2 mediates the bicomponent leukocidin cytotoxicity by promoting cell membrane receptor sulfation modification that facilitates its binding to leukocidin S component.

Structural Insights into Protein-Ligand Interactions of Small Leucine Rich Repeat Proteoglycans with a Large Number of Binding Partners: An Overview

Small leucine rich repeat proteoglycans (SLRPs) exist in the extracellular matrix. SLRPs contain tandem arrays of LRRs flanked by cysteine clusters at the both N- and C-termini. The extreme N- and/or C-termini contain low complexity sequences, glycosaminoglycan (GAG) chain and/or sulfated tyrosine residues in some members of SLRPs. The LRR solenoid structure may be divided into four parts consisting of a concave surface, an ascending surface, a convex surface, and a descending surface. SLRPs share many biological functions including collagen fibrillogenesis, signaling, innate immunity, and inflammation through the binding of various ligands. We undertake a comprehensive literature search of publications in order to make a list of ligands of SLRPs. We describe and discuss the interacting sites of SLRPs to binding partners. The protein-ligand interactions occur on not only the concave surface but also the ascending surface and the N- or C-terminal capping regions. In addition, the extreme N- and/or C-terminal regions with the GAG chains or sulfated tyrosine residues participate in ligand-interactions.

Simulated Docking of alpha-Conotoxin Interactions with Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptors (nAChRs) are linked to several biological processes and neurological conditions. The α-conotoxins represent an essential resource for ligands with which to explore structure-function correlations for a variety of nAChR subtypes. With the multitude of nAChR subtypes occurring in vivo, it is imperative to search for subtype-selective ligands to effectively utilize their modulatory functions for their target receptors. In this work,docking simulations using PatchDock showed that either the replacement of Ala10 with the more hydrophobic Leu in PnIA to PnIA[A10L] or the presence of a sulfated tyrosine in the structural scaffold of PnIA[Y15Y] or PnIB[Y15Y] (Y stands for sulfated tyrosine) is sufficient to alter the interactions of α-conotoxins PnIA and PnIB for their target nAChR receptor subtypes – α7and α3β2. The employed protocol can be utilized to screen for ligand docking and amino acid residue interactions of α-conotoxins to certain nAChR subtypes. This was tested on α-conotoxinLoIA, which was predicted to preferentially bind to the α3β2 nAChR over α7 nAChR. The present work is supportive of the idea that the conserved α-conotoxin structural scaffold can be further investigated to guide the synthesis of novel nAChR ligands with greater specificity.The results of this study will be useful in the rational design of selective nAChR antagonists for potential peptide therapeutics.

Chemokine binding to PSGL-1 is controlled by O-glycosylation and tyrosine sulfation

Protein glycosylation influences cellular recognition and regulates protein interactions, but how glycosylation functions alongside other common posttranslational modifications (PTMs), like tyrosine sulfation (sTyr), is unclear. We produced a library of 53 chemoenzymatically synthesized glycosulfopeptides representing N-terminal domains of human and murine P-selectin glycoprotein ligand-1 (PSGL-1), varying in sTyr and O-glycosylation (structure and site). Using these, we identified key roles of PSGL-1 O-glycosylation and sTyr in controlling interactions with specific chemokines. Results demonstrate that sTyr positively affects CCL19 and CCL21 binding to PSGL-1N terminus, whereas O-glycan branching and sialylation reduced binding. For murine PSGL-1, interference between PTMs is greater, attributed to proximity between the two PTMs. Using fluorescence polarization, we found sTyr is a positive determinant for some chemokines. We showed that synthetic sulfopeptides are potent in decreasing chemotaxis of human dendritic cells toward CCL19 and CCL21. Our results provide new research avenues into the interplay of PTMs regulating leukocyte/chemokine interactions.

ADY tripeptide is a minimum sequence for Tyrosylprotein sulfotransferases 1 and 2 substrate recognition

Tyrosylprotein sulfotransferases (TPSTs) catalyze the transfer of a sulphonate moiety from 3′-Phosphoadenosine 5′-Phosphosulfate (PAPS) to the hydroxyl group of a tyrosine residue in substrate proteins. The positively charged substrate binding region of TPST homodimer interacts with acidic residues located in N-terminal region from the sulfated tyrosine in substrates. However, the sequence pattern in TPST substrate recognition remains unclear. Therefore, we aimed to determine the minimum recognition chain length required for tyrosine sulfation. We prepared His-tagged polypeptide, His-TPST143-370 and His-TPST243-377, form 43–370 of TPST1 and 43–377 of TPST2. Next, we prepared a series of synthesized ADYAE peptides and used a combination of reverse phase high-performance liquid chromatography (RP-HPLC) and mass spectrometric analysis to show that the tripeptide amino acid sequence, ADY, was sulfated by TPST1 and TPST2. Furthermore, we found that the acidic residue, located two residues C-terminal region from the tyrosine residue, may be involved in the TPST-induced sulfation regulation. The results in our study propose that proteins with the ADY sequence may be useful for searching the novel TPST tyrosine sulfated substrates.

