Protein Cysteine Research Articles

Understanding Polysulfide-Mediated Papain Inhibition and Differentiating between Disulfide vs Persulfide Formation.

Protein cysteine residues are sensitive to redox-regulating molecules, including reactive sulfur species (RSS). As an important member of the RSS family, polysulfides are known to react with protein cysteines to form persulfides and disulfides, both affecting protein functions. In this work, we studied how polysulfides could impact cysteine proteases through careful mechanistic and kinetic studies. The model protein papain was treated with different polysulfides to elucidate the efficacy of polysulfides as inhibitors for this protein. We also explored the effects of different reductants that could regenerate papain activity after polysulfide-mediated inhibition. A triarylphosphine reagent, TXPTS, was found to be efficient in differentiating between papain persulfidation and disulfide formation.

Disrupted Nitric Oxide Homeostasis Impacts Fertility through Multiple Processes Including Protein Quality Control.

Plant fertility is fundamental to plant survival and requires the coordinated interaction of developmental pathways and signaling molecules. Nitric oxide (NO) is a small, gaseous signaling molecule that plays crucial roles in plant fertility as well as other developmental processes and stress responses. NO influences biological processes through S-nitrosation, the posttranslational modification of protein cysteines to S-nitrosocysteine (R-SNO). NO homeostasis is controlled by S-nitrosoglutathione reductase (GSNOR), which reduces S-nitrosoglutathione (GSNO), the major form of NO in cells. GSNOR mutants (hot5-2/gsnor1) have defects in female gametophyte development along with elevated levels of reactive nitrogen species and R-SNOs. To better understand the fertility defects in hot5-2, we investigated the in vivo nitrosoproteome of Arabidopsis (Arabidopsis thaliana) floral tissues coupled with quantitative proteomics of pistils. To identify protein-SNOs, we used an organomercury-based method that involves direct reaction with S-nitrosocysteine, enabling specific identification of S-nitrosocysteine-containing peptides and S-nitrosated proteins. We identified 1102 endogenously S-nitrosated proteins in floral tissues, of which 1049 were unique to hot5-2. Among the identified proteins, 728 were novel S-nitrosation targets. Notably, specific UDP-glycosyltransferases and argonaute proteins are S-nitrosated in floral tissues and differentially regulated in pistils. We also discovered S-nitrosation of subunits of the 26S proteasome together with increased abundance of proteasomal components and enhanced trypsin-like proteasomal activity in hot5-2 pistils. Our data establish a method for nitrosoprotein detection in plants, expand knowledge of the plant S-nitrosoproteome, and suggest that nitro-oxidative modification and NO homeostasis are critical to protein quality control in reproductive tissues.

Endoplasmic Reticulum Oxidative Stress Promotes Glutathione-Dependent Oxidation of Collagen-1A1 and Promotes Lung Fibroblast Activation.

Changes in the oxidative (redox) environment accompany idiopathic pulmonary fibrosis (IPF). S-glutathionylation of reactive protein cysteines is a post-translational event that transduces oxidant signals into biological responses. We recently demonstrated that increases in S-glutathionylation promote pulmonary fibrosis, which was mitigated by the deglutathionylating enzyme glutaredoxin (GLRX). However, the protein targets of S-glutathionylation that promote fibrogenesis remain unknown. In the present study we addressed whether the extracellular matrix is a target for S-glutathionylation. We discovered increases in COL1A1 (collagen 1A1) S-glutathionylation (COL1A1-SSG) in lung tissues from subjects with IPF compared with control subjects in association with increases in ERO1A (endoplasmic reticulum [ER] oxidoreductin 1) and enhanced oxidation of ER-localized PRDX4 (peroxiredoxin 4), reflecting an increased oxidative environment of the ER. Human lung fibroblasts exposed to TGFB1 (transforming growth factor-β1) show increased secretion of COL1A1-SSG. Pharmacologic inhibition of ERO1A diminished the oxidation of PRDX4, attenuated COL1A1-SSG and total COL1A1 concentrations, and dampened fibroblast activation. Absence of Glrx enhanced COL1A1-SSG and overall COL1A1 secretion and promoted the activation of mechanosensing pathways. Remarkably, COL1A1-SSG resulted in marked resistance to collagenase degradation. Compared with COL1, lung fibroblasts plated on COL1-SSG proliferated more rapidly and increased the expression of genes encoding extracellular matrix crosslinking enzymes and genes linked to mechanosensing pathways. Overall, these findings suggest that glutathione-dependent oxidation of COL1A1 occurs in settings of IPF in association with enhanced ER oxidative stress and may promote fibrotic remodeling because of increased resistance to collagenase-mediated degradation and fibroblast activation.

