RESUMEN
mRNAs continually change their protein partners throughout their lifetimes, yet our understanding of mRNA-protein complex (mRNP) remodeling is limited by a lack of temporal data. Here, we present time-resolved mRNA interactome data by performing pulse metabolic labeling with photoactivatable ribonucleoside in human cells, UVA crosslinking, poly(A)+ RNA isolation, and mass spectrometry. This longitudinal approach allowed the quantification of over 700 RNA binding proteins (RBPs) across ten time points. Overall, the sequential order of mRNA binding aligns well with known functions, subcellular locations, and molecular interactions. However, we also observed RBPs with unexpected dynamics: the transcription-export (TREX) complex recruited posttranscriptionally after nuclear export factor 1 (NXF1) binding, challenging the current view of transcription-coupled mRNA export, and stress granule proteins prevalent in aged mRNPs, indicating roles in late stages of the mRNA life cycle. To systematically identify mRBPs with unknown functions, we employed machine learning to compare mRNA binding dynamics with Gene Ontology (GO) annotations. Our data can be explored at chronology.rna.snu.ac.kr.
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ARN Mensajero , Proteínas de Unión al ARN , Humanos , ARN Mensajero/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Unión Proteica , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Células HeLa , Factores de Tiempo , Aprendizaje AutomáticoRESUMEN
The RING-type E3 ligase has been known for over two decades, yet its diverse modes of action are still the subject of active research. Plant homeodomain (PHD) finger protein 7 (PHF7) is a RING-type E3 ubiquitin ligase responsible for histone ubiquitination. PHF7 comprises three zinc finger domains: an extended PHD (ePHD), a RING domain, and a PHD. While the function of the RING domain is largely understood, the roles of the other two domains in E3 ligase activity remain elusive. Here, we present the crystal structure of PHF7 in complex with the E2 ubiquitin-conjugating enzyme (E2). Our structure shows that E2 is effectively captured between the RING domain and the C-terminal PHD, facilitating E2 recruitment through direct contact. In addition, through in vitro binding and functional assays, we demonstrate that the N-terminal ePHD recognizes the nucleosome via DNA binding, whereas the C-terminal PHD is involved in histone H3 recognition. Our results provide a molecular basis for the E3 ligase activity of PHF7 and uncover the specific yet collaborative contributions of each domain to the PHF7 ubiquitination activity.
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Histonas , Ubiquitina-Proteína Ligasas , Histonas/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Dedos de Zinc , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.
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Autoantígenos/genética , COVID-19/genética , ARN Viral/genética , Ribonucleoproteínas/genética , SARS-CoV-2/genética , COVID-19/virología , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/patogenicidad , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión al ARN/genética , SARS-CoV-2/patogenicidad , Serina-Treonina Quinasas TOR/genética , Factores de Transcripción/genética , Transcriptoma/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Replicación Viral/genética , Antígeno SS-BRESUMEN
PIWI-interacting RNAs (piRNAs) are small RNAs that play a conserved role in genome defense. The piRNA processing pathway is dependent on the sequestration of RNA precursors and protein factors in specific subcellular compartments. Therefore, a highly resolved spatial proteomics approach can help identify the local interactions and elucidate the unknown aspects of piRNA biogenesis. Herein, we performed TurboID proximity labeling to investigate the interactome of Zucchini (Zuc), a key factor of piRNA biogenesis in germline cells and somatic follicle cells of the Drosophila ovary. Quantitative mass spectrometry analysis of biotinylated proteins defined the Zuc-proximal proteome, including the well-known partners of Zuc. Many of these were enriched in the outer mitochondrial membrane (OMM), where Zuc was specifically localized. The proximal proteome of Zuc showed a distinct set of proteins compared with that of Tom20, a representative OMM protein, indicating that chaperone function-related and endomembrane system/vesicle transport proteins are previously unreported interacting partners of Zuc. The functional relevance of several candidates in piRNA biogenesis was validated by derepression of transposable elements after knockdown. Our results present potential Zuc-interacting proteins, suggesting unrecognized biological processes.
