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1.
Pharmacol Res ; 204: 107208, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38729587

RESUMEN

Cancer cell line is commonly used for discovery and development of anti-cancer drugs. It is generally considered that drug response remains constant for a certain cell line due to the identity of genetics thus protein patterns. Here, we demonstrated that cancer cells continued dividing even after reaching confluence, in that the proteomics was changed continuously and dramatically with strong relevance to cell division, cell adhesion and cell metabolism, indicating time-dependent intrinsically reprogramming of cells during expansion. Of note, the inhibition effect of most anti-cancer drugs was strikingly attenuated in culture cells along with cell expansion, with the strongest change at the third day when cells were still expanding. Profiling of an FDA-approved drug library revealed that attenuation of response with cell expansion is common for most drugs, an exception was TAK165 that was a selective inhibitor of mitochondrial respiratory chain complex I. Finally, we screened a panel of natural products and identified four pentacyclic triterpenes as selective inhibitors of cancer cells under prolonged growth. Taken together, our findings underscore that caution should be taken in evaluation of anti-cancer drugs using culture cells, and provide agents selectively targeting overgrowth cancer cells.


Asunto(s)
Antineoplásicos , Proliferación Celular , Proteómica , Humanos , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Factores de Tiempo , Productos Biológicos/farmacología , Triterpenos Pentacíclicos/farmacología
2.
Rapid Commun Mass Spectrom ; 38(1): e9673, 2024 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-38073198

RESUMEN

RATIONALE: Thermal proteome profiling (TPP) has been widely used for the identification of drug targets for several years, and TMTpro-16plex has recently been evaluated for TPP of vehicle- and drug-treated samples in a single labeling process to reduce missing values and save instrument time. A novel isobaric labeling reagent, IBT-16plex, was developed with slightly better performance in protein identification and quantification than the commercially available TMTpro-16plex. METHODS: In this study, we applied the newly developed IBT-16plex for target identification of methotrexate and panobinostat using TPP. RESULTS: The known targets of these two drugs were successfully identified with elevated melting temperatures, and some known off-targets and potential new off-targets were also identified. CONCLUSIONS: IBT-16plex can be a cost-effective replacement for TMTpro-16plex for TPP applications.


Asunto(s)
Proteoma , Proteómica , Proteoma/metabolismo , Panobinostat
3.
Nature ; 563(7730): 249-253, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30401835

RESUMEN

N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.


Asunto(s)
Adenina/análogos & derivados , Hipocampo/citología , Hipocampo/fisiología , Memoria/fisiología , Neuronas/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenina/metabolismo , Animales , Sitios de Unión , Femenino , Masculino , Metiltransferasas/deficiencia , Metiltransferasas/genética , Metiltransferasas/metabolismo , Ratones , Ratones Noqueados , Plasticidad Neuronal , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Aprendizaje Espacial/fisiología , Transmisión Sináptica
4.
Bioorg Chem ; 133: 106435, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36841049

RESUMEN

Herein, we synthesized an affinity-based probe of myricanol (pMY) with a photo-affinity cross-linker to initiate a bioconjugation reaction, which was applied for target identification in live C2C12 myotubes. Pull-down of biotinylated pMY coupled with mass spectroscopy and Western blotting revealed that pMY can bind with nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in the nicotinamide adenine dinucleotide salvage pathway. Cellular thermal shift assay, drug affinity responsive target stability assay and recombinant protein labeling further validated the direct interaction between myricanol and Nampt. Myricanol did not affect the protein expression of Nampt, but enhanced its activity. Knock-down of Nampt totally abolished the promoting effect of myricanol on insulin-stimulated glucose uptake in C2C12 myotubes. Taken together, myricanol sensitizes insulin action in myotubes through binding with and activating Nampt.


