The multifunctional protein Y-box binding protein 1 (YBX1), is a critical regulator of transcription and translation, and is widely recognized as an oncogenic driver in several solid tumors, ...including colorectal cancer (CRC). However, very little is known about the upstream or downstream factors that underlie YBX1's regulation and involvement in CRC. Previously, we demonstrated that YBX1 overexpression correlated with potent activation of nuclear factor κB (NF-κB), a well-known transcription factor believed to be crucial in CRC progression. Here, we report a novel interaction between NF-κB, YBX1 and protein arginine methyltransferase 5 (PRMT5). Our findings reveal for the first time that PRMT5 catalyzes methylation of YBX1 at arginine 205 (YBX1-R205me2), an event that is critical for YBX1-mediated NF-κB activation and its downstream target gene expression. Importantly, when WT-YBX1 is overexpressed, this methylation exists under basal (unstimulated) conditions and is further augmented upon interleukin-1β (IL-1β) stimulation. Mechanistically, co-immunoprecipitation studies reveal that the R205 to alanine (A) mutant of YBX1 (YBX1-R205A) interacted less well with the p65 subunit of NF-κB and attenuated the DNA binding ability of p65. Importantly, overexpression of YBX1-R205A significantly reduced cell growth, migration and anchorage-independent growth of CRC cells. Collectively, our findings shed important light on the regulation of a novel PRMT5/YBX1/NF-κB axis through PRMT5-mediated YBX1-R205 methylation. Given the fact that PRMT5, YBX1 and NF-κB are all among top crucial factors in cancer progression, pharmacological disruption of this pivotal axis could serve as the basis for new therapeutics for CRC and other PRMT5/YBX1/NF-κB-associated cancers.
This paper introduces in detail the development process of full screen camera technology and products in the past two years, carefully compares the TFT inside method represented by Samsung Fold3 ...product and the TFT outside method represented by ZTE Axon30 product, and proposes the FDC (Full Display with Camera) technology developed by BOE that has been used in ZTE Axon40 product. Compared with the one‐ to‐multi array technology launched by Visionox, BOE's one‐to‐one driving method realized by TFT compression design is optimal in both display quality and photographing, which can be really called full screen technology.
Cysteine (Cys) residues are major causes of crystallin disulfide formation and aggregation in aging and cataractous human lenses. We recently found that disulfide linkages are highly and partly ...conserved in β- and γ-crystallins, respectively, in human age-related nuclear cataract and glutathione depleted LEGSKO mouse lenses, and could be mimicked by in vitro oxidation. Here we determined which Cys residues are involved in disulfide-mediated crosslinking of recombinant human γD-crystallin (hγD). In vitro diamide oxidation revealed dimer formation by SDS-PAGE and LC-MS analysis with Cys 111-111 and C111-C19 as intermolecular disulfides and Cys 111-109 as intramolecular sites. Mutation of Cys111 to alanine completely abolished dimerization. Addition of αB-crystallin was unable to protect Cys 111 from dimerization. However, Cu2+-induced hγD-crystallin aggregation was suppressed up to 50% and 80% by mutants C109A and C111A, respectively, as well as by total glutathionylation. In contrast to our recently published results using ICAT-labeling method, manual mining of the same database confirmed the specific involvement of Cys111 in disulfides with no free Cys111 detectable in γD-crystallin from old and cataractous human lenses. Surface accessibility studies show that Cys111 in hγD is the most exposed Cys residue (29%), explaining thereby its high propensity toward oxidation and polymerization in the aging lens.
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•In vitro oxidation of human γD crystallin leads to dimerization that is abolished by C111A mutation.•ICAT labelling of γD-crystallin from old and cataractous human lenses confirms Cys111 involved in disulfides in vivo.•Cys111 plays a key role in Cu2+ induced hγD-crystallin aggregation and precipitation.•Glutathionylation of Cys 111 protects against dimerization and aggregation.
αA-crystallin is abundant in the lens of the eye and acts as a molecular chaperone by preventing aggregation of denaturing proteins. We previously found that chemical modification of the guanidino ...group of selected arginine residues by a metabolic α-dicarbonyl compound, methylglyoxal (MGO), makes human αA-crystallin a better chaperone. Here, we examined how the introduction of additional guanidino groups and modification by MGO influence the structure and chaperone function of αA-crystallin. αA-crystallin lysine residues were converted to homoarginine by guanidination with o-methylisourea (OMIU) and then modified with MGO. LC-ESI-mass spectrometry identified homoargpyrimidine and homohydroimidazolone adducts after OMIU and MGO treatment. Treatment with 0.25 M OMIU abolished most of the chaperone function. However, subsequent treatment with 1.0 mM MGO not only restored the chaperone function but increased it by ∼40% and ∼60% beyond that of unmodified αA-crystallin, as measured with citrate synthase and insulin aggregation assays, respectively. OMIU treatment reduced the surface hydrophobicity but after MGO treatment, it was ∼39% higher than control. FRET analysis revealed that αA-crystallin subunit exchange rate was markedly retarded by OMIU modification, but was enhanced after MGO modification. These results indicate a pattern of loss and gain of chaperone function within the same protein that is associated with introduction of guanidino groups and their neutralization. These findings support our hypothesis that positively charged guanidino group on arginine residues keeps the chaperone function of αA-crystallin in check and that a metabolic α-dicarbonyl compound neutralizes this charge to restore and enhance chaperone function.
Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl ...radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a “one-pot” reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois’ reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as “one-pot” reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.