Root-knot nematodes produce functional mimics of tyrosine-sulfated plant peptides

Root-knot nematodes (Meloidogyne spp.) are highly evolved obligate parasites threatening global food security. These parasites have a remarkable ability to establish elaborate feeding sites in roots, which are their only source of nutrients throughout their life cycle. A wide range of nematode effectors have been implicated in modulation of host pathways for defense suppression and/or feeding site development. Plants produce a diverse array of peptide hormones including PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides, which promote root growth via cell expansion and proliferation. A sulfated PSY-like peptide RaxX (required for activation of XA21 mediated immunity X) produced by the biotrophic bacterial pathogen (Xanthomonas oryzae pv. oryzae) has been previously shown to contribute to bacterial virulence. Here, we report the identification of genes from root-knot nematodes predicted to encode PSY-like peptides (MigPSYs) with high sequence similarity to both bacterial RaxX and plant PSYs. Synthetic sulfated peptides corresponding to predicted MigPSYs stimulate root growth in Arabidopsis. MigPSY transcript levels are highest early in the infection cycle. Downregulation of MigPSY gene expression reduces root galling and egg production, suggesting that the MigPSYs serve as nematode virulence factors. Together, these results indicate that nematodes and bacteria exploit similar sulfated peptides to hijack plant developmental signaling pathways to facilitate parasitism.

#4941 A METABOLOMIC FINGERPRINT PERSPECTIVE OF GUT-DERIVED METABOLITES ON EARLY DIABETIC KIDNEY DISEASE IN TYPE 2 DIABETES MELLITUS PATIENTS

Abstract Background and Aims Type 2 diabetes mellitus (T2DM) is the first cause of end stage renal disease worldwide. Metabolite profiling is an emerging field of great significance, as T2DM prevalence is continuously rising. The subtle dynamics of gut microbiota derived metabolites may have important implications in diabetic kidney disease (DKD) development. Nitrogen metabolic pathway and retinoic acid signaling pathway seem to be involved in early pathogenic mechanisms of DKD. The aim of study was to discover new putative gut-derived biomarkers that interfere with early physio-pathological mechanisms in DKD, regarding the podocyte, tubule, and the renal endothelium. Method The serum and urine of 90 T2DM patients (P group) and 20 healthy subjects (C group), were assessed in a cross-sectional study, by ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-QTOF-ESI+-MS) techniques. P group was divided in 3 subgroups (normoalbuminuria-P1, microalbuminuria-P2, macroalbuminuria-P3). Statistical analysis was performed by untargeted multivariate (PLSDA, VIP scores, Random Forest, Biomarker analysis) and univariate (One Way ANOVA, Biomarker Analysis) methods. Results Multivariate analysis, by which the P group vs. C group were compared, displayed: (1) in serum samples: increased levels (P>C) of all-trans retinoic acid, cysteine-S sulfate, oleoylglicine, threonylglycine and decreased levels (P<C) of phenylalanine, tyrosine, kynurenic acid; (2) in urine samples, there were increased levels of: all-trans retinoic acid, indoxyl suflate, serotonin sulfate and glycylprolylarginine. Subsequently, by applying univariate analysis, the biomarkers derived from gut microbiota were selected: (1) in serum: all-trans retinoic acid (C<P1<P2<3), phenylalanine, tyrosine, and kynurenic acid with levels decreasing progressively from C to P1-P2-P3 (2) in urine: all trans retinoic acid, indoxyl sulfate, and serotonin sulfate, showing good differentiation between C and P1, and slight differences between P1∼P2∼P3 subgroups. The study suggests that ATRAs dynamic is disturbed in the normoalbuminuric stage of DKD. While its urinary levels correlated significantly with albuminuria, the changes observed in P1 subgroup, in serum and urine, may be correlated to the loss of renal tubular tight junction as a result of claudin downregulation. Phenylalanine and tyrosine, a part of nitrogen metabolic pathway, are indicators of the new-onset DKD. Their levels were upregulated in serum vs. urine, indicating their possible involvement in early DKD when comparing C vs. P1-P2-P3. Kynurenic acid and indoxyl-sulfate result from tryptophan metabolism. Low levels in serum of kynurenic acid may suggest its possible implication in incipient endothelial dysfunction in DKD. Indoxyl-sulfate (IS) urinary excretion is dependent on albuminuria. The study shows an elevated excretion of IS, in the normoalbuminuric group compared to controls and may suggest a tubular damage that precedes the glomerular modifications in DKD. Conclusion UHPLC-QTOF-ESI+-MS untargeted analysis reveals a particular metabolite fingerprint in early DKD. The study describes all-trans retinoic acid, phenylalanine, tyrosine, kynurenic acid, and indoxyl sulfate, as potential key biomarkers involved in early DKD pathogenesis, their levels being expressed in P1 subgroup compared with C group, and P2, P3 subgroups.

