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Quantitative proteome analysis of pluripotent cells by iTRAQ mass tagging reveals post-transcriptional regulation of proteins required for ES cell self-renewal.

ABSTRACT: Embryonic stem cells and embryonal carcinoma cells share two key characteristics: pluripotency (the ability to differentiate into endoderm, ectoderm, and mesoderm) and self-renewal (the ability to grow without change in an untransformed, euploid state). Much has been done to identify and characterize transcription factors that are necessary or sufficient to maintain these characteristics. Oct-4 and Nanog are necessary to maintain pluripotency; they are down-regulated at the mRNA level by differentiation. There may be additional regulatory genes whose mRNA levels are unchanged but whose proteins are destabilized during differentiation. We generated proteome-wide, quantitative profiles of ES and embryonal carcinoma cells during differentiation, replicating a microarray-based study by Aiba et al. (Aiba, K., Sharov, A. A., Carter, M. G., Foroni, C., Vescovi, A. L., and Ko, M. S. (2006) Defining a developmental path to neural fate by global expression profiling of mouse embryonic stem cells and adult neural stem/progenitor cells. Stem Cells 24, 889-895) who triggered differentiation by treatment with 1 μM all-trans-retinoic acid. We identified several proteins whose levels decreased during differentiation in both cell types but whose mRNA levels were unchanged. We confirmed several of these cases by RT-PCR and Western blot. Racgap1 (also known as mgcRacgap) was particularly interesting because it is required for viability of preimplantation embryos and hematopoietic stem cells, and it is also required for differentiation. To confirm our observation that RACGAP-1 declines during retinoic acid-mediated differentiation, we used multiple reaction monitoring, a targeted mass spectrometry-based quantitation method, and determined that RACGAP-1 levels decline by half during retinoic acid-mediated differentiation. We knocked down Racgap-1 mRNA levels using a panel of five shRNAs. This resulted in a loss of self-renewal that correlated with the level of knockdown. We conclude that RACGAP-1 is post-transcriptionally regulated during blastocyst development to enable differentiation by inhibiting ES cell self-renewal.

SUBMITTER: O'Brien RN

PROVIDER: S-EPMC2953917 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHuman saliva is a protein-rich, easily accessible source of potential biomarkers for the diagnosis of oral and systemic diseases. However, little is known about the changes in salivary proteome associated with aging of patients with dental caries. Here, we applied isobaric tags for relative and absolute quantitation (iTRAQ) in combination with multiple reaction monitoring mass spectrometry (MRM-MS) to characterize the salivary proteome profiles of subjects of different ages, presenting with and without caries, with the aim of identifying age-related biomarkers for dental caries.MethodsUnstimulated whole saliva samples were collected from 40 caries-free and caries-susceptible young adults and elderly individuals. Salivary proteins were extracted, reduced, alkylated, digested with trypsin and then analyzed using iTRAQ-coupled LC-MS/MS, followed by GO annotation, biological pathway analysis, hierarchical clustering analysis, and protein-protein interaction analysis. Candidate verification was then conducted using MRM-MS.ResultsAmong 658 salivary proteins identified using tandem mass spectrometry, 435 proteins exhibited altered expression patterns in different age groups with and without caries. Of these proteins, 96 displayed age-specific changes among caries-susceptible adults and elderly individuals, and were mainly associated with salivary secretion pathway, while 110 age-specific proteins were identified among healthy individuals. It was found that the age factor caused significant variations and played an important role in both healthy and cariogenic salivary proteomes. Subsequently, a total of 136 target proteins with complex protein-protein interactions, including 14 age-specific proteins associated with caries, were further successfully validated using MRM analysis. Moreover, non-age-specific proteins (histatin-1 and BPI fold-containing family B member 1) were verified to be important candidate biomarkers for common dental caries.ConclusionsOur proteomic analysis performed using the discovery-through-verification pipeline revealed distinct variations caused by age factor in both healthy and cariogenic salivary proteomes, highlighting the significance of age in the great potential of saliva for caries diagnosis and biomarker discovery.

