Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of ...complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific
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-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for
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-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the
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-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.
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In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of ...separate Cys residues, which are allosterically linked. S-Oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4. In isolated SR (SR vesicles), an average of six to eight Cys thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys residues/RyR1 monomer is unknown. Here we use isotope-coded affinity tag labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys residues) to identify 13 Cys residues subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys residues are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance NADPH oxidase 4 activity. pO2-sensitive Cys residues were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (three of 21) or as S-nitrosylated. Cys residues subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1 in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca(2+) channel (dihydropyridine receptor) and FK506-binding protein 12 as well as in "hot spot" regions containing sites of mutation implicated in malignant hyperthermia and central core disease. pO2-coupled disulfide formation was identified, whereas neither S-glutathionylated nor sulfenamide-modified Cys residues were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.
Abstract
Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the US with a staggeringly less than 14% survival rate for patients with metastatic disease. This poor ...clinical outcome for CRC patients is largely attributed to the lack of understanding of the factors that drive CRC progression, leading to a dearth of available treatment strategies for this disease. Today, treatment modalities and clinical management of colorectal cancer (CRC) are fundamentally based on the success of controlling specific tumorigenic pathways. Interestingly, aberrant activation of the transcription factor nuclear factor ĸB (NF-ĸB) is increasingly recognized as a crucial player in CRC progression. Therefore, understanding the mechanisms underlying regulation of NF-ĸB holds great promise for devising new therapeutic strategies for CRC. Recently, we identified the oncogenic protein, Y-box binding protein 1 (YBX1), as a novel activator of NF-κB. Since YBX1 overexpression has also been linked to poor patient outcome in CRC, we sought to further understand whether this YBX1/NF-ĸB axis could be a potential target for CRC treatment. In this study, we discovered that YBX1 is methylated on arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-ĸB activation and target gene expression. Additionally, co-immunoprecipitation studies revealed that the R205 to alanine (A) mutant (YBX1-R205A) also significantly diminished the interaction between YBX1 and the p65 subunit of the transcriptionally active NF-ĸB, demonstrating a novel mechanism by which methylation of YBX1 mediates protein-protein interactions. Overexpression of YBX1-R205A significantly attenuated the migration, proliferation, and anchorage-independent growth of a panel of CRC cells, suggesting that YBX1-R205me2 is essential to the oncogenic functions exerted by the YBX1/NF-ĸB axis in CRC. Furthermore, we revealed that protein arginine methyltransferase 5 (PRMT5) is responsible for the methylation of YBX1-R205. Collectively, our novel findings present a complex picture of the sophisticated regulation of NF-ĸB through PRMT5-mediated YBX1-R205 methylation and suggest that pharmacological disruption of the YBX1/NF-κB axis using PRMT5 inhibitors could serve as the basis for new therapeutics that impede YBX1/NF-κB-driven CRC progression.
Citation Format: Antja-Voy Hartley, Benlian Wang, Masaru Miyagi, Rasika Mundade, James Hamilton, Tao Lu. PRMT5-mediated methylation of YBX1 regulates NF-kB activity in colorectal cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3346.
Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human ...cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract.
Γ-Crystallins play a major role in age-related lens transparency. Their destabilization by mutations and physical chemical insults are associated with cataract formation. Therefore, drugs that ...increase their stability should have anticataract properties. To this end, we screened 2560 Federal Drug Agency–approved drugs and natural compounds for their ability to suppress or worsen H2O2 and/or heat-mediated aggregation of bovine γ-crystallins. The top two drugs, closantel (C), an antihelminthic drug, and gambogic acid (G), a xanthonoid, attenuated thermal-induced protein unfolding and aggregation as shown by turbidimetry fluorescence spectroscopy dynamic light scattering and electron microscopy of human or mouse recombinant crystallins. Furthermore, binding studies using fluorescence inhibition and hydrophobic pocket–binding molecule bis-8-anilino-1-naphthalene sulfonic acid revealed static binding of C and G to hydrophobic sites with medium-to-low affinity. Molecular docking to HγD and other γ-crystallins revealed two binding sites, one in the “NC pocket” (residues 50–150) of HγD and one spanning the “NC tail” (residues 56–61 to 168–174 in the C-terminal domain). Multiple binding sites overlap with those of the protective mini αA-crystallin chaperone MAC peptide. Mechanistic studies using bis-8-anilino-1-naphthalene sulfonic acid as a proxy drug showed that it bound to MAC sites, improved Tm of both H2O2 oxidized and native human gamma D, and suppressed turbidity of oxidized HγD, most likely by trapping exposed hydrophobic sites. The extent to which these drugs act as α-crystallin mimetics and reduce cataract progression remains to be demonstrated. This study provides initial insights into binding properties of C and G to γ-crystallins.