SPARC activates p38γ signaling to promote PFKFB3 protein stabilization and contributes to keloid fibroblast glycolysis

BackgroundKeloids are currently challenging to treat because they recur after resection which may affect patients’ quality of life. At present, no universal consensus on treatment regimen has been established. Thus, finding new molecular mechanisms underlying keloid formation is imminent. This study aimed to explore the function of secreted protein acidic and cysteine rich (SPARC) on keloids and its behind exact mechanisms.MethodsThe expression of SPARC, p38γ, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), α-SMA, and Ki67 in patients with keloid and bleomycin (BLM)-induced fibrosis mice was assessed utilizing western blot, qRT-PCR, and immunohistochemical staining. After transfected with pcDNA-SPARC, si-SPARC-1#, si-SPARC-2#, and si-p38γ, and treated with glycolytic inhibitor (2-DG) or p38 inhibitor (SB203580), CCK-8, EdU, transwell, and western blot were utilized for assessing the proliferation, migration, and collagen production of keloid fibroblasts (KFs).ResultsSPARC, p38γ, and PFKFB3 were highly expressed in patients with keloid and BLM-induced fibrosis mice. SPARC promoted the proliferation, migration, and collagen production of KFs via inducing glycolysis. Moreover, SPARC could activate p38γ signaling to stabilize PFKFB3 protein expression in KFs. Next, we demonstrated that SPARC promoted the proliferation, migration, collagen production, and glycolysis of KFs via regulating p38γ signaling. In addition, in BLM-induced fibrosis mice, inhibition of p38γ and PFKFB3 relieved skin fibrosis.ConclusionsOur findings indicated that SPARC could activate p38γ pathway to stabilize the expression of PFKFB3, and thus promote the glycolysis of KFs and the progression of keloid.

Identification and Verification of Key Genes in Colorectal Cancer Liver Metastases Through Analysis of Single-Cell Sequencing Data and TCGA Data

BackgroundColorectal cancer (CRC) is highly prevalent worldwide, with more patients experiencing colorectal cancer liver metastases (CRLM). This study aimed to identify key genes in CRLM through single-cell sequencing data reanalysis and experimental validation.MethodsThe study analyzed single-cell RNA-sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for gene functional enrichment analysis. The Cancer Genome Atlas (TCGA) data enabled bulk-RNA expression and survival prognosis analysis. Real-time polymerase chain reaction (qPCR) detected mRNA expression, whereas Western blot determined protein levels. Cell function experiments assessed SPARC’s impact on CRC cell behavior.ResultsCluster analysis showed 23 classes among 17 CRLM samples, representing six cell types. A GO and KEGG analysis identified interleukin-1 beta (IL1B), CD2 molecule (CD2), and C-X-C motif chemokine ligand 8 (CXCL8) as significant prognostic factors in CRC. Secreted protein acidic and cysteine rich (SPARC) was one of the top differentially expressed genes (DEGs) in tissue stem cells, confirmed in primary and metastatic lesions. Metastatic lesions showed higher expression of SPARC and CRC stem cell marker leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), which was significantly correlated positively with LGR5 expression. Knockdown of SPARC reduced CRC cell sphere- and colony-formation, invasion, and migration abilities. Overexpression of SPARC significantly increased the malignancy of CRC cells.ConclusionsSeveral key genes were identified in the process of CRLM. In CRLM samples and those corresponding to CRC stem cells, SPARC was significantly upregulated. In the therapy of CRLM, SPARC might be a potential target.