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Proteínas de Drosophila , Drosophila , Animales , Femenino , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteoma/metabolismo , Ovario/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Elementos Transponibles de ADN , ARN de Interacción con Piwi , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Targeting proximity-labeling enzymes to specific cellular locations is a viable strategy for profiling subcellular proteomes. Here, we generated transgenic mice (MAX-Tg) expressing a mitochondrial matrix-targeted ascorbate peroxidase. Comparative analysis of matrix proteomes from the muscle tissues showed differential enrichment of mitochondrial proteins. We found that reticulon 4-interacting protein 1 (RTN4IP1), also known as optic atrophy-10, is enriched in the mitochondrial matrix of muscle tissues and is an NADPH oxidoreductase. Interactome analysis and in vitro enzymatic assays revealed an essential role for RTN4IP1 in coenzyme Q (CoQ) biosynthesis by regulating the O-methylation activity of COQ3. Rtn4ip1-knockout myoblasts had markedly decreased CoQ9 levels and impaired cellular respiration. Furthermore, muscle-specific knockdown of dRtn4ip1 in flies resulted in impaired muscle function, which was reversed by dietary supplementation with soluble CoQ. Collectively, these results demonstrate that RTN4IP1 is a mitochondrial NAD(P)H oxidoreductase essential for supporting mitochondrial respiration activity in the muscle tissue.
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Oxidorreductasas , Ubiquinona , Animales , Ratones , Drosophila melanogaster , Ratones Transgénicos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteoma , Ubiquinona/metabolismo , Proteínas PortadorasRESUMEN
The signal peptidase complex (SPC) mediates processing of signal peptides of secretory precursors. But, recent studies show that the eukaryotic SPC also cleaves internal transmembrane segments of some membrane proteins, and its non-catalytic subunit, Spc1/SPCS1 plays a critical role in this process. To assess the impact of Spc1 on membrane proteostasis, we carried out quantitative proteomics of yeast cells with and without Spc1. Our data show that the abundance of the membrane proteome in yeast cells lacking Spc1 is in general reduced compared to that in wild-type cells, implicating its role in controlling the cellular levels of membrane proteins.
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Proteínas de la Membrana , Proteoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteómica/métodos , Membrana Celular/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Ácido Aspártico Endopeptidasas/genética , Serina EndopeptidasasRESUMEN
Ubiquitin-specific protease 4 (USP4) is highly overexpressed in colon cancer and acts as a potent protooncogenic protein by deubiquitinating ß-catenin. However, its prominent roles in tumor formation and migration in cancer cells are not fully understood by its deubiquitinating enzyme (DUB) activity on ß-catenin. Thus, we investigated an additional role of USP4 in cancer. In this study, we identified cortactin (CTTN), an actin-binding protein involved in the regulation of cytoskeleton dynamics and a potential prognostic marker for cancers, as a new cellular interacting partner of USP4 from proximal labeling of HCT116 cells. Additionally, the role of USP4 in CTTN activation and promotion of cell dynamics and migration was investigated in HCT116 cells. We confirmed that interacting of USP4 with CTTN increased cell movement. This finding was supported by the fact that USP4 overexpression in HCT116 cells with reduced expression of CTTN was insufficient to promote cell migration. Additionally, we observed that USP4 overexpression led to a significant increase in CTTN phosphorylation, which is a requisite mechanism for cell migration, by regulating Src/focal adhesion kinase (FAK) binding to CTTN and its activation. Our results suggest that USP4 plays a dual role in cancer progression, including stabilization of ß-catenin as a DUB and interaction with CTTN to promote cell dynamics by inducing CTTN phosphorylation. Therefore, this study demonstrates that USP4 is important for cancer progression and is a good target for treating or preventing cancer.
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Neoplasias del Colon , beta Catenina , Humanos , Células HCT116 , beta Catenina/metabolismo , Cortactina/metabolismo , Movimiento Celular/fisiología , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
BACKGROUND: As lip enhancement using filler has grown in popularity, practitioners have sought to identify methods of injection that achieve aesthetically pleasing results while avoiding adverse events such as arterial injury due to intravascular injection. OBJECTIVES: The primary objective of this study is to establish a safe injection technique for creating appealing, proportionate, and aesthetically pleasing lips while elevating the lip corners using filler. The author investigated the pathway of the labial artery using sonography and devised a 9-point injection technique (9-PIT) to reliably achieve fashionable lips. METHODS: Before injection, the locations of the superior (SLA) and inferior labial arteries (ILA) were assessed using sonography. Particle HA filler was administered to 50 patients using the 9-PIT and followed up for 3 months. The extent of lip corner elevation and the angle of lip corners were quantified using the 3D-analysis, while changes in the length and curvature along the upper peristomal lines were evaluated after 1 week. RESULTS: SLA and ILA originated from the deep lateral aspect of the lip and gradually traversed toward the midline in the superficial layer. Superficial arterial branches were identified in the submucosal layer near the midline. All patients expressed satisfaction with the lip shaping and corner elevation, without any adverse effects or vascular complications. The angle of lip corners decreased by 8.80%, and lip corners were lifted by 1.02mm. The upper lip exhibited a more pronounced S-shape, with elongation of the upper lip line by 6.5%. This accentuated S-shape contributed to the appearance of lifted lip corners. CONCLUSIONS: The 9-PIT facilitated safe and aesthetically pleasing lip volumization with corner elevation in a consistent manner, while elucidating vascular pathways. Lip corner elevation was achieved solely using HA filler.