Computational Characterization of the Binding Properties of the HIV1-Neutralizing Antibody PG16 and Design of PG16-Derived CDRH3 Peptides.

PG16 is a broadly neutralizing antibody that binds to the gp120 subunit of the HIV-1 Env protein. The major interaction site is formed by the unusually long complementarity determining region (CDR) H3. The CDRH3 residue Tyr100H is known to represent a tyrosine sulfation site; however, this modification is not present in the experimental complex structure of PG16 with full-length HIV-1 Env. To investigate the role of sulfation for this complex, we modeled the sulfation of Tyr100H and compared the dynamics and energetics of the modified and unmodified complex by molecular dynamics simulations at the atomic level. Our results show that sulfation does not affect the overall conformation of CDRH3, but still enhances gp120 interactions both at the site of modification and for the neighboring residues. This stabilization affects not only protein-protein contacts, but also the interactions between PG16 and the gp120 glycan shield. Furthermore, we also investigated whether PG16-CDRH3 is a suitable template for the development of peptide mimetics. For a peptide spanning residues 93-105 of PG16, we obtained an experimental EC50 value of 3nm for the binding of gp120 to the peptide. This affinity can be enhanced by almost one order of magnitude by artificial disulfide bonding between residues 99 and 100F. In contrast, any truncation results in significantly lower affinity, suggesting that the entire peptide segment is involved in gp120 recognition. Given their high affinity, it should be possible to further optimize the PG16-derived peptides as potential inhibitors of HIV invasion.

Sulfo-peptide CLEL6 inhibits anthocyanin biosynthesis in Arabidopsis thaliana.

Post-translationally modified peptides are now recognized as important regulators of plant stress responses. Here, we identified the small sulfated CLE-LIKE6 (CLEL6) peptide as a negative regulator of anthocyanin biosynthesis in etiolated and in light-stressed Arabidopsis (Arabidopsis thaliana) seedlings. CLEL6 function depends on proteolytic processing of the CLEL6 precursor by subtilisin-like serine proteinase 6.1 (SBT6.1) and on tyrosine sulfation by tyrosylprotein sulfotransferase (TPST). Loss-of-function mutants of either sbt6.1 or tpst showed significantly higher anthocyanin accumulation than the wild type upon light stress. The anthocyanin overaccumulation phenotype of sbt6.1 and tpst was suppressed by application of mature CLEL6. Overexpression and external application of CLEL6 inhibited the expression of anthocyanin biosynthesis genes in etiolated and light-stressed seedlings, confirming the role of CLEL6 as an inhibitor of anthocyanin biosynthesis. Small post-translationally modified peptides are perceived by leucine-rich-repeat receptor like kinases. Using a quintuple mutant of ROOT MERISTEM GROWTH FACTOR 1 INSENSITIVE (RGI) receptors, we showed the essential function of the RGI receptor family in CLEL6 signaling. Our data indicate that overexpression or application of CLEL6 inhibit anthocyanin biosynthesis through RGI receptors. We propose that CLEL6 inhibits anthocyanin biosynthesis in etiolated seedlings, and that anthocyanin biosynthesis is derepressed when CLEL6 expression is down-regulated upon light exposure. Hyperaccumulation of anthocyanins in light-stressed tpst and sbt6.1 mutant seedlings suggests that CLEL6, or related sulfopeptides, continue to act as negative regulators to limit pigment accumulation in the light.

Tyrosylprotein Sulfotransferase suppresses ABA Signaling via Sulfation of SnRK2.2/2.3/2.6.

Phytohormone ABA plays vital roles in stress tolerance, while long-term overactivation of ABA signaling suppresses plant growth and development. However, the braking mechanism of ABA responses is not clear. Protein tyrosine sulfation catalyzed by tyrosylprotein sulfotransferase (TPST) is a critical post-translational modification. Through genetic screening, we identified a tpst mutant in Arabidopsis that was hypersensitive to ABA. In-depth analysis revealed that TPST could interact with and sulfate SnRK2.2/2.3/2.6, which accelerated their degradation and weakened the ABA signaling. Taken together, these findings uncovered a novel mechanism of desensitizing ABA responses via protein sulfation. This article is protected by copyright. All rights reserved.