Investigating the expression profiles of cysteine string proteins (CSPs) in cochlear tissue

ObjectiveThis study aims to explore the expression patterns of cysteine string protein alpha (CSPα) and cysteine string protein beta (CSPβ) in the mammalian inner ear, with an emphasis on their temporal dynamics during the developmental stages of C57BL/6 mice. MethodsWe utilized immunofluorescence staining to assess the localization and distribution of CSPα and CSPβ within the inner ears of C57BL/6 mice and miniature pigs. Additionally, this method facilitated the investigation of their temporal expression profiles. ResultsIn adult C57BL/6 mice and miniature pigs, CSPα and CSPβ were identified in the cytoplasm of inner hair cells and spiral ganglion cells, yet were absent in outer hair cells. Both proteins were found to colocalize with Ctbp2 on the basal side of the cytoplasm in inner hair cells' basilar membrane. Expression of CSPα was observed at the nerve fiber termini at the basilar membrane's base of inner and outer hair cells 10 days postnatally in C57BL/6 mice. Notably, expression of both CSPα and CSPβ in the cytoplasm of inner hair cells emerged on the 12th day post-birth, aligning with the timeline for registering cochlear potentials. The expression levels of both proteins increased with age, but were consistently absent in outer hair cells. Contrastingly, expression of CSPα and CSPβ was present in the cytoplasm of inner hair cells in miniature pigs as early as one day post-birth, yet remained absent in the three rows of outer hair cells. ConclusionCSPα and CSPβ exhibit predominant and specific expression in inner hair cells and spiral ganglion cells. A unique expression pattern was observed for CSPα, which was also present at the nerve fiber endings of both inner and outer hair cells. The developmental expression trajectory of CSPα and CSPβ in mouse inner hair cells is characterized by an initial absence, followed by a gradual increase. Moreover, the timing of expression onset between mice and miniature pigs indicates distinct temporal dynamics, suggesting a potential role in auditory development.

Quantification of persulfidation on specific proteins: are we nearly there yet?

Hydrogen sulfide (H2S) played a pivotal role in the early evolution of life on Earth before the predominance of atmospheric oxygen. The legacy of a persistent role for H2S in life's processes recently emerged through its discovery in modern biochemistry as an endogenous cellular signalling modulator involved in numerous biological processes. One major mechanism through which H2S signals is protein cysteine persulfidation, an oxidative post-translational modification. In recent years, chemoproteomic technologies have been developed to allow the global scanning of protein persulfidation targets in mammalian cells and tissues, providing a powerful tool to elucidate the broader impact of altered H2S in organismal physiological health and human disease states. While hundreds of proteins were confirmed to be persulfidated by global persulfidome methodologies, the targeting of specific proteins of interest and the investigation of further mechanistic studies are still underdeveloped due to a lack of stringent specificity of the methods and the inherent instability of persulfides. This review provides an overview of the processes of endogenous H2S production, oxidation, and signalling and highlights the application and limitations of current persulfidation labelling approaches for investigation of this important evolutionarily conserved biological switch for protein function.

S-Nitrosylation of p39 promotes its degradation and contributes to synaptic dysfunction induced by β-amyloid peptide

Alzheimer’s disease (AD), characterized by cognitive decline, is increasingly recognized as a disorder marked by synaptic loss and dysfunction. Despite this understanding, the underlying pathophysiological mechanisms contributing to synaptic impairment remain largely unknown. In this study, we elucidate a previously undiscovered signaling pathway wherein the S-nitrosylation of the Cdk5 activator p39, a post-translational modification involving the addition of nitric oxide to protein cysteine residues, plays a crucial role in synaptic dysfunction associated with AD. Our investigation reveals heightened p39 S-nitrosylation in the brain of an amyloid precursor protein (APP)/presenilin 1 (PS1) transgenic mouse model of AD. Additionally, soluble amyloid-β oligomers (Aβ), implicated in synaptic loss in AD, induce p39 S-nitrosylation in cultured neurons. Notably, we uncover that p39 protein level is regulated by S-nitrosylation, with nitric oxide S-nitrosylating p39 at Cys265 and subsequently promoting its degradation. Furthermore, our study demonstrates that S-nitrosylation of p39 at Cys265 significantly contributes to amyloid-β (Aβ) peptide-induced dendrite retraction and spine loss. Collectively, our findings highlight S-nitrosylation of p39 as a novel aberrant redox protein modification involved in the pathogenesis of AD, suggesting its potential as a therapeutic target for the disease.