Project description:BACKGROUND: In the mouse, culture of embryonic stem (ES) cells may decrease their pluripotency and give rise to foetal abnormalities in recipient embryos. These abnormalities are frequently associated with both, chromosome abnormalities or epigenetic alteration of imprinting genes; however, little is known about the epigenetic stability of endogenous retrotransposable elements (REs). In our laboratory, we came across a R1 ES cell line, which at passage 27, lost the ability of germline transmission and started inducing the kinky tail phenotype in all chimeric animals produced with it. METHODS: In order to investigate whether this phenotype was associated with chromosome alteration, inadvertent differentiation, or epigenetic modification, we characterized and compared this R1 ES cell line at passage 27 with an early passage and with a second ES cell line C57/CBAF1 generated in our laboratory. We assessed: i) karyotype; ii) expression of pluripotent and differentiation markers, iii) mRNA transcription by qRT-PCR of two REs, intracisternal-A particle (IAP) and murine endogenous-retrovirus-L (MuERV-L), and iv) methylation of IAP and MuERV-L. RESULTS: The R1 ES cell at passage 27, presented normal morphology, karyotype, and expression of genetic markers characteristic of pluripotent; however, it was detected an altered mRNA transcription of sense and antisense RNA strands of both REs, concomitantly with an altered methylation pattern for the IAP element but not for MuERV-L. These results indicate that besides methylation, other post-transcriptional processes are involved in gene silencing of some REs; and that culture of ES cells may decrease their pluripotency by producing inadvertent alterations in the expression of REs without significantly affecting the morphology, chromosome structure, and expression of pluripotent or differentiation markers. CONCLUSION: Inadvertent REs instability may have important consequences for the use of ES cells in transgenesis (chimera formation) or in cell therapy.

Project description:BACKGROUND:The degradation of the steppe of Inner Mongolia, due to overgrazing, has resulted in ecosystem damage as well as extensive reductions in sheep production. The growth performance of sheep is greatly reduced because of overgrazing, which triggers massive economic losses every year. The liver is an essential organ that has very important roles in multiple functions, such as nutrient metabolism, immunity and others, which are closely related to animal growth. However, to our knowledge, no detailed studies have evaluated hepatic metabolism adaption in sheep due to overgrazing. The molecular mechanisms that underlie these effects remain unclear. METHODS:In the present study, our group applied isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis to investigate changes in the protein profiles of sheep hepatic tissues when nutrition was reduced due to overgrazing (12.0 sheep/ha), with the goal of characterizing the molecular mechanisms of hepatic metabolism adaption in sheep in an overgrazing condition. RESULTS:The body weight daily gain of sheep was greatly decreased due to overgrazing. Overall, 41 proteins were found to be differentially abundant in the hepatic tissue between a light grazing group and an overgrazing group. Most of the differentially expressed proteins identified are involved in protein metabolism, transcriptional and translational regulation, and immune response. In particular, the altered abundance of kynureninase (KYNU) and HAL (histidine ammonia-lyase) involved in protein metabolic function, integrated with the changes of serum levels of blood urea nitrogen (BUN) and glucose (GLU), suggest that overgrazing triggers a shift in energy resources from carbohydrates to proteins, causing poorer nitrogen utilization efficiency. Altogether, these results suggest that the reductions in animal growth induced by overgrazing are associated with liver proteomic changes, especially the proteins involved in nitrogen compounds metabolism and immunity. CONCLUSIONS:This provides new information that can be used for nutritional supplementation to improve the growth performance of sheep in an overgrazing condition.

Project description:BACKGROUND: Wheat (Triticum aestivum L.) is an economically important grain crop. Two-dimensional gel-based approaches are limited by the low identification rate of proteins and lack of accurate protein quantitation. The recently developed isobaric tag for relative and absolute quantitation (iTRAQ) method allows sensitive and accurate protein quantification. Here, we performed the first iTRAQ-based quantitative proteome and phosphorylated proteins analyses during wheat grain development. RESULTS: The proteome profiles and phosphoprotein characterization of the metabolic proteins during grain development of the elite Chinese bread wheat cultivar Yanyou 361 were studied using the iTRAQ-based quantitative proteome approach, TiO2 microcolumns, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among 1,146 non-redundant proteins identified, 421 showed at least 2-fold differences in abundance, and they were identified as differentially expressed proteins (DEPs), including 256 upregulated and 165 downregulated proteins. Of the 421 DEPs, six protein expression patterns were identified, most of which were up, down, and up-down expression patterns. The 421 DEPs were classified into nine functional categories mainly involved in different metabolic processes and located in the membrane and cytoplasm. Hierarchical clustering analysis indicated that the DEPs involved in starch biosynthesis, storage proteins, and defense/stress-related proteins significantly accumulated at the late grain development stages, while those related to protein synthesis/assembly/degradation and photosynthesis showed an opposite expression model during grain development. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of 12 representative genes encoding different metabolic proteins showed certain transcriptional and translational expression differences during grain development. Phosphorylated proteins analyses demonstrated that 23 DEPs such as AGPase, sucrose synthase, Hsp90, and serpins were phosphorylated in the developing grains and were mainly involved in starch biosynthesis and stress/defense. CONCLUSIONS: Our results revealed a complex quantitative proteome and phosphorylation profile during wheat grain development. Numerous DEPs are involved in grain starch and protein syntheses as well as adverse defense, which set an important basis for wheat yield and quality. Particularly, some key DEPs involved in starch biosynthesis and stress/defense were phosphorylated, suggesting their roles in wheat grain development.

