Fluorometric determination of the CCAAT/enhancer binding protein alpha by using gold nanoparticles and a labeled protein-binding DNA

2019 ◽  
Vol 187 (1) ◽  
Author(s):  
Jiehua Ma ◽  
Jinlong Li ◽  
Xianwei Cui ◽  
Lianghui You ◽  
Yun Li ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Protein Binding ◽  
Binding Protein ◽  
Fluorometric Determination ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Post-Translational Modification and Differential Protein Binding Partners of CCAAT Enhancer Binding Protein Alpha (C/EBPα) p42 Versus p30 May Contribute to Aberrant C/EBPα Activity in Acute Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3577-3577
Author(s):  
Matthew Silver ◽  
Nirmalee Abayasekara ◽  
Dylan Perry ◽  
Hong Sun ◽  
Nancy Berliner ◽  
...  
Keyword(s):  
Protein Binding ◽  
Transcriptional Activity ◽  
Binding Protein ◽  
Myeloid Cells ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Differential Protein ◽  
Binding Partners ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is the founding member of a family of basic region/leucine zipper (bzip) transcription factors and has been shown to be a master regulator of granulopoiesis It is expressed at high levels throughout myeloid differentiation and has been shown to bind to the promoters of multiple myeloid- specific gene promoters at different stages of myeloid maturation. Profound hematopoietic abnormalities have been reported for mice nullizygous for including a selective early block in the differentiation of C/EBPα, granulocytes. Mutations in C/EBPα have been demonstrated in a subset of patients with AML presenting with a normal karyotype. These mutations can result in the expression of a 30kD dominant negative C/EBPα isoform which contributes to loss of C/EBPα function. We have sought to understand the molecular basis for this observation. We and others have demonstrated that C/EBPα is post-translationally modified by small ubiquitin-related modifier (SUMO) at a lysine residue (K159) that lies within a region of the C/EBPα protein that can negatively affect transcriptional activity. We have demonstrated that the levels of sumoylated p42C/EBPα decrease upon normal neutrophil maturation and that transactivation of the myeloid-specific lactoferrin (LF) promoter reporter is significantly enhanced by a p42 sumoylation mutant of C/EBPα (K159A). Additionally, in oligonucleotide pull down assays, we show that sumoylated p42C/EBPα binds to the C/EBP site in the LF promoter in immature myeloid cells (which do not express LF) while loss binding and LF of sumoylation correlates with loss of p42C/EBPα expression in more mature cells. Based on these observations we is associated with the negative conclude that sumoylated p42C/EBPα regulation of LF in early myeloid cells. We further demonstrate that sumoylated p42C/EBPα remains bound to the LF promoter following ATRA induction of the leukemic NB4 cells, which do not express LF despite induction of morphologic maturation. Based on these observations we conclude that during normal myeloid differentiation, sumoylated p42C/EBPα is associated with the negative regulation of LF in early myeloid cells, and that LF expression upon maturation is associated with loss of binding of sumoylated p42 C/EBPα In leukemic cells induced toward mature neutrophils, sumoylated p42C/EBPα remains bound to the LF promoter, contributing to the lack of expression of LF in these cells. We show in addition, that p30 C/EBPα can also be sumoylated. In transactivation assays, however, sumoylated p42C/EBPα suppresses LF promoter activity more efficiently than p30C/EBPα in 293 cells. In order to identify differential protein binding partners of p30 and p42C/EBPα that could account for the differential transcriptional activity of the two isoforms, we have used a one step purification method that allows isolation of biotinylated C/EBPα p30 and p42- containing complexes using magnetic-streptavidin beads. The K562 myelomonocytic cell line stably expressing a biotin ligase (BirA) plasmid was transfected with p30C/EBPα or p42C/EBPα each containing a 23 amino acid tag at the N-terminus that allows for in vivo biotinylation. Proteins complexed with the two C/EBP isofoms have been isolated and are currently being identified by LC- MS MS analysis. Their differential association with the two isofoms of C/EBPα will be confimed by coimmunoprecipitation assays in normal myeloid and in leukemic cells. The identification of differentially bound proteins to p30 and p42 C/EBPα may identify molecular targets for future drug development.

scholarly journals Dysregulation of CCAAT/enhancer binding protein-alpha (CEBPA) expression in the bone marrow of acute myeloid leukemia patients

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Naglaa M. Hassan ◽  
Fadwa Said ◽  
Roxan E. Shafik ◽  
Mona S. Abdellateif
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Binding Protein ◽  
Response To Treatment ◽  
Pathological Features ◽  
Poor Prognostic Factor ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein ◽  
Acute Myeloid

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous malignant disease characterized by accumulation of different types of mutations commonly the CCAAT/enhancer binding protein-alpha (CEBPA). However, the dysregulations of CEBPA expression in AML is still a debatable issue. The aim of the current study was to assess CEBPA gene expression in bone marrow (BM) aspiration specimens of 91 AML patients, compared to 20 control donors of bone marrow transplantation (BMT), using RT-PCR. Data were correlated with patients’ clinico-pathological features, response to treatment, progression-free survival (PFS), and overall survival (OS) rates. Results There was overexpression of CEBPA gene in AML patients compared to normal control [1.7 (0.04–25.6) versus 0.17 (0–4.78), respectively, P < 0.001]. Upregulation of CEBPA expression associated significantly with increased BM hypercellularity, total leucocyte counts, peripheral blood blast cell count, and poor PFS (P < 0.001, 0.002, 0.001, and 0.013, respectively). There was no significant association between CEBPA expression and any other relevant clinico-pathological features or OS rates (P = 0.610) of the patients. ROC analysis for biological relevance of CEBPA expression with AML showed that sensitivity and specificity of CEBPA expression at a cut-off value of 0.28 are 92.3% and 78.6%, respectively (P < 0.001). All patients who had CEBPA overexpression and mutant FLT3 showed BM hypercellularity, adverse cytogenetic risk, increased TLC, and PB blast cells count (P = 0.007, P < 0.001, 0.016, and 0.002, respectively). Conclusion CEBPA overexpression could be used as a genetic biological marker for AML diagnosis, as well as a poor prognostic factor for disease progression. It has no impact on OS rates of the patients.

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