Asunto(s)
Insulinas , Nicotinamida Fosforribosiltransferasa , Nicotinamida Fosforribosiltransferasa/metabolismo , Nicotinamida Fosforribosiltransferasa/farmacología , Fibras Musculares Esqueléticas , Diarilheptanoides/farmacología , Citocinas/metabolismo , Insulinas/metabolismo , Insulinas/farmacología , NAD/metabolismo
5.
BMC Biol ; 19(1): 194, 2021 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-34493279

RESUMEN

BACKGROUND: KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. RESULTS: We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. CONCLUSIONS: These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Aparato de Golgi , Receptores de Péptidos , Membrana Celular , Proteínas Quinasas Dependientes de AMP Cíclico
6.
Br J Haematol ; 195(2): 267-275, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34409610

RESUMEN

The expression of GGCT (γ-glutamyl cyclotransferase) is upregulated in various human cancers. γ-glutamyl cyclotransferase enzyme activity was originally purified from human red blood cells (RBCs), but the physiological function of GGCT in RBCs is still not clear. Here we reported that Ggct deletion in mice leads to splenomegaly and progressive anaemia phenotypes, due to elevated oxidative damage and the shortened life span of Ggct-/- RBCs. Ggct-/- RBCs have increased reactive oxygen species (ROS), and are more sensitive to H2 O2 -induced damage compared to control RBCs. Glutathione (GSH) and GSH synthesis precursor l-cysteine are decreased in Ggct-/- RBCs. Our study suggests a critical function of Ggct in RBC redox balance and life span maintenance through regulating GSH metabolism.


Asunto(s)
Eritrocitos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , gamma-Glutamilciclotransferasa/metabolismo , Anemia/genética , Animales , Antioxidantes/metabolismo , Cisteína/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Eritropoyetina/metabolismo , Femenino , Eliminación de Gen , Glutatión/metabolismo , Masculino , Metabolómica/métodos , Ratones , Modelos Animales , Fenotipo , Esplenomegalia/genética , Regulación hacia Arriba/genética
7.
Small ; 17(23): e2100837, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33893713

RESUMEN

Efficient cell internalization of framework nucleic acid nanostructures free of transfection agents provides new opportunities for developing biocompatible and intelligent nanoprobes and drug delivery carriers. Here, a proteomic identification method to screen target proteins that interact with tetrahedral DNA nanostructures (TDNs) during the process of endocytosis by combining drug affinity responsive target stability (DARTS) with liquid chromatography/tandem mass spectrometry (LC-MS/MS) techniques, is reported. It is found that that caveolin-1 (CAV1) and macropinocytosis-related protein sorting nexin5 (SNX5) are associated with the endocytosis of TNDs, which is further validated by microscale thermophoresis (MST) analysis. CAV1- and SNX5- knockout experiments reveal that both caveolae-mediated endocytosis and macropinocytosis mediate the cellular uptake of TDNs, which complement previous findings with fluorescence tracing methods. This method provides a generic strategy to analyze cellular internalization process of DNA nanostructures for biomedical applications.


Asunto(s)
Ácidos Nucleicos , Cromatografía Liquida , Endocitosis , Proteómica , Espectrometría de Masas en Tándem
8.
Nat Methods ; 15(9): 715-722, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30104635

RESUMEN

The communication between cells and between cellular organelles is often controlled by the interaction of membrane proteins. Although many methods for the detection of protein-protein interactions (PPIs) exist, membrane PPIs remain difficult to detect. Here we developed a proximity-based tagging system, PUP-IT (pupylation-based interaction tagging), to identify membrane protein interactions. In this approach, a small protein tag, Pup, is applied to proteins that interact with a PafA-fused bait, enabling transient and weak interactions to be enriched and detected by mass spectrometry. Pup does not diffuse from the enzyme, which allows high-specificity labeling. We applied this approach to CD28, a critical costimulatory receptor for T lymphocyte activation, and identified known CD28 binding partners and multiple potential interacting proteins. In addition, we demonstrated that this method can identify the interaction between a cell surface receptor and its ligand.


Asunto(s)
Proteínas de la Membrana/metabolismo , Mapas de Interacción de Proteínas , Antígenos CD28/metabolismo , Humanos , Espectrometría de Masas , Unión Proteica , Proteolisis
9.
Proc Natl Acad Sci U S A ; 115(17): E4051-E4060, 2018 04 24.
Artículo en Inglés | MEDLINE | ID: mdl-29632189

RESUMEN

Despite decades of research, mechanisms controlling T cell activation remain only partially understood, which hampers T cell-based immune cancer therapies. Here, we performed a genome-wide CRISPR screen to search for genes that regulate T cell activation. Our screen confirmed many of the known regulators in proximal T cell receptor signaling and, importantly, also uncovered a previously uncharacterized regulator, FAM49B (family with sequence similarity 49 member B). FAM49B deficiency led to hyperactivation of Jurkat T cells following T cell receptor stimulation, as indicated by enhancement of CD69 induction, PAK phosphorylation, and actin assembly. FAM49B directly interacted with the active form of the small GTPase Rac, and genetic disruption of the FAM49B-Rac interaction compromised FAM49B function. Thus, FAM49B inhibits T cell activation by repressing Rac activity and modulating cytoskeleton reorganization.