Project description:Heat stress (HS) is one of the main environmental factors affecting the efficiency of poultry production. The yellow-feather chickens (YFC) as an indigenous strain of chicken is a popular poultry breed in China. Our previous study used the RNA-seq to analyze the gene expression profiles of male YFC under HS and showed that the lipid and energy metabolism pathways are activated in livers of YFC exposed to acute HS (38°C, 4 h and 25°C recovery 2 h). In this study, we used quantitative proteome analysis based on iTRAQ to study the liver response of YFC to cycle chronic HS (38 ± 1°C, 8 h/d, 7 d, CyCHS). The male YFCs treatment used the CyCHS from 22 to 28 days of age. The liver tissue samples were collected at 28 d old. A total of 39,327 unique peptides matches were detected using iTRAQ analysis and 4,571 proteins exhibited a false discovery rate of 1% or less. Forty-six significant differentially expressed proteins (DEPs) were detected in the CyCHS group compared with the control group for the liver samples, including up- and down-regulated DEPs were 18 and 28, respectively. We found that the enriched biological process terms of the DEPs expressed in the liver were related to DNA metabolic process, oxidation-reduction process, oxidative stress and gluconeogenesis. In KEGG pathway analysis. Most of the hepatic DEPs were annotated to glutathione metabolism and TCA cycle in response to CyCHS. The up-regulation of 5 DEPs (GPX1, GSTT1, GSTT1L, RRM2, and LOC100859645) in the glutathione metabolism pathway likely reflects an attempt to deal with oxidative damage by CyCHS. The down-regulation of 3 DEPs (Isocitrate dehydrogenase [IDH3A], IDH3B, and phosphoenolpyruvate carboxykinase 1) in the TCA cycle pathway contributes to the regulation mechanism of energy metabolism and probably to cope with the balance of heat production and dissipation during CyCHS in order to adapt to high temperature environments. Our results provide insights into the potential molecular mechanism in heat-induced oxidative stress and energy in YFCs and future studies will investigate the functional genes associated with the response to HS.

Project description:BackgroundBovine viral diarrhea (BVD) which is caused by Bovine viral diarrhea virus (BVDV), is an acute, contagious disease. In spite of the use of vaccines and elimination projects, BVDV still causes severe economic losses to the cattle industry for the past few years. The current study presents a preliminary analysis of the pathogenic mechanisms from the perspective of protein expression levels in infected host cells at different points in time to elucidate the infection process associated with BVDV.MethodsWe used the isobaric tags for relative and absolute quantitation (iTRAQ) technology coupled with liquid chromatography-tandem mass spectrometric (LC-MS/MS) approach for a quantitative proteomics comparison of BVDV NADL-infected MDBK cells and non-infected cells. The functions of the proteins were deduced by functional annotation and their involvement in metabolic processes explored by KEGG pathway analysis to identify their interactions.ResultsThere were 357 (47.6% downregulated, 52.4% upregulated infected vs. control), 101 (52.5% downregulated, 47.5% upregulated infected vs. control), and 66 (21.2% downregulated, 78.8% upregulated infected vs. control) proteins were differentially expressed (fold change > 1.5 or < 0.67) in the BVDV NADL-infected MDBK cells at 12, 24, and 48 h after infection. GO analysis showed that the differentially expressed proteins (DEPs) are mainly involved in metabolic processes, biological regulation and localization. KEGG enrichment analysis showed that some signaling pathways that involved in the regulation of BVDV NADL-infection and host resistance are significantly (P < 0.05) enriched at different stages of the BVDV NADL-infection, such as Endocytosis signaling pathway, FoxO signaling pathway, Homologous recombination signaling pathway and Lysosome pathway.ConclusionsThese results revealed that the DEPs in BVDV NADL-infected MDBK cells have a wide range of regulatory effects; in addition, they provide a lot of resources for the study of host cell proteomics after BVDV infection.

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Quantitative proteome analysis of pluripotent cells by iTRAQ mass tagging reveals post-transcriptional regulation of proteins required for ES cell self-renewal.

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