Protein phosphatase PP2Cα S-glutathionylation regulates cell migration

Redox signaling is a fundamental mechanism that controls all major biological processes partly via protein cysteine oxidations, including S-glutathionylation. Despite over 2,000 cysteines identified to form S-glutathionylation in databases, the identification of redox cysteines functionally linked to a biological process of interest remains challenging. Here, we demonstrate a strategy combining glutathionylation proteomic database, bioinformatics, and biological screening, which resulted in the identification of S-glutathionylated proteins, including PP2Cα, as redox players of cell migration. We showed that PP2Cα, a prototypical magnesium-dependent serine/threonine phosphatase, is susceptible to S-glutathionylation selectively at non-conserved C314. PP2Cα glutathionylation causes increased migration and invasion of breast cancer cell lines in oxidative stress or upon hydrogen peroxide production. Mechanistically, PP2Cα glutathionylation modulates its protein-protein interactions, activating c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) pathways to elevate migration and invasion. In addition, PP2Cα glutathionylation occurs in response to epidermal-growth factor, supporting a serine/threonine phosphatase PP2Cα as a new redox player in growth factor signal transduction.

The impact of κ-carrageenan on the pea protein gelation by high pressure processing and the gelling mechanisms study

The transition toward sustainable and health-conscious diets has intensified plant-based proteins, particularly pea protein. This research explored the gelation mechanisms of pea protein with κ-carrageenan by high-pressure processing (HPP) as an alternative to traditional thermal methods. Suspensions containing 15% pea protein with 0.1–1% κ-carrageenan were subjected to HPP at 100–600 MPa for 5–30 min. Increasing κ-carrageenan concentration reduced the pressure required to obtain good gels, rendering HPP more energy efficient. Pea protein gels with 1% κ-carrageenan at 600 MPa demonstrated a 27-fold increase in compressive strength compared to 15% pea protein alone by HPP and over 5 times the strength of heat-induced counterparts. Gel textures could be tailored by modulating HPP conditions and κ-carrageenan concentrations. Fourier transform infrared spectroscopy, confocal laser scanning microscopy, and scanning electron microscopy characterized protein unfolding, phase separation, and network formation to study the gelation mechanisms. Results revealed that HPP induced protein tertiary structure unfolding, facilitating phase separation and uniform submicron κ-carrageenan particle distribution in the protein matrix. This, along with a more compact protein network induced by HPP, enhanced gel strength, compensating for limited cysteine in pea protein for disulfide bonds. Higher pressures intensified protein unfolding and aggregation, with κ-carrageenan particles evenly distributing into smaller particles, further strengthening gel structures. Conversely, heating-induced gelation caused more random protein aggregation, leading to coarser and weaker gel networks. This research highlights HPP's effectiveness over traditional heating methods in preparing strong pea protein gels with minimal κ-carrageenan and provides preliminary insights into tailoring HPP and κ-carrageenan for desirable gel textures.

The structure of Chlamydomonas LOV1 as revealed by time-resolved serial synchrotron crystallography.

The photo-reaction of the LOV1 domain of the Chlamydomonas reinhardtii phototropin is investigated by room-temperature time-resolved serial crystallography. A covalent adduct forms between the C4a atom of the central flavin-mononucleotide chromophore and a protein cysteine. The structure of the adduct is very similar to that of LOV2 determined 23 years ago from the maidenhair fern Phy3.

Peptide and Protein Cysteine Modification Enabled by Hydrosulfuration of Ynamide.