RESUMEN
Signal peptidase (SPase) cleaves the signal sequences (SSs) of secretory precursors. It contains an evolutionarily conserved membrane protein subunit, Spc1, that is dispensable for the catalytic activity of SPase and whose role remains unknown. In this study, we investigated the function of yeast Spc1. First, we set up an in vivo SPase cleavage assay using variants of the secretory protein carboxypeptidase Y (CPY) with SSs modified in the N-terminal and hydrophobic core regions. When comparing the SS cleavage efficiencies of these variants in cells with or without Spc1, we found that signal-anchored sequences became more susceptible to cleavage by SPase without Spc1. Furthermore, SPase-mediated processing of model membrane proteins was enhanced in the absence of Spc1 and was reduced upon overexpression of Spc1. Spc1 co-immunoprecipitated with proteins carrying uncleaved signal-anchored or transmembrane (TM) segments. Taken together, these results suggest that Spc1 protects TM segments from SPase action, thereby sharpening SPase substrate selection and acting as a negative regulator of the SPase-mediated processing of membrane proteins.
Asunto(s)
Péptido Hidrolasas , Proteínas de Saccharomyces cerevisiae , Serina Endopeptidasas , Proteínas de la Membrana/genética , Señales de Clasificación de Proteína , Saccharomyces cerevisiae , Serina Endopeptidasas/metabolismoRESUMEN
RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.
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Proteómica/métodos , Ribonucleoproteínas/análisis , Animales , Reactivos de Enlaces Cruzados , Embrión no Mamífero/metabolismo , Formaldehído , Células HeLa , Humanos , Hígado/metabolismo , Masculino , Ratones , Oocitos/metabolismo , Péptidos , Ribonucleoproteínas/metabolismo , Rayos Ultravioleta , Xenopus laevisRESUMEN
The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.
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Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteoma/análisis , Proteínas de Unión a Tacrolimus/metabolismo , Calcio/metabolismo , Humanos , Biogénesis de Organelos , Proteoma/metabolismo , Transducción de SeñalRESUMEN
Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC-MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Esenciales , Holoenzimas/metabolismo , Lisina/metabolismo , Factor sigma/metabolismo , Streptomyces/metabolismo , Acetilación , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Mutación/genética , Regiones Promotoras Genéticas , Unión Proteica , Factor sigma/química , Streptomyces/genética , Streptomyces/crecimiento & desarrolloRESUMEN
The microprocessor complex cleaves the primary transcript of microRNA (pri-miRNA) to initiate miRNA maturation. Microprocessor is known to consist of RNase III DROSHA and dsRNA-binding DGCR8. Here, we identify Enhancer of Rudimentary Homolog (ERH) as a new component of Microprocessor. Through a crystal structure and biochemical experiments, we reveal that ERH uses its hydrophobic groove to bind to a conserved region in the N-terminus of DGCR8, in a 2:2 stoichiometry. Knock-down of ERH or deletion of the DGCR8 N-terminus results in a reduced processing of suboptimal pri-miRNAs in polycistronic miRNA clusters. ERH increases the processing of suboptimal pri-miR-451 in a manner dependent on its neighboring pri-miR-144. Thus, the ERH dimer may mediate 'cluster assistance' in which Microprocessor is loaded onto a poor substrate with help from a high-affinity substrate in the same cluster. Our study reveals a role of ERH in the miRNA biogenesis pathway.
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Proteínas de Ciclo Celular/metabolismo , Procesamiento Postranscripcional del ARN , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Células HCT116 , Células HEK293 , Humanos , Células K562 , MicroARNs/metabolismo , Unión Proteica , Conformación ProteicaRESUMEN
Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.
