RESUMO
The identification of readers, an important class of proteins that recognize modified residues at specific sites, is essential to uncover the biological roles of post-translational modifications. Photoreactive crosslinkers are powerful tools for investigating readers. However, existing methods usually employ synthetically challenging photoreactive warheads, and their high-energy intermediates generated upon irradiation, such as nitrene and carbene, may cause substantial non-specific crosslinking. Here we report dimethylsulfonium as a methyllysine mimic that binds to specific readers and subsequently crosslinks to a conserved tryptophan inside the binding pocket through single-electron transfer under ultraviolet irradiation. The crosslinking relies on a protein-templated σ-π electron donor-acceptor interaction between sulfonium and indole, ensuring excellent site selectivity for tryptophan in the active site and orthogonality to other methyllysine readers. This method could escalate the discovery of methyllysine readers from complex cell samples. Furthermore, this photo crosslinking strategy could be extended to develop other types of microenvironment-dependent conjugations to site-specific tryptophan.
Assuntos
Lisina , Compostos de Sulfônio , Triptofano , Triptofano/química , Triptofano/análogos & derivados , Compostos de Sulfônio/química , Lisina/química , Lisina/análogos & derivados , Transporte de Elétrons , Raios Ultravioleta , Reagentes de Ligações Cruzadas/química , Processos Fotoquímicos , Humanos , Proteínas/químicaRESUMO
Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Proteínas NLR , Nicotiana , Imunidade Vegetal , Proteínas de Plantas , Multimerização Proteica , Solanum lycopersicum , Nicotiana/metabolismo , Nicotiana/imunologia , Proteínas NLR/metabolismo , Proteínas NLR/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/imunologia , Ácido Fítico/metabolismo , Ácido Fítico/química , Morte Celular , Ligação Proteica , MutaçãoRESUMO
The reverse cholesterol transport pathway is responsible for the maintenance of human cholesterol homeostasis, an imbalance of which usually leads to atherosclerosis. As a key component of this pathway, the ATP-binding cassette transporter ABCG1 forwards cellular cholesterol to the extracellular acceptor nascent high-density lipoprotein (HDL). Here, we report a 3.26-Å cryo-electron microscopy structure of cholesterol-bound ABCG1 in an inward-facing conformation, which represents a turnover condition upon ATP binding. Structural analyses combined with functional assays reveals that a cluster of conserved hydrophobic residues, in addition to two sphingomyelins, constitute a well-defined cholesterol-binding cavity. The exit of this cavity is closed by three pairs of conserved Phe residues, which constitute a hydrophobic path for the release of cholesterol in an acceptor concentration-dependent manner. Overall, we propose an ABCG1-driven cholesterol transport cycle initiated by sphingomyelin-assisted cholesterol recruitment and accomplished by the release of cholesterol to HDL.
Assuntos
Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/química , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Colesterol/metabolismo , Microscopia Crioeletrônica , Humanos , Esfingomielinas/metabolismoRESUMO
The supercritical fluid chromatography coupled with mass spectrometry (SFC-MS) method and liquid chromatography coupled with mass spectrometry (LC-MS) method were developed for the separation and characterization of poly (ethylene oxide) methyl glucose sesquistearate (PEO-Glu-sesquistearate). The products of PEO-Glu-sesquistearate are composed of complex oligomers. The relationship between molecular structure of these oligomers and chromatographic retention behavior in both SFC and LC were discussed and compared. As compared with LC, hydrophobic moieties of compounds favor the fast elution in SFC. The different series can be better separated by LC, while the homologues compounds in same series can be better separated by SFC, and SFC-MS provided more comprehensive structural information. Different series such as PEO-distearate, PEO-stearate, PEO, PEO-Glu-tetrastearate, PEO-Glu-tristearate, PEO-Glu-distearate, PEO-Glu-stearate, and PEO-Glu were identified by MS/MS.
RESUMO
RATIONALE: Pentaerythritol fatty acid esters are an excellent lubricant oil additive, due to their good biodegradability, thermal ability, anti-wear, and friction properties. However, to meet the application requirements, fatty acids with different alkyl chain lengths are reacted with pentaerythritol, resulting in complex ester compositions. To reveal the relation between the functionalities and the composition of esters, it is important to develop a method for their analysis. METHODS: We developed a method using ultra-high-performance supercritical fluid chromatography combined with quadrupole time-of-flight mass spectrometry (UHPSFC/QTOF-MS) to separate and characterize pentaerythritol fatty acid esters. This method has the advantages such as short analysis time and high separation efficiency for such weakly polar compounds; high-resolution mass spectrometry (HRMS) provides exact mass information, enabling the identification of the structure of the pentaerythritol fatty acid esters. RESULTS: Based on the exact masses and characteristic ions, the pentaerythritol fatty acid esters and their main fragmentation pathways were identified; the fatty acid composition was also deduced from characteristic product ions. A dihydrogen rearrangement reaction caused the neutral loss of fatty acid fragment; [M + Na-FA]+ product ions (a stable six-member ring structure) were produced due to the absence of a γ hydrogen in pentaerythritol fatty acid esters. CONCLUSIONS: A UHPSFC/QTOF-MS method was successfully employed for the separation of pentaerythritol fatty acid esters. Exact masses and product ion information were determined using HRMS. The composition of the fatty acids was effectively deduced by characteristic ions and their relative abundances. This method is an effective means for the quality control and process optimization of this type of product, serving as a positive reference for further study on pentaerythritol fatty acid esters.