Efficient functionalization of peptides and proteins has widespread applications in chemical biology and drug discovery. However, the chemoselective and site-selective modification of proteins remains a daunting task. Herein, a highly efficient chemo-, regio-, and stereoselective hydrosulfuration of ynamide was identified as an efficient method for the precise modification of peptides and proteins by uniquely targeting the thiol group of cysteine (Cys) residues. This novel method could be facilely operated in aqueous buffer and was fully compatible with a wide range of proteins, including small model proteins and large full-length antibodies, without compromising their integrity and functions. Importantly, this reaction provides the Z-isomer of the corresponding conjugates exclusively with superior stability, offering a precise approach to peptide and protein therapeutics. The potential application of this method in peptide and protein chemical biology was further exemplified by Cys-bioconjugation with a variety of ynamide-bearing functional molecules such as small molecule drugs, fluorescent/affinity tags, and PEG polymers. It also proved efficient in redox proteomic analysis through Cys-alkenylation. Overall, this study provides a novel bioorthogonal tool for Cys-specific functionalization, which will find broad applications in the synthesis of peptide/protein conjugates.

Interrogating the Presence of RNA Phosphorothioate in Nature by a Highly Selective and Sensitive Fluorescence Method.

In nature, DNA phosphorothioate (PT) is found in the genomic materials of some prokaryotes. In contrast, whether there is natural RNA PT is still a question under debate. A groundbreaking study reported the discovery of RNA PT in cellular RNA samples from both prokaryotes and eukaryotes at contents of >100 PT per million nucleotides (PPM-nt) according to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. However, this finding was challenged by a later work showing that other RNA modifications, such as 2'-O-methylation, could give almost the same LC-MS/MS signal patterns as RNA PT. As the LC-MS/MS technique led to contradicting conclusions, another independent method is thus needed to interrogate the presence of RNA PT in nature. In this work, we have developed a highly selective and sensitive fluorescence method for RNA PT quantification based on a new RNA PT-specific conversion reaction. It can detect as low as 2.8 PPM-nt in RNA without interference from RNA thiobases or protein cysteines. We measured the total RNA samples from some bacteria and human cells using this method. None of these samples gave any RNA PT signal above the detection limit (2.8 PPM-nt), suggesting that the widespread presence of natural RNA PT at the 100 PPM-nt level or above is highly unlikely. Nevertheless, due to the limited number of cell species tested in this work, the possible existence of natural RNA PT cannot be excluded. The fluorescence method reported here is simple and low-cost; therefore, it should be an ideal assay for broadly screening various types of cells to search for the clue of RNA PT in nature.

Co-cold extrusion synergized with cysteine for enhancing physicochemical, rheological characteristics and in vitro digestibility of whey protein isolate

Impacts of co-cold extrusion (≤50 °C) of whey protein isolate (WPI) and cysteine (Cys, 0, 20, 40, 60, 80 and 100 mmol/L) on its physicochemical, in vitro digestion and rheological properties were investigated. As Cys concentration increased, the emulsifying properties and in vitro digestibility of co-extruded WPI-Cys products showed an increasing trend. Specifically, when Cys reached 100 mmol/L, surface hydrophobicity, emulsification activity index (EAI), emulsification stability index (ESI) and in vitro stomach digestibility of the co-extruded WPI-Cys products increased by 205.07%, 77.51%, 193.95% and 71.81% compared with WPI, respectively. Principal component analysis (PCA) results further indicated that co-extruded WPI-Cys at a concentration of 100 mmol/L had the best functional properties. In addition, co-extruded WPI-Cys exhibited the strongest Péclet number (Pe) value and apparent viscosity at a Cys concentration of 100 mmol/L among all samples. Therefore, co-extrusion would be an effective method for modifying WPI, providing whey protein-based ingredients with excellent functional properties for food processing.

Abstract Tu128: p22 phox is a critical factor for the prevention of oxidation and stabilization of SERCA2a in the heart.

