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Differential proteome and phosphoproteome signatures in human T-lymphoblast cells induced by sirolimus.

ABSTRACT: OBJECTIVES:The present study was designed to investigate early proteome and phosphoproteome changes during inhibition of lymphocyte proliferation induced by sirolimus (SRL). MATERIALS AND METHODS:Proliferation assays were conducted using human CCRF-CEM T lymphoblasts under different SRL concentrations. Total protein lysates after SRL treatment were used to identify significantly regulated proteins and phosphorylated proteins by 2-DE and Q-TOF Ultima Global mass spectrometer. RESULTS AND CONCLUSIONS:Incubation with 2.5 micromol/l SRL resulted in a approximately 70% inhibition of cell proliferation. Cells incubated with 2.5 micromol/l for 30 min showed a differential phosphorylation pattern with one higher (TCPQ) and six lower phosphorylation signals (TBA1B, VIME, HNRPD, ENPL, SEPT9, PLSL). On investigating the differential protein expression, five proteins were found to be up-regulated (ECHB, PSB3, MTDC, LDHB and NDKA) and four were down-regulated (EHD1, AATC, LMNB1 and MDHC). Nine of these differentially regulated proteins/phosphoproteins (TCPQ, TBA1B, VIME, HNRPD, ENPL, ECHB, PSB3, LDHB and LMNB1) showed significant interaction potential, through binding protein YWHAZ using MINT software. CONCLUSIONS:We report for the first time the simultaneous early influence of SRL on phosphorylation status and on protein expression in the total proteome of CCRF-CEM T lymphoblasts and predict that 56% of the proteins interact with each other, highlighting significance of these results.

SUBMITTER: Schultze FC

PROVIDER: S-EPMC6496580 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Differential proteome and phosphoproteome signatures in human T-lymphoblast cells induced by sirolimus.

Schultze F C FC Petrova D T DT Oellerich M M Armstrong V W VW Asif A R AR

Cell proliferation 20100801 4

<h4>Objectives</h4>The present study was designed to investigate early proteome and phosphoproteome changes during inhibition of lymphocyte proliferation induced by sirolimus (SRL).<h4>Materials and methods</h4>Proliferation assays were conducted using human CCRF-CEM T lymphoblasts under different SRL concentrations. Total protein lysates after SRL treatment were used to identify significantly regulated proteins and phosphorylated proteins by 2-DE and Q-TOF Ultima Global mass spectrometer.<h4>Re ...[more]

PMID: 20590665

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Project description:Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important in cellular signaling and recognition, and their function and activity are frequently regulated by post-translational modifications such as phosphorylation and glycosylation. To obtain information about membrane-associated proteins and their modified amino acids potentially involved in changes of hESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well as changes in phosphorylation and sialylation between hESCs and NSCs. Combining proteomics and modification specific proteomics we identified a total of 5105 proteins whereof 57% contained transmembrane domains or signal peptides. The enrichment strategy yielded a total of 10,087 phosphorylated peptides in which 78% of phosphopeptides were identified with ?99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development. In the latter group of proteins, we could identify potential NSC markers as Crumbs 2 and several novel proteins. A motif analysis of the altered phosphosites showed a sequence consensus motif (R-X-XpS/T) significantly up-regulated in NSC. This motif is among other kinases recognized by the calmodulin-dependent protein kinase-2, emphasizing a possible importance of this kinase for this cell stage. Collectively, this data represent the most diverse set of post-translational modifications reported for hESCs and NSCs. This study revealed potential markers to distinguish NSCs from hESCs and will contribute to improve our understanding on the differentiation process.

Dataset Information

Differential proteome and phosphoproteome signatures in human T-lymphoblast cells induced by sirolimus.

Publications

Differential proteome and phosphoproteome signatures in human T-lymphoblast cells induced by sirolimus.

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