Asunto(s)
Activación de Linfocitos , Proteínas de Neoplasias/inmunología , Linfocitos T/inmunología , Actinas/genética , Antígenos CD/genética , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Sistemas CRISPR-Cas , Citoesqueleto/genética , Citoesqueleto/inmunología , Estudio de Asociación del Genoma Completo , Humanos , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Linfocitos T/citología
10.
J Proteome Res ; 19(12): 4857-4866, 2020 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-33210925

RESUMEN

Since the Chromosome-Centric Human Proteome Project (C-HPP) was launched in 2010, many techniques have been adopted for the discovery of missing proteins (MPs). Because of these efforts, only 1481 MPs remained as of July 2020; however, by relying only on technique optimization, researchers have reached a bottleneck in MP discovery. Protein expression is tissue- or cell-type-dependent. The tissues of the human testis and brain have been reported to harbor a large number of tissue-specific genes and proteins; however, few studies have been performed on human brain tissue or cells to identify MPs. Herein a metastatic cell line derived from brain cancer, D283 Med, was used to search for MPs. With a traditional and simple shotgun workflow to separate the peptides into 20 fractions, 12 MPs containing at least two unique non-nested peptides (amino acid length ≥9) were identified in this cell line with a protein false discovery rate of <1%. Following the same experimental protocol, only one MP was found in a nonmetastatic brain cancer cell line, U-118 MG. Furthermore, 12 MPs were verified as having two non-nested unique peptides by matching them with corresponding chemically synthesized peptides through parallel reaction monitoring. These results clearly demonstrate that the appropriate selection of experimental materials, either tissues or cell lines, is imperative for MP discovery. The data obtained in this study are available via ProteomeXchange (PXD021482) and PeptideAtlas (PASS01627).


Asunto(s)
Neoplasias Cerebelosas , Meduloblastoma , Línea Celular , Humanos , Masculino , Meduloblastoma/genética , Péptidos , Proteómica
11.
J Proteome Res ; 19(1): 401-408, 2020 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-31773964

RESUMEN

The mission of the Chromosome-Centric Human Proteome Project (C-HPP) to discover missing proteins (MPs) has become increasingly difficult due to the remaining low-abundance, high-hydrophobicity, or low-molecular-weight MPs. We have reported two approaches to resolve these identification problems for the low-abundance and high-hydrophobicity MPs, respectively. In this study, to improve the identification of low-abundance MPs with high hydrophobicity, we combined two approaches and obtained MPs from several different cancer cell lines. Their membrane fractions were isolated by ultracentrifugation, and the low-abundance proteins were enriched at the protein level with the ProteoMiner kit. After that, the peptides from the enriched proteins were separated by high concentrations of organic solvents according to their hydrophobicity as the first dimension of separation at the peptide level, and the second and third dimensions of separation involved a high pH reversed-phase and an acid reversed-phase column, respectively. In total, 16 MPs (at least two non-nested unique peptides with ≥9 amino acids) with 61 unique peptides were identified from four human cancer cell lines, including 2, 8, 2, and 7 MPs from HeLa, HCT116, SNU-1, and HepG2 cells, respectively. Furthermore, all MPs were verified with two non-nested unique peptides through parallel reaction monitoring (PRM) by matching the peptides with their chemically synthesized peptides. Interestingly, two additional MPs were verified from the same cell line by PRM assay, although the two non-nested unique peptides with ≥9 amino acids for each MP were identified from different MS injections or cell lines by data-dependent acquisition (DDA). Thus, a total of 18 MPs were dug out in this study. The data are available via ProteomeXchange (PXD014058) and PeptideAtlas (PASS01388).