Cholecystokinin receptors in GtoPdb v.2023.1

Cholecystokinin receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on CCK receptors [90]) are activated by the endogenous peptides cholecystokinin-8 (CCK-8), CCK-33, CCK-58 and gastrin (gastrin-17). There are only two distinct subtypes of CCK receptors, CCK1 and CCK2 receptors [64, 124], with some alternatively spliced forms most often identified in neoplastic cells. The CCK receptor subtypes are distinguished by their peptide selectivity, with the CCK1 receptor requiring the carboxyl-terminal heptapeptide-amide that includes a sulfated tyrosine for high affinity and potency, while the CCK2 receptor requires only the carboxyl-terminal tetrapeptide shared by each CCK and gastrin peptides. These receptors have characteristic and distinct distributions, with both present in both the central nervous system and peripheral tissues.

Chemical Synthesis of Glycopeptides containing l-Arabinosylated Hydroxyproline and Sulfated Tyrosine.

Post-translationally modified peptides are important regulating molecules for living organisms. Here, we report the stereoselective total synthesis of β-1,2-linked l-arabinosylated Fmoc-protected hydroxyproline building blocks and their incorporation, together with sulfated tyrosine and hydroxyproline, into the plant peptide hormone PSY1. Clean glycopeptides were obtained by performing acetyl removal from the l-arabinose groups prior to deprotection of the neopentyl-protected sulfated tyrosine.

Histone tyrosine sulfation by SULT1B1 regulates H4R3me2a and gene transcription.

Tyrosine sulfation is a common posttranslational modification in mammals. To date, it has been thought to be limited to secreted and transmembrane proteins, but little is known about tyrosine sulfation on nuclear proteins. Here we report that SULT1B1 is a histone sulfotransferase that can sulfate the tyrosine 99 residue of nascent histone H3 in cytosol. The sulfated histone H3 can be transported into the nucleus and majorly deposited in the promoter regions of genes in chromatin. While the H3Y99 residue is buried inside octameric nucleosome, dynamically regulated subnucleosomal structures provide chromatin-H3Y99sulf the opportunity of being recognized and bound by PRMT1, which deposits H4R3me2a in chromatin. Disruption of H3Y99sulf reduces PRMT1 binding to chromatin, H4R3me2a level and gene transcription. These findings reveal the mechanisms underlying H3Y99 sulfation and its cross-talk with H4R3me2a to regulate gene transcription. This study extends the spectrum of tyrosine sulfation on nuclear proteins and the repertoire of histone modifications regulating chromatin functions.

A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin

Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EμB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.

“Glyco-sulfo barcodes” regulate chemokine receptor function

Chemokine ligands and receptors regulate the directional migration of leukocytes. Post-translational modifications of chemokine receptors including O-glycosylation and tyrosine sulfation have been reported to regulate ligand binding and resulting signaling. Through in silico analyses, we determined potential conserved O-glycosylation and sulfation sites on human and murine CC chemokine receptors. Glyco-engineered CHO cell lines were used to measure the impact of O-glycosylation on CC chemokine receptor CCR5, while mutation of tyrosine residues and treatment with sodium chlorate were performed to determine the effect of tyrosine sulfation. Changing the glycosylation or tyrosine sulfation on CCR5 reduced the receptor signaling by the more positively charged CCL5 and CCL8 more profoundly compared to the less charged CCL3. The loss of negatively charged sialic acids resulted only in a minor effect on CCL3-induced signal transduction. The enzymes GalNAc-T1 and GalNAc-T11 were shown to be involved in the process of chemokine receptor O-glycosylation. These results indicate that O-glycosylation and tyrosine sulfation are involved in the fine-tuning and recognition of chemokine interactions with CCR5 and the resulting signaling.

Directly mapping the O-sulfation sites within CCR2 via an engineered nanopore.

