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Functional characterization of BCL11B-a human transcription factor and UBR1 ligase aided by proteomic analysis-a human protein ubquitin

  • In the post genomic era, novel “Omics” technologies like genomics and proteomics can be used in powerful screening approaches to provide unbiased lists of candidate genes and proteins and thus facilitate a comprehensive analysis of complex diseases such as cancer, which would not have been possible applying traditional genetic and biochemical approaches alone. During my PhD tenure I applied functional genomics screening technologies including proteomics in combination with traditional biochemical and cell biology approaches in two disease oriented projects: 1. Characterization of the role of BCL11b in Human T cell lymphomas (and) 2. Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines. 1.Characterization of the role of BCL11b in Human T cell lymphomas : The Bcl11b protein belongs to the C2H2-family of Krueppel-like zinc finger proteins and thus is a member of the largest family of transcription factors in eukaryotes. It was shown to be important for a variety of functions such as T cell differentiation, normal development of central nervous system and DNA damage response. Malignant T cells undergo apoptotic cell death upon BCL11B down-regulation. However, the detailed mechanism of this cell death is not fully understood. Two dimensional difference in-gel electrophoresis (2D-DIGE), mass spectrometry and cell biological experiments were employed to investigate the functional impact of knock down of BCL11B in malignant T cell lines such as Jurkat and huT78. To further confirm the findings of these experiments, changes in protein patterns were also recorded after down-regulation of BCL11B expression in Jurkat cells over expressing BcL-xL and in Jurkat cells over expressing BCL11B. These experiments provide evidence for the involvement of the mitochondrial apoptotic pathway and increased levels of cleavage fragments of known caspase targets such as myosin, spectrin and vimentin were observed after BCL11B knockdown. The findings suggest an involvement of ERM proteins, which were up-regulated and phosphorylated upon BCL11B down-regulation. Besides ERM proteins, PDCD5, a key regulator of apoptosis, was also found at increased levels upon down regulation of BCL11B. Moreover, the levels of several proteins implicated in cell cycle entry, including DUT-N, UCK2, MAT1, CDK6, MCM4 and MCM6 were elevated, which might lead to uncontrolled cell cycle progression, uracil misincorporation and cell death. Interestingly, an inverse regulation pattern, i.e. decreased levels of ERM proteins, DUT-N, UCK2 and PDCD5 was seen upon over expression of BCL11B in Jurkat cells. In summary, proteome analyses revealed several previously unidentified mechanisms which could significantly contribute to the cell death following BCL11B knockdown. 2.Elucidation of the mechanism of pathophysiology of Johanson Blizzard Syndrome using UBR1 knockout mice and JBS patients’ lymphoblasts cell lines : Johanson-Blizzard syndrome (JBS; OMIM 243,800), which was first described in 1971, is a rare autosomal recessively inherited genetic disorder with a unique combination of congenital abnormalities. The most constant clinical feature of JBS is the loss of exocrine pancreatic function due to progressive destruction of pancreatic acini. Genome wide linkage analysis identified the disease associated locus in the 15q14-q21 chromosome region and high-throughput sequencing of this region revealed several truncated and some missense mutations in the UBR1 gene. UBR1 gene contains 47 exons and spans over 161 kilobases. The UBR1 protein belongs to the E3 ubiquitin ligase family and is an important component of the N-end rule pathway of ubiquitous protein degradation. It was hypothesized that stabilization of direct and unique substrates of UBR1 could be the main cause of the JBS pathophysiology. So far sequencing of the UBR1 gene is the only available diagnostic procedure. However, sequencing might not always allow precise prediction of residual UBR1 activity. Hence, this study was started to develop a protein based diagnostic assay for the detection of subclinical cases of JBS and to identify signalling pathways contributing to the pathophysiology of this complex disorder using a murine UBR1 knockout model. 