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5-Metilcitosina/metabolismo , Eccema/genética , Trastornos del Crecimiento/genética , Discapacidad Intelectual/genética , Metiltransferasas/genética , Microcefalia/genética , Procesamiento Postranscripcional del ARN , ARN Mitocondrial/genética , ARN de Transferencia/genética , Animales , Sistemas CRISPR-Cas , Eccema/metabolismo , Eccema/patología , Facies , Fibroblastos/metabolismo , Fibroblastos/patología , Edición Génica , Técnicas de Inactivación de Genes , Trastornos del Crecimiento/metabolismo , Trastornos del Crecimiento/patología , Células HEK293 , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Metilación , Metiltransferasas/deficiencia , Ratones , Ratones Noqueados , Microcefalia/metabolismo , Microcefalia/patología , Mitocondrias/genética , Mitocondrias/metabolismo , Conformación de Ácido Nucleico , Fosforilación Oxidativa , Cultivo Primario de Células , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Mitocondrial/metabolismo , ARN de Transferencia/metabolismoRESUMEN
BACKGROUND: Hyaluronic acid (HA) is a key contributor to skin moisture (hydration), and MicroBotox demonstrates improvements for fine wrinkles of the face. OBJECTIVES: The author sought to evaluate the safety and efficacy of intradermal injection of hydrotoxin (combined mixture of MicroHA and MicroBotox) for the treatment of skin roughness and dryness on facial dermis. METHODS: Fifty women who had thin, dry skin with fine wrinkles throughout the whole face, especially in the crow's feet and forehead areas, were enrolled in the study. Two cc stabilized-HA filler and 1 cc (40 U) of botulinumtoxinA were mixed in the novel combined hydrotoxin mixture. Intended to hydrate the dermis and treat fine wrinkles of the face, the mixture was injected into the real dermal layer of the face. The volume of HA per site was 0.002 cc and toxin was 0.04 U. Skin roughness and stratum corneum hydration were measured at 1, 2, 3, and 6 months. RESULTS: One month post-treatment, skin roughness was reduced to 50.19% in topographic computer analysis utilizing 10× dermascope photos. Stratum corneum hydration on crow's feet improved to 81.34% at 1 month and 56.12% at 2 months from pre-treatment baseline (P < 0.0001). Global Aesthetic improvement scale of Skin hydration and fine wrinkle improved. CONCLUSIONS: The combination injection method of MicroHA and MicroBotox is not associated with side effects and showed significant synergic effect in improvement of skin roughness and moisturizing. Neuramix-hydrotoxin injection method is an easy and reproducible procedure to make constant injection depth and amount.
Asunto(s)
Técnicas Cosméticas , Envejecimiento de la Piel , Técnicas Cosméticas/efectos adversos , Dermis , Cara , Femenino , Humanos , Piel , Resultado del TratamientoRESUMEN
Protein expression levels are regulated through both translation and degradation mechanisms. Levels of degradation intermediates, that is, partially degraded proteins, cannot be distinguished from those of intact proteins by global proteomics analysis, which quantify total protein abundance levels. This study aimed to develop a tool for assessing the aspects of degradation regulation via proteolytic processing through a new multiplexed N-terminomics method involving selective isobaric labeling of protein N-termini and immunoaffinity capture of the labeled N-terminal peptides. Our method allows for not only identification of proteolytic cleavage sites, but also highly multiplexed quantification of proteolytic processing. We profiled a number of potential cleavage sites by signal peptidase and provided experimental confirmation of predicted cleavage sites of signal peptide. Furthermore, the present method uniquely represents the landscape of proteomic proteolytic processing rate during early embryogenesis in Drosophila melanogaster, revealing the underlying mechanism of stringent decay regulation of zygotically expressed proteins during early stages of embryogenesis.
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Proteínas de Drosophila/análisis , Péptidos/análisis , Animales , Drosophila melanogaster/embriología , Desarrollo Embrionario , Proteolisis , ProteómicaRESUMEN
Quantitative proteomic platforms based on precursor intensity in mass spectrometry (MS1-level) uniquely support in vivo metabolic labeling with superior quantification accuracy but suffer from limited multiplexity (≤3-plex) and frequent missing quantities. Here we present a new MS1-level quantification platform that allows maximal multiplexing with high quantification accuracy and precision for the given labeling scheme. The platform currently comprises 6-plex in vivo SILAC or in vitro diethylation labeling with a dedicated algorithm and is also expandable to higher multiplexity (e.g., nine-plex for SILAC). For complex samples with broad dynamic ranges such as total cell lysates, our platform performs highly accurately and free of missing quantities. Furthermore, we successfully applied our method to measure protein synthesis rate under heat shock response in human cells by 6-plex pulsed SILAC experiments, demonstrating the unique biological merits of our in vivo platform to disclose translational regulations for cellular response to stress.