Tyrosine O-sulfation is one of the significant post-translational modifications that participates in and mediates various life activities in cells. However, until now, the detailed functions of tyrosine O-sulfation have hardly been understood. Although mass spectrometry, ELISA, and radioactive isotope-label have been explored to study tyrosine O-sulfation, the in-depth characterization of this post-translational modification is still rare due to the lack of general, label-free methods for accurately mapping and quantifying the tyrosine O-sulfation sites. Specifically, for mass spectrometry, sulfotyrosine is easily broken down at high temperatures and low pH, usually leading to the loss of sulfated modification in both positive and negative ion MS/MS signals. Moreover, since sulfotyrosine and phosphotyrosine have a similar mass change around 80 Da (sulfotyrosine: 79.9568 Da; phosphotyrosine: 79.9663 Da), it is quite difficult to directly discriminate them unless using ultra-high accuracy mass measurements. Therefore, mass spectrometry still faces huge limitations in rapid sulfotyrosine identification and quantification. Recently, nanopore has raised its potential for single protein sequencing and achieved the detection of various post-translational modifications, such as phosphorylation, glycosylation, methylation, etc. Here, mutant aerolysin nanopores are used for the detection of sulfotyrosine and the mapping of its site without labeling. It should be noted that the experiments are performed under mild conditions without impact on the stability of sulfotyrosine. The results show that phosphorylated, sulfated and nonmodified peptides can be clearly distinguished by differences in the relative current blockades. Furthermore, the mapping of the sites of sulfated tyrosine on CCR2 was achieved by further improving the sensitivity of aerolysin nanopore. These findings offer a path to the single-molecule, label-free identification of sulfotyrosine.

Inhibition of the SLC35B2–TPST2 Axis of Tyrosine Sulfation Attenuates the Growth and Metastasis of Pancreatic Ductal Adenocarcinom

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for invitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth invivo. Mouse PDAC cells derived from the KrasLSL-G12D/+;Tp53L/+;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis invivo. High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration invitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin β4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin β4 protein, which may have accounted for the suppression of metastasis. Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.

Analysis of Peptide Hormone Maturation and Processing Specificity Using Isotope-Labeled Peptides.

Many peptide hormones and growth factors in plants, particularly the small posttranslationally modified signaling peptides, are synthesized as larger precursor proteins. Proteolytic processing is thus required for peptide maturation, and additional posttranslational modifications may contribute to bioactivity. To what extent these posttranslational modifications impact on processing is largely unknown. Likewise, it is poorly understood how the cleavage sites within peptide precursors are selected by specific processing proteases, and whether or not posttranslational modifications contribute to cleavage site recognition. Here, we describe a mass spectrometry-based approach to address these questions. We developed a method using heavy isotope labeling to directly compare cleavage efficiency of different precursor-derived synthetic peptides by mass spectrometry. Thereby, we can analyze the effect of posttranslational modifications on processing and the specific sequence requirements of the processing proteases. As an example, we describe how this method has been used to assess the relevance of tyrosine sulfation for the processing of the Arabidopsis CIF4 precursor by the subtilase SBT5.4.

Peptide ligand-mediated trade-off between plant growth and stress response.

Deciding whether to grow or to divert energy to stress responses is a major physiological trade-off for plants surviving in fluctuating environments. We show that three leucine-rich repeat receptor kinases (LRR-RKs) act as direct ligand-perceiving receptors for PLANT PEPTIDE CONTAINING SULFATED TYROSINE (PSY)-family peptides and mediate switching between two opposing pathways. By contrast to known LRR-RKs, which activate signaling upon ligand binding, PSY receptors (PSYRs) activate the expression of various genes encoding stress response transcription factors upon depletion of the ligands. Loss of PSYRs results in defects in plant tolerance to both biotic and abiotic stresses. This ligand-deprivation-dependent activation system potentially enables plants to exert tuned regulation of stress responses in the tissues proximal to metabolically dysfunctional damaged sites where ligand production is impaired.

Uncommon posttranslational modifications in proteomics: ADP-ribosylation, tyrosine nitration, and tyrosine sulfation.

Posttranslational modifications (PTMs) are covalent modifications of proteins that modulate the structure and functions of proteins and regulate biological processes. The development of various mass spectrometry-based proteomics workflows has facilitated the identification of hundreds of PTMs and aided the understanding of biological significance in a high throughput manner. Improvements in sample preparation and PTM enrichment techniques, instrumentation for liquid chromatography-tandem mass spectrometry (LC-MS/MS), and advanced data analysis tools enhance the specificity and sensitivity of PTM identification. Highly prevalent PTMs like phosphorylation, glycosylation, acetylation, ubiquitinylation, and methylation are extensively studied. However, the functions and impact of less abundant PTMs are not as well understood and underscore the need for analytical methods that aim to characterize these PTMs. This review focuses on the advancement and analytical challenges associated with the characterization of three less common but biologically relevant PTMs, specifically, adenosine diphosphate-ribosylation, tyrosine sulfation, and tyrosine nitration. The advantages and disadvantages of various enrichment, separation, and MS/MS techniques utilized to identify and localize these PTMs are described.