Introduction: Elevated oxidative stress linked with heart failure is a major cause for the downregulation of sarcoplasmic/endoplasmic reticulum (SR/ER) Ca2+ ATPase 2a (SERCA2a). NADPH oxidase (NOX) complexes are a key source of reactive oxygen species (ROS) in cardiomyocytes. p22 phox , a transmembrane partner of NOX1-4, plays an essential role in mediating ROS production in multiple NOX complexes. We investigated the role of p22 phox in oxidative stress and SERCA2a levels during pressure overload (PO). Question: Does loss of p22 phox alleviate heart failure through reduction of oxidative stress and stabilization of SERCA2a levels during PO? Methods/approach: Cardiac-specific p22 phox knockout ( p22 phox -cKO) mice were subjected to sham operation or transverse aortic constriction (TAC). The p22 phox interactome was analyzed by co-immunoprecipitation and mass spectrometry. Protein cysteine oxidation was probed by biotinylated-iodoacetamide (BIAM) labelling. Results: The p22 phox -cKO mouse heart showed a 30% reduction in Dityrosine levels compared to WT after 4-week TAC (p<0.05) and 50% lower total tissue H 2 O 2 levels compared to WT after 1-week TAC (p<0.01). Unexpectedly, p22 phox -cKO mice had higher mortality (+20%, p<0.05), lung congestion (2-fold, p<0.05), and fibrosis (>40%, p<0.01) and a lower (40%, p<0.01) left ventricle ejection fraction than WT after TAC. p22 phox directly interacted with SERCA2a. Lack of p22 phox led to oxidation of SERCA2a (50% reduction in BIAM labelling, p<0.01) at cysteine 498 and promoted proteasome-mediated degradation of SERCA2a. The relative SERCA2a ATPase activity was unaltered in both WT and p22 phox -cKO mice. Furthermore, in the absence of p22 phox , SERCA2a interacted strongly with HMG-CoA reductase degradation protein 1 (Hrd1), an endoplasmic reticulum-associated degradation (ERAD)-specific E3 ubiquitin ligase. Knockdown of Hrd1 in the presence of p22 phox knockdown restored SERCA2a to 70% of WT levels (ns and p<0.5 vs p22 phox knockdown). SERCA2a C498S knock-in mice exhibited better cardiac function and stable SERCA2a protein levels after TAC compared to WT mice. Conclusion: The loss of p22 phox exacerbated heart failure through increased SERCA2a cysteine 498 oxidation and degradation through ERAD.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 μg of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g., H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 μg of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamline the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Evidence of Gas Phase Glucosyl Transfer and Glycation in the CID/HCD-Spectra of S-Glucosylated Peptides.

Protein cysteine S-glycosylation is a relatively rare and less well characterized post-translational modification (PTM). Creating reliable model proteins that carry this modification is challenging. The lack of available models or natural S-glycosylated proteins significantly hampers the development of mass-spectrometry-based (MS-based) methodologies for detecting protein cysteine S-glycosylation in real-world proteomic studies. There is also limited MS-sequencing data describing it as easier to create synthetic S-glycopeptides. Here, we present the results of an in-depth manual analysis of automatically annotated CID/HCD spectra for model S-glucopeptides. The CID spectra show a long series of y/b-fragment ions with retained S-glucosylation, regardless of the dominant m/z signals corresponding to neutral loss of 1,2-anhydroglucose from the precursor ions. In addition, the spectra show signals manifesting glucosyl transfer from the cysteine position onto lysine, arginine (Lys, Arg) side chains, and a peptide N-terminus. Other spectral evidence indicates that the N-glucosylated initial products of transfer are converted into N-fructosylated (i.e., glycated) structures due to Amadori rearrangement. We discuss the peculiar transfer of the glucose oxocarbenium ion (Glc+) to positively charged guanidinium residue (ArgH+) and propose a mechanism for the gas-phase Amadori rearrangement involving a 1,2-hydride ion shift.

Analysis of Protein Cysteine Acylation Using a Modified Suspension Trap (Acyl-Trap).