Y box protein 1 (YBX1) is a well known oncoprotein that has tumor-promoting functions. YBX1 is widely considered to be an attractive therapeutic target in cancer. To develop novel therapeutics to ...target YBX1, it is of great importance to understand how YBX1 is finely regulated in cancer. Previously, we have shown that YBX1 could function as a tumor promoter through phosphorylation of its Ser-165 residue, leading to the activation of the NF-κB signaling pathway (1). In this study, using mass spectrometry analysis, we discovered a distinct phosphorylation site, Ser-176, on YBX1. Overexpression of the YBX1-S176A (serine-to-alanine) mutant in either HEK293 cells or colon cancer HT29 cells showed dramatically reduced NF-κB-activating ability compared with that of WT-YBX1, confirming that Ser-176 phosphorylation is critical for the activation of NF-κB by YBX1. Importantly, the mutant of Ser-176 and the previously reported Ser-165 sites regulate distinct groups of NF-κB target genes, suggesting the unique and irreplaceable function of each of these two phosphorylated serine residues. Our important findings could provide a novel cancer therapy strategy by blocking either Ser-176 or Ser-165 phosphorylation or both of YBX1 in colon cancer.
Summary
Age‐related cataractogenesis is associated with disulfide‐linked high molecular weight (HMW) crystallin aggregates. We recently found that the lens crystallin disulfidome was evolutionarily ...conserved in human and glutathione‐depleted mouse (LEGSKO) cataracts and that it could be mimicked by oxidation in vitro (Mol. Cell Proteomics, 14, 3211‐23 (2015)). To obtain a comprehensive blueprint of the oxidized key regulatory and cytoskeletal proteins underlying cataractogenesis, we have now used the same approach to determine, in the same specimens, all the disulfide‐forming noncrystallin proteins identified by ICAT proteomics. Seventy‐four, 50, and 54 disulfide‐forming proteins were identified in the human and mouse cataracts and the in vitro oxidation model, respectively, of which 17 were common to all three groups. Enzymes with oxidized cysteine at critical sites include GAPDH (hGAPDH, Cys247), glutathione synthase (hGSS, Cys294), aldehyde dehydrogenase (hALDH1A1, Cys126 and Cys186), sorbitol dehydrogenase (hSORD, Cys140, Cys165, and Cys179), and PARK7 (hPARK7, Cys46 and Cys53). Extensive oxidation was also present in lens‐specific intermediate filament proteins, such as BFSP1 and BFSP12 (hBFSP1 and hBFSP12, Cys167, Cys65, and Cys326), vimentin (mVim, Cys328), and cytokeratins, as well as microfilament and microtubule filament proteins, such as tubulin and actins. While the biological impact of these modifications for lens physiology remains to be determined, many of these oxidation sites have already been associated with either impaired metabolism or cytoskeletal architecture, strongly suggesting that they have a pathogenic role in cataractogenesis. By extrapolation, these findings may be of broader significance for age‐ and disease‐related dysfunctions associated with oxidant stress.
We studied the ionization equilibria of the COOH groups bound to the copolymer and to the cross-linked copolymer network (i.e., gel) consisting of N-isopropylacrylamide (NIPA) and acrylic acid (AAc) ...by means of potentiometric titration with NaOH. Microgel particles (average hydrodynamic diameter ∼ 120 nm in the acid form and at 25 °C; COOH content ∼ 30 mol %) were employed, in addition to the usual bulk gel (of a fine grind) with the same COOH content. The microgel was prepared by aqueous redox polymerization using N,N ‘-methylenebis(acrylamide) as a cross-linker and sodium dodecylbenzenesulfonate as a surfactant. The titration was carried out at 25 and 35 °C in the absence and the presence of NaCl. The dependence of the apparent dissociation constant (pK a) on the degree of dissociation (αd) was investigated. It was found that, on the whole, pK a increased with increasing αd but decreased by addition of NaCl. Over a wide αd range there was little difference in pK a between both gels, as well as between the gel and copolymer. At 35 °C and at αd < 0.15, however, an increase in αd brought about a distinct fall in pK a for the microgel as well as the copolymer. At αd < 0.15, pK a was larger at 35 °C than at 25 °C. To discuss these results in connection with the swelling behavior of the gel, size changes of the microgel particles were studied using the photon correlation spectroscopy. The size of the microgel increased with increasing the degree of neutralization (αn) but decreased by screening the COO- charges with counterions. Over the αn range of 0.6 to 1.0, there was little or no increase in the particle size. These aspects were analogous to those generally observed in the chain expansion of poly(AAc) in solutions, suggesting that the “counterion binding effect” plays an important role in the swelling of NIPA/AAc gels at high charge densities. It was also suggestive that hydrogen bonding as well as hydrophobic interaction affects the ionization of COOH and thereby the swelling degree at 35 °C and at αd < 0.15. Therefore, it may be concluded that our ionic NIPA gels swell through an increase in the net charge density due to the COO- ions rather than a rise in the osmotic pressure arising from mobile counterions within the gel phase.
Interphotoreceptor retinoid‐binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and ...polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo‐electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N‐linked carbohydrate post‐translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three‐dimensional architecture not seen among other retinoid‐binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
CD318 is a ligand for CD6 Enyindah-Asonye, Gospel; Li, Yan; Ruth, Jeffrey H. ...
Proceedings of the National Academy of Sciences - PNAS,
08/2017, Letnik:
114, Številka:
33
Journal Article
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It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune ...conditions suggest that the CD6–CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.
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