Asunto(s)
Proteínas/análisis , Proteínas/química , Proteómica/métodos , Línea Celular Tumoral , Electroforesis en Gel de Poliacrilamida , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/métodos
12.
J Biol Chem ; 294(48): 18306-18323, 2019 11 29.
Artículo en Inglés | MEDLINE | ID: mdl-31653698

RESUMEN

Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/ß-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/ß-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of ß-catenin on Tyr-142, a key site controlling the transcriptional activity of ß-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, ß-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating ß-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.


Asunto(s)
Neoplasias Ováricas/genética , Proteínas Tirosina Fosfatasas Clase 7 Similares a Receptores/genética , Vía de Señalización Wnt/genética , beta Catenina/genética , Animales , Línea Celular , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Humanos , Ratones Desnudos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosforilación , Interferencia de ARN , Tratamiento con ARN de Interferencia/métodos , Proteínas Tirosina Fosfatasas Clase 7 Similares a Receptores/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , beta Catenina/metabolismo
13.
Anal Chem ; 92(5): 3913-3922, 2020 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-31992042

RESUMEN

We describe an affinity purification-mass spectrometry (AP-MS) method for probing the interactome of a special targeting protein. The AP was implemented with monolithic micro immobilized metal ion affinity chromatography columns (m-IMAC) which were prepared by photoinitiated polymerization in the tip of a pipet (spin-tip columns). The recombinant His6-tagged protein (bait protein) was reversibly immobilized on the affinity column through the chelating group nitrilotriacetic acid (NTA)-Ni2+. The bait protein and its interacting partners can be easily eluted from the affinity matrix. The pulled-down cellular proteins were then analyzed with label-free quantitative proteomics. We used this method for probing the interactome concerning the GOLD (Golgi dynamics) domain of the autophagy-associated adaptor protein FYCO1. Totally, 96 proteins including seven literature-reported FYCO1-associating proteins were identified. Among them CCZ1 and MON1A were further biochemically validated, and the direct interaction between the FYCO1 GOLD domain with CCZ1 was confirmed by co-immunoprecipitation experiments.


Asunto(s)
Cromatografía de Afinidad/métodos , Mapas de Interacción de Proteínas/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Cromatografía Líquida de Alta Presión , Histidina/química , Histidina/genética , Histidina/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Ácido Nitrilotriacético/química , Oligopéptidos/química , Oligopéptidos/genética , Oligopéptidos/metabolismo , Péptidos/análisis , Unión Proteica , Proteómica/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Espectrometría de Masas en Tándem
14.
Rapid Commun Mass Spectrom ; 34(10): e8733, 2020 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-32031715

RESUMEN

High specificity of trypsin is a prerequisite for accurate identification and quantification of proteins in shotgun proteomics. It is important to minimize nonspecific enzymatic cleavages during proteomic sample preparation. METHODS: In this study, protein extraction and trypsin digestion conditions were extensively evaluated using the less-complex Escherichia coli lysates to improve the sensitivity of detecting low-abundance nonspecific peptides by liquid chromatography/tandem mass spectrometry. RESULTS: Trypsin digestion buffers and digestion times were proved to have a significant effect on nonspecific cleavages. The triethylammonium bicarbonate buffer induces significantly lower nonspecific cleavages than the other two buffers, but a freshly prepared urea solution does not induce more than sodium dodecyl sulfate. Because prolonged trypsin digestion resulted in a considerable number of nonspecific cleavages, an optimized 2-h protocol was developed with 45.2% less semispecific tryptic peptides but 18.5% more unmodified peptides identified than the commonly used 16-h protocol. CONCLUSIONS: The significant decrease in nonspecific cleavages and artificial modifications improves the accuracy of protein quantification and the identification of low-abundance proteins, and it is especially useful for studying protein posttranslational modifications. For trypsin digestion, the proposed 2-h protocol can potentially be a replacement for the traditional 16-h protocol.