Complexation between human serum albumin (HSA) and poly(ethylene glycol) (PEG) was studied using different experimental techniques: quasi-elastic light scattering (QELS), static light scattering ...(SLS), electrophoretic light scattering (ELS), dialysis, and fluorescence spectroscopy. The QELS study for aqueous HSA−PEG mixtures at different levels of pH and ionic strength (NaCl) showed the formation of a water-soluble complex, the size of which varied depending on both the ionic strength and the molecular weight of PEG but remained unaltered when the mixing ratio of PEG to HSA was varied. The study of the complexation in the presence and absence of 1 M urea as a function of pH by QELS and fluorescence spectroscopy strongly suggested that hydrogen bonding plays an important role in the complex formation. A combination of SLS and dialysis at pH 2 and at the ionic strength 0.1 demonstrated that the complexation yielded an “intrapolymer” complex in which several HSA molecules bound to a PEG chain. In addition, ELS indicated that the resulting intrapolymer complex behaves like a free draining coil during electrophoresis.
The β2-adrenergic receptor (β2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of β-agonist drugs in asthma is limited by ...desensitization and tachyphylaxis. We find that during activation, the β2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive β2AR internalization in the absence of traditional agonist. Mutant β2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in β2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.
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•β2AR is S-nitrosylated at Cys265, mediating receptor desensitization•S-nitrosylation drives β2AR internalization via caveolae•β2AR lacking S-nitrosylation exhibits prolonged signaling to cAMP, Erk, and NO•Mice with β2AR refractory to S-nitrosylation are protected from asthma
Post-translational modification of the β2AR by S-nitrosylation, resulting from receptor activation or direct modification, promotes receptor desensitization and caveolar internalization. Preventing β2AR S-nitrosylation leads to sustained receptor signaling, thereby increasing cAMP, Erk, and NO. Mice with mutant β2AR refractory to S-nitrosylation are resistant to bronchoconstriction, inflammation, and development of asthma, suggesting that this newly identified desensitization mechanism may be targeted therapeutically in obstructive airway diseases.
α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous ...studies have identified the peptide 70KFVIFLDVKHFSPEDLTVK88 in αA-crystallin and the peptide 73DRFSVNLDVKHFSPEELKVK92 in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys70) of αA and Lys92 of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.
Background: Peptides derived from the core domain of human α-crystallin act as molecular chaperones.
Results: Chaperone peptides of α-crystallin inhibit stress-induced apoptosis in cultured cells and prevent experimental cataracts in rats.
Conclusion: Chaperone peptides of α-crystallin are anti-apoptotic and retain biological activity when injected into animals.
Significance: α-Crystallin peptides could be used as therapeutic agents to inhibit protein aggregation and apoptosis in diseases.
Nuclear factor κB (NF-κB) is a central coordinator in immune and inflammatory responses. Constitutive NF-κB is often found in some types of cancers, contributing to oncogenesis and tumor progression. ...Therefore, knowing how NF-κB is regulated is important for its therapeutic control. Post-translational modification of the p65 subunit of NF-κB is a well known approach for its regulation. Here, we reported that in response to interleukin 1β, the p65 subunit of NF-κB is phosphorylated on the novel serine 316. Overexpression of S316A (serine 316 → alanine) mutant exhibited significantly reduced ability to activate NF-κB and decreased cell growth as compared with wtp65 (wild type p65). Moreover, conditioned media from cells expressing the S316A-p65 mutant had a considerably lower ability to induce NF-κB than that of wtp65. Our data suggested that phosphorylation of p65 on Ser-316 controls the activity and function of NF-κB. Importantly, we found that phosphorylation at the novel Ser-316 site and other two known phosphorylation sites, Ser-529 and Ser-536, either individually or cooperatively, regulated distinct groups of NF-κB-dependent genes, suggesting the unique role of each individual phosphorylation site on NF-κB-dependent gene regulation. Our novel findings provide an important piece of evidence regarding differential regulation of NF-κB-dependent genes through phosphorylation of different p65 serine residues, thus shedding light on novel mechanisms for the pathway-specific control of NF-κB. This knowledge is key to develop strategies for prevention and treatment of constitutive NF-κB-driven inflammatory diseases and cancers.
Background: Post-translational modification is an important approach to regulate NF-κB activity.
Results: Serine 316 (Ser-316) is a novel phosphorylation site on p65.
Conclusion: Phosphorylation of Ser-316 on p65 is essential for NF-κB activation and its related biological functions.
Significance: Our data shed light on how NF-κB transcriptional specificity is achieved through site-specific phosphorylation.
The prefrontal cortex (PFC) has been implicated as a key brain region responsible for age-related cognitive decline. Little is known about aging-related molecular changes in PFC that may mediate ...these effects. To date, no studies have used untargeted discovery methods with integrated analyses to determine PFC molecular changes in healthy female primates. We quantified PFC changes associated with healthy aging in female baboons by integrating multiple omics data types (transcriptomics, proteomics, metabolomics) from samples across the adult age span. Our integrated omics approach using unbiased weighted gene co-expression network analysis to integrate data and treat age as a continuous variable, revealed highly interconnected known and novel pathways associated with PFC aging. We found Gamma-aminobutyric acid (GABA) tissue content associated with these signaling pathways, providing 1 potential biomarker to assess PFC changes with age. These highly coordinated pathway changes during aging may represent early steps for aging-related decline in PFC functions, such as learning and memory, and provide potential biomarkers to assess cognitive status in humans.
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