Proteins undergo reversible S-acylation via a thioester linkage in vivo. S-palmitoylation, modification by C16:0 fatty acid, is a common S-acylation that mediates critical protein-membrane and protein-protein interactions. The most widely used S-acylation assays, including acyl-biotin exchange and acyl resin-assisted capture, utilize blocking of free Cys thiols, hydroxylamine-dependent cleavage of the thioester and subsequent labeling of nascent thiol. These assays generally require >500 micrograms of protein input material per sample and numerous reagent removal and washing steps, making them laborious and ill-suited for high throughput and low input applications. To overcome these limitations, we devised "Acyl-Trap", a suspension trap-based assay that utilizes a thiol-reactive quartz to enable buffer exchange and hydroxylamine-mediated S-acyl enrichment. We show that the method is compatible with protein-level detection of S-acylated proteins (e.g. H-Ras) as well as S-acyl site identification and quantification using "on trap" isobaric labeling and LC-MS/MS from as little as 20 micrograms of protein input. In mouse brain, Acyl-Trap identified 279 reported sites of S-acylation and 1298 previously unreported putative sites. Also described are conditions for long-term hydroxylamine storage, which streamlines the assay. More generally, Acyl-Trap serves as a proof-of-concept for PTM-tailored suspension traps suitable for both traditional protein detection and chemoproteomic workflows.

Sulfur dioxide inhibits mast cell degranulation by sulphenylation of galectin-9 at cysteine 74.

Mast cell (MC) degranulation is a key process in allergic reactions and inflammatory responses. Aspartate aminotransferase 1 (AAT1)-derived endogenous sulfur dioxide (SO2) is an important regulator of MC function. However, the mechanism underlying its role in MC degranulation remains unclear. This study aimed to investigate the mechanism by which endogenous SO2 controlled MC degranulation. HMC-1 and Rat basophilic leukemia cell MC line (RBL-2H3) were used in the cell experiments. SO2 content was detected by in situ fluorescent probe. MC degranulation represented by the release rate of MC β-hexosaminidase was determined using a colorimetric assay. Sulfenylation of galectin-9 (Gal-9) in MCs and purified protein was detected using a biotin switch assay. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the exact sulfenylation sites of Gal-9 by SO2. Animal models of passive cutaneous anaphylaxis (PCA) and hypoxia-driven pulmonary vascular remodeling were used to investigate the effect of SO2 on mast cell activation in vivo. Site-directed mutation of Gal-9 was conducted to confirm the exact site of SO2 and support the significance of SO2/Gal-9 signal axis in the regulation of MC degranulation. Degranulation was increased in AAT1-knockdowned MCs, and SO2 supplementation reversed the increase in MC degranulation. Furthermore, deficiency of endogenous SO2 contributed to IgE-mediated degranulation in vitro. Besides, SO2 inhibited IgE-mediated and hypoxia-driven MC degranulation in vivo. Mechanistically, LC-MS/MS analysis and site-directed mutation results showed that SO2 sulfenylated Gal-9 at cysteine 74. Sulfenylation of the 74th cysteine of Gal-9 protein was required in the SO2-inhibited MC degranulation under both physiological and pathophysiological conditions. These findings elucidated that SO2 inhibited MC degranulation via sulfenylating Gal-9 under both physiological and pathophysiological conditions, which might provide a novel treatment approach for MC activation-related diseases.

The Dual Roles of S-Nitrosylation of Proteins in Cancer: Molecular Mechanisms and Recent Advancements

<p class="MsoNormal" style="text-align: justify;"><span lang="EN-US" style="font-family: Nunito; color: #212529; background: white;">Protein S-nitrosylation (SNO), emerging as an important posttranslational modification, involves covalent addition of nitric oxide (NO) to the sulfur atom of cysteine in proteins. Accumulated evidence suggests that protein SNO plays crucial roles in pathophysiological mechanisms in cancer, which is attracting great attention. However, there are still controversies about whether S-nitrosylated proteins act as oncogenic proteins or tumor suppressors in cancer. In this review, we provide an overview of the early and latest evidence regarding the underlying mechanism and dual roles of SNO in cancer, in an effort to clarify its contribution in tumor progression. It has been well established that S-nitrosylated proteins restrain tumor progression in several types of cancer, while they have exhibited activities in promoting cell proliferation and inhibiting apoptosis in some other kinds of cancer. Interestingly, emerging evidence also has highlighted both its anti-cancer and pro-tumorigenic roles in several other cancer diseases. Finally, current limitations and future research prospects are presented. The overview of targeting SNO in cancer will provide new opportunities for drug development through in-depth exploration of SNO-mediated signaling pathways.</span></p>