Asunto(s)
Péptidos/análisis , Proteínas/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Tripsina/química , Células A549 , Animales , Bovinos , Cromatografía Liquida/métodos , Escherichia coli/química , Proteínas de Escherichia coli/análisis , Humanos , Proteolisis
15.
J Proteome Res ; 18(1): 461-468, 2019 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-30394099

RESUMEN

Quantitative proteomics has been extensively applied in the screening of differentially regulated proteins in various research areas for decades, but its sensitivity and accuracy have been a bottleneck for many applications. Every step in the proteomics workflow can potentially affect the quantification of low-abundance proteins, but a systematic evaluation of their effects has not been done yet. In this work, to improve the sensitivity and accuracy of label-free quantification and tandem mass tags (TMT) labeling in quantifying low-abundance proteins, multiparameter optimization was carried out using a complex 2-proteome artificial sample mixture for a series of steps from sample preparation to data analysis, including the desalting of peptides, peptide injection amount for LC-MS/MS, MS1 resolution, the length of LC-MS/MS gradient, AGC targets, ion accumulation time, MS2 resolution, precursor coisolation threshold, data analysis software, statistical calculation methods, and protein fold changes, and the best settings for each parameter were defined. The suitable cutoffs for detecting low-abundance proteins with at least 1.5-fold and 2-fold changes were identified for label-free and TMT methods, respectively. The use of optimized parameters will significantly improve the overall performance of quantitative proteomics in quantifying low-abundance proteins and thus promote its application in other research areas.


Asunto(s)
Proteínas/análisis , Proteómica/métodos , Cromatografía Liquida , Análisis de Datos , Programas Informáticos , Manejo de Especímenes/métodos , Espectrometría de Masas en Tándem , Flujo de Trabajo
16.
J Proteome Res ; 18(12): 4180-4188, 2019 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-31592669

RESUMEN

Identifying more missing proteins (MPs) is an important mission of C-HPP. With the number of identified MPs being attenuated year by year (2,949 to 2,129 MPs from 2016 to 2019), we have realized that the difficulty of exploring the remaining MPs is a challenge in technique. Herein, we propose a comprehensive strategy to effectively enrich, separate, and identify proteins with low molecular weights, aiming at the discovery of MPs. Basically, a protein extract from human placenta was passed through a C18 SPE column, and the bound proteins that were eluted were further separated with an SDS-PAGE gel or a 50 kDa cutoff filter. The separated proteins were subjected to trypsin digestion, and the MS/MS signals were searched against data sets with two different digestion modes (full-trypsin and semitrypsin). The strategy was adopted, resulting in the identification of 4 MPs with 8 unique peptides (≥2 non-nested unique peptides with ≥9 amino acids). Importantly, the identification of 6 out of 8 of the unique peptides derived from the MPs was further supported by parallel reaction monitoring, which confirmed the identification of 3 MPs from human placenta tissues (Q6NT89: TMF-regulated nuclear protein 1; A0A183: late cornified envelope protein 6A; and Q6UWQ7: insulin growth factor-like family member 2, mapped to chromosomes 1, 1, and 19, respectively). The three proteins ranged in length from 80 aa to 227 aa. The study not only establishes a feasible strategy for analyzing proteins with low molecular weights but also fills a small part of a large gap in the list of MPs. The data obtained in this study are available via ProteomeXchange (PXD014083) and PeptideAtlas (PASS01389).


Asunto(s)
Péptidos/análisis , Placenta/química , Proteómica/métodos , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Peso Molecular , Péptidos/química , Embarazo , Espectrometría de Masas en Tándem/métodos , Tripsina/química
17.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-31671542

RESUMEN

Understanding the fundamental role of the stroma in normal development and cancer progression has been an emerging focus in recent years. The receptor tyrosine kinase (RTK) signaling pathway has been reported playing critical roles in regulating the normal and cancer microenvironment, but the underlying mechanism is still not very clear. By applying the quantitative phosphoproteomic analysis of Sprouty proteins (SPRYs), generic modulators of RTK signaling and deleted mouse mammary fibroblasts, we quantified a total of 11,215 unique phosphorylation sites. By contrast, 554 phosphorylation sites on 425 proteins had SPRY-responsive perturbations. Of these, 554 phosphosites, 362 sites on 277 proteins, were significantly increased, whereas 192 sites on 167 proteins were decreased. Among the regulated proteins, we identified 31 kinases, 7 phosphatases, and one phosphatase inhibitor that were not systematically characterized before. Furthermore, we reconstructed a phosphorylation network centered on RTK signaling regulated by SPRY. Collectively, this study uncovered a system-wide phosphorylation network regulated by SPRY, providing an additional insight into the complicated RTK signaling pathways involved in the mammary gland microenvironment.