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Proteínas de Neoplasias/análisis , Proteoma/análisis , Células HeLa , Humanos , Espectrometría de Masas , Células Tumorales CultivadasRESUMEN
BACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease affecting the axial skeleton and peripheral joints. The etiology of this disease remains poorly understood, but interactions between genetic and environmental factors have been implicated. The present study identified differentially expressed proteins in the synovial fluid (SF) of AS patients to elucidate the underlying cause of AS. METHODS: A cohort of 40 SF samples from 10 AS and 10 each of rheumatoid arthritis (RA), gout, and osteoarthritis (OA) patients were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins specific to AS. The label-free LC-MS/MS results were verified by western blotting. RESULTS: We identified 8 proteins that were > 1.5-fold upregulated in the SF of AS patients compared to that of the disease control groups, including HP, MMP1, MMP3, serum amyloid P-component (APCS), complement factor H-related protein 5 (CFHR5), mannose-binding lectin 2 (MBL2), complement component C9 (C9), and complement C4-A (C4A). CFHR5 and C9 were previously found in serum from AS patients, while APCS was previously found in SF as well as in serum. However, the present study has identified C4A, and MBL2 as potential AS biomarkers for the first time. The expression levels of MMP3, C9, and CFHR5 were verified in AS SF using western blotting. CONCLUSION: We performed quantitative comparative proteomic analysis using by LC-MS/MS of the SF from four disease states: RA, gout, and OA. This systematic comparison revealed novel differentially expressed proteins in AS SF, as well as two previously reported candidate biomarkers. We further verified the expression of MMP3, C9 and CFHR5 by western blot. These proteins may serve as diagnostic or prognostic biomarkers in patients with AS, and may thus improve the clinical outcomes of this serious disease.
RESUMEN
The deuterium, a frequently used stable isotope in isotopic labeling for quantitative proteomics, could deteriorate the accuracy and precision of proteome quantification owing to the retention time shift of deuterated peptides from the hydrogenated counterpart. We introduce a novel three-plexed peptide "diethylation" using only 13C isotopologues of acetaldehyde and demonstrate that the accuracy and precision of our method in proteome quantification are significantly superior to the conventional deuterium-based dimethylation labeling in both a single-shot and multidimensional LC-MS/MS analysis of the HeLa proteome. Furthermore, in time-resolved profiling of Xenopus laevis early embryogenesis, our 3-plexed diethylation outperformed isobaric labeling approaches in terms of the quantification accuracy or the number of protein identifications, generating more than two times more differentially expressed proteins. Our cost-effective and highly accurate 3-plexed diethylation method could contribute to various types of quantitative proteomics applications in which three of multiplexity would be sufficient.
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Desarrollo Embrionario/genética , Proteoma/genética , Proteómica/métodos , Xenopus laevis/genética , Animales , Cromatografía Liquida , Deuterio/química , Regulación del Desarrollo de la Expresión Génica/genética , Células HeLa , Humanos , Marcaje Isotópico , Espectrometría de Masas en Tándem , Xenopus laevis/crecimiento & desarrolloRESUMEN
Dyskerin pseudouridine synthase 1 (DKC1) is a conserved gene encoding the RNA-binding protein dyskerin, which is an essential component of the telomerase holoenzyme. DKC1 up-regulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatographic-tandem mass spectrometric analysis after isolating the DKC1 regulatory region binding proteins. PDIA3 repressed DKC1 expression in HCC cells by recognizing the G-quadruplex DNA at the DKC1 location. However, oxidative modification of PDIA3 induced by ROS redistributed this protein into the cytosolic regions, which stimulated DKC1 expression. We also identified Met338 in PDIA3 as the oxidatively modified residue and validated the effect of oxidative modification using an ectopic expression system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 knock-in system, and a xenograft mouse model. We observed that oxidatively modified PDIA3 promoted DKC1-mediated malignancy and survival of HCC cells in vitro and in vivo. HCC tissues showed a positive association with ROS, cytoplasmic PDIA3, and nuclear DKC1 levels. HCC patients with high PDIA3 protein and DKC1 mRNA levels also displayed reduced recurrence-free survival rates. Cumulatively, the results showed that cytoplasmic PDIA3 activity could be essential in raising DKC1 expression in HCC progression and predicting poor prognoses in HCC patients. Conclusion: Our study indicates that the elevated ROS levels in HCC modulate cytoplasmic PDIA3 levels, resulting in HCC cell survival through DKC1 up-regulation.