RESUMO
Elimination of harmful antibodies from organisms is a valuable approach for the intervention of antibody-associated diseases, such as Dengue hemorrhagic fever and autoimmune diseases. Since thousands of antibodies with different epitopes are circulating in blood, no universal method, except for the dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF), was reported to target specific harmful antibodies. The development of DCAF molecules makes significant contribution to the progress of targeted therapy, which were demonstrated to eliminate the antibody dependent enhancement (ADE) effect in a Dengue virus (DENV) infection model and to boost the acetylcholine receptor activity in a myasthenia gravis model. Here, we describe a protocol for the synthesis of a DCAF molecule (DCAF1), which can selectively block 4G2 antibody to attenuate ADE effect during Dengue virus infection, and illustrate the binding of DCAF1 to 4G2 antibody by an ELISA assay. In our method, DCAF1 is synthesized by the conjugation of a hydrazine derivative of a Fc-III peptide and a recombinant expressed long α-helix with antigenic sequence through native chemical ligation (NCL). This protocol has been successfully applied to DCAF1 as well as other DCAF molecules for targeting their cognate antibodies.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Materiais Biomiméticos/farmacologia , Vírus da Dengue/imunologia , Fragmentos Fc das Imunoglobulinas/imunologia , Peptídeos/química , Animais , Materiais Biomiméticos/químicaRESUMO
The synthesis of atomically precise thiolate-stabilized silver (Ag) nanoclusters is the subject of intense research interest, yet the formation mechanism of such nanoclusters remains obscure. Here, electrospray ionization mass spectrometry is successfully applied to monitor the reaction intermediates formed during the sodium-borohydride-reduction of silver 4-tert-butylbenzenethiolate (AgSPh-tBu). We demonstrate a unique evolution route to thiolate-stabilized Ag nanoclusters mediated by Ag-thiolate clusters. The Ag-thiolate clusters form in the initial stage of reduction contain tens of Ag atoms and similar number of ligands, and they are transformed into Ag17(SPh-tBu)123- and Ag44(SPh-tBu)304- nanoclusters in the later reduction process. The number of Ag atoms in the Ag-thiolate clusters determines the reaction path to each final nanocluster product. A similar mechanism is found when silver 2,4-dimethylbenzenethiolate (AgSPhMe2) is used as precursor. This mechanism differs markedly from the long-established bottom-up evolution process, providing valuable new insights into the synthesis of metal nanoclusters.
RESUMO
Polysorbate 80, as a nonionic surfactant, is widely used in the food, personal care, and pharmaceutical industries due to the advantages of high surface activity, low toxicity, etc. In fact, the products of polysorbate 80 are complex mixtures of oligomers. In this work, a novel and fast method was developed to characterize the commercial polysorbate 80 by ultra-high performance supercritical fluid chromatography (UHPSFC) combined with quadrupole time-of-flight mass spectrometry (QTOF-MS). Some crucial parameters, such as temperature, back pressure and flow rate were optimized. UHPSFC could distinguish n-mer from (n-1)-mer and (n+1)-mer in the same series, which provided the high separation resolution needed for quantitative determination of each oligomer in same series. It was not achieved in previous studies. Furthermore, the characteristic ion fragments were found in MS/MS experiment and used to identify different series. The results revealed that main components of this nonionic surfactant comprise polyethylene oxide (PEO), PEO-monooleate, PEO-isosorbide, PEO-isosorbide monooleate, PEO-isosorbide dioleate, PEO-sorbitan, PEO-sorbitan monooleate, PEO-sorbitan dioleate and PEO-sorbitan trioleate, etc. The separation was performed using BEH stationary phase, so the relationship between molecular structure of these oligomers and chromatographic retention behavior in supercritical fluid chromatography were also investigated for first time. The whole analytical process only takes 8min for one sample. Therefore, UHPSFC-QTOF-MS is a simple, novel and efficient tool to analyze polysorbate 80.