2D-DIGE proteome analysis was carried out for a comparative evaluation of lymphoblast samples of 14 patients and 11 controls. Principal component Analysis (PCA) clearly discriminated JBS patients from controls. However, 4 JBS patients differed from the rest and resembled controls more closely. Western-blot analysis revealed residual UBR1 levels in these patients, which were linked to a milder phenotype. Hierarchical clustering of the three groups (controls, patients with residual UBR1 levels and patients without UBR1) showed group-specific characteristic differences in the abundance of differentially regulated proteins. Quantification of a panel of five selected protein spots encompassing Interferon-induced GTP binding protein, HLA class II histocompatibility antigen, Annexin A6, FK506-binding protein 4 and GRP78 permitted discrimination of controls and JBS patients with mild phenotypes. Of note, the molecular chaperones GRP78 (BiP) and FK506BP were consistently altered in level in JBS patients and probably constitute UBR1 dependent substrates. This suggested JBS as an ER-stress related disease also indicating a possible way of therapeutic intervention. Comparative proteome analysis of UBR1 knockout and wild type animals after caerulein treatment revealed a significant accumulation of pancreatic proteases such as chymotrypsin B, anionic trypsin and pancreatic elastase in animals lacking UBR1. Furthermore, an up-regulation of ER-stress proteins and inflammation related proteins was observed. Phenotypic characterisation revealed in UBR1 knockout animals significantly increased lipase levels, a significantly increased histological score and significantly increased elastase activity 8h after the onset of pancreatitis. In isolated pancreatic acini of UBR1 knockout animals we found a significant increase in intracellular elastase activation upon supramaximal CCK stimulation, which was associated with a significant rise in the rate of necrosis explaining the more severe phenotype in the UBR1 knock-out animals. A TUNEL assay showed that there was more apoptosis in wild type compared to UBR1 knockout mice. Another set of experiments was designed to identify physiologically important substrates of UBR1. Inhibition of such substrates might then in turn allow reversion or prevention of the severe form of pancreatitis in UBR1 knockout mice. However, using the trypsin specific and reversible inhibitor S-124 it was shown that impaired trypsin degradation and thereby prolonged activation of this protease did not critically influence the phenotype. Calcium analysis after physiological stimulation revealed an increase of pathological Ca2+ signalling events, i.e. significant decrease of spike number and significant increase of spike duration. Of the candidates potentially influencing Ca2+ signalling RGS4 turned out to be of particular importance. Pre-incubation of pancreatic acini of UBR1 knockout animals with a specific RGS4 inhibitor (CCG-4986, 10 µM) normalized Ca2+ patterns, did not affect trypsin activity itself but prevented Ca2+-triggered premature trypsin activation and thus acinar disintegration. In summary, using lymphoblasts samples of JBS patients we were able to deduce a protein panel which could be developed as a possible diagnostic tool for confirmation of JBS syndrome. Furthermore, using UBR1 knockout mice in an experimental model we were able to elucidate the vital function of UBR1 and its direct substrate RGS4 in the defense against pathologic pancreatic damage thereby manifesting JBS as an inflammatory disorder due to an inadequate UBR1 mediated defense.
  • Funktionelle Charakterisierung des humanen Transkriptionsfaktors BCL11B und der humanen Ubiquitin Ligase UBR1 mit Hife von Proteomanalysen

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Author: Narasimha Kumar. Karanam
Title Additional (English):Functional characterization of BCL11B-a human transcription factor and UBR1 ligase aided by proteomic analysis-a human protein ubquitin
Title Additional (German):Funktionelle Charakterisierung des humanen Transkriptionsfaktors BCL11B und der humanen Ubiquitin Ligase UBR1 mit Hife von Proteomanalysen
Advisor:Prof. Dr. Uwe Völker
Document Type:Doctoral Thesis
Date of Publication (online):2010/11/25
Granting Institution:Ernst-Moritz-Arndt-Universität, Mathematisch-Naturwissenschaftliche Fakultät (bis 31.05.2018)
Date of final exam:2010/11/22
Release Date:2010/11/25
Tag:BCL11b, Pancreatitis, Proteomics, UBR1, Uracil misincorporation
GND Keyword:Proteomanalyse
Faculties:Universitätsmedizin / Arbeitsgruppe "Funktionelle Genomforschung"
DDC class:500 Naturwissenschaften und Mathematik / 570 Biowissenschaften; Biologie