Asunto(s)
Glándulas Mamarias Animales/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteómica/métodos , Animales , Cromatografía Liquida , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Glándulas Mamarias Animales/citología , Ratones , Mapas de Interacción de Proteínas , Transducción de Señal , Espectrometría de Masas en Tándem
18.
J Proteome Res ; 17(12): 4152-4159, 2018 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-30362765

RESUMEN

Following an enormous effort by the global scientific community coordinated by HUPO's Human Proteome Project, the number of proteins without high-quality MS or other evidence (colloquially termed missing proteins) has substantially decreased; however, some highly hydrophobic MPs remain on the list. We believe that efficient peptide separation is an approach that can be used to improve the identification of these hydrophobic MPs. We propose that peptides prepared from the membrane fractions of human cell lines and placental tissue can be well separated from hydrophilic peptides in organic solvents at high concentrations due to the precipitation of hydrophilic peptides with lower solubility. Using a combination strategy of peptide separation in 98% acetonitrile prior to traditional 2D reverse-phase liquid chromatography, more hydrophobic peptides were detected in the supernatants of the organic solvent extractions than were found in the pellets. When this strategy was adopted, 30 MPs (≥2 non-nested unique peptides with ≥9 amino acids) with 114 unique peptides were identified at protein false discovery rate (FDR) < 1%, including 7, 12, and 13 MPs obtained from membrane preparations derived from K562, HeLa cells, and human placenta, respectively. Of the 30 MPs identified in this study, 19 were categorized as membrane proteins or extracellular matrix proteins. Furthermore, 20 were verified to possess two non-nested unique peptides through parallel reaction monitoring with the corresponding chemically synthesized peptides. The use of organic solvents at high concentrations was shown to be an efficient way to improve the exploration of hydrophobic MPs. The data obtained in this study are available via ProteomeXchange (PXD010630) and PeptideAtlas (PASS01218).


Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de la Membrana/análisis , Péptidos/análisis , Línea Celular , Femenino , Células HeLa , Humanos , Células K562 , Péptidos/aislamiento & purificación , Placenta/citología , Embarazo , Proteómica/métodos , Solventes/química
19.
Mass Spectrom Rev ; 36(6): 677-692, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-26763661

RESUMEN

Protein deamidation has been proposed to represent a "molecular clock" that progressively disrupts protein structure and function in human degenerative diseases and natural aging. Importantly, this spontaneous process can also modify therapeutic proteins by altering their purity, stability, bioactivity, and antigenicity during drug synthesis and storage. Deamidation occurs non-enzymatically in vivo, but can also take place spontaneously in vitro, hence artificial deamidation during proteomic sample preparation can hamper efforts to identify and quantify endogenous deamidation of complex proteomes. To overcome this, mass spectrometry (MS) can be used to conduct rigorous site-specific characterization of protein deamidation due to the high sensitivity, speed, and specificity offered by this technique. This article reviews recent progress in MS analysis of protein deamidation and discusses the strengths and limitations of common "top-down" and "bottom-up" approaches. Recent advances in sample preparation methods, chromatographic separation, MS technology, and data processing have for the first time enabled the accurate and reliable characterization of protein modifications in complex biological samples, yielding important new data on how deamidation occurs across the entire proteome of human cells and tissues. These technological advances will lead to a better understanding of how deamidation contributes to the pathology of biological aging and major degenerative diseases. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:677-692, 2017.


Asunto(s)
Envejecimiento/metabolismo , Espectrometría de Masas/métodos , Proteínas/análisis , Proteínas/química , Proteómica/métodos , Envejecimiento/fisiología , Ácido Aspártico/análisis , Ácido Aspártico/química , Bases de Datos de Proteínas , Humanos , Enfermedades Neurodegenerativas/metabolismo , Péptidos/análisis , Péptidos/química , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/química , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismo , Reproducibilidad de los Resultados
20.
Cell Microbiol ; 19(9)2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28409866

RESUMEN

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.


Asunto(s)
Basigina/metabolismo , Señalización del Calcio/fisiología , Proteínas Portadoras/metabolismo , Eritrocitos/fisiología , Merozoítos/patogenicidad , Plasmodium falciparum/patogenicidad , Anticuerpos Monoclonales/inmunología , Antígenos de Protozoos/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/inmunología , Línea Celular , Citoesqueleto/parasitología , Citoesqueleto/patología , Eritrocitos/parasitología , Interacciones Huésped-Parásitos/fisiología , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/biosíntesis
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