Modulation of the deubiquitinating system in viral infection, lymphoid cell activation and malignant transformation
Abstract: Most cellular proteins are affected by the conjugation of Ubiquitin (Ub), which may regulate their stability, activity or localization. The family of deubiquitinating enzymes (DUBs) removes Ub from conjugates and regulates the production and recycling of Ub. DUBs are thereby critically involved in the control of important functions such as cell growth, differentiation and apoptosis. Increasing evidence implicates deregulation of DUBs in malignant transformation. The human genome contains many putative DUB-encoding genes but little is known about their tissue distribution, pattern of expression, activity and substrate specificity. This thesis describes the use of a chemistry-based functional proteomics approach to identify active DUBs in human tumor cells of different tissue origin, in primary resting and mitogen stimulated cells, in Epstein-Barr virus (EBV) infected B lymphocytes, as well as in human papilloma virus (HPV) E6/E7 immortalized keratinocytes. The role of the ubiquitin C-terminal hydrolase (UCH)-L1 in the EBV-related B cell tumor Burkitt´s lymphoma (BL) is further investigated. Both tumor specific and tissue specific patterns of DUB activity were identified in a panel of tumor cell lines of different tissue origin. HPV carrying cervical carcinomas showed a diverse activity pattern when compared to the adjacent normal tissue, suggesting a role for some of the DUBs in the development of this tumor. The activity of specific enzymes, including USP5, -7, -9, -13, -15 and -22, was upregulated by mitogen activation or virus infection in normal T- and B-lymphocytes. UCH-L1 was highly expressed in tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen activated cells. Upregulation of this DUB was a late event in the establishment of EBV immortalized lymphoblastoid cell lines (LCLs) and correlated with enhanced proliferation suggesting a possible role in growth transformation. UCH-L1 was shown to regulate lymphocyte function-associated antigen (LFA)-1 dependent homotypic adhesion in B-lymphocytes. Integrin-mediated cell adhesion is essential in the development and activation of B-cells, and tight LFA-1 mediated adhesion may hinder malignant cell growth and invasion. LFA-1 dependent homotypic adhesion was activated following UCH-L1 knockdown in BL cells and this correlated with slow-down of BL cell proliferation in suspension and in semisolid agar and an accumulation in the G1 phase of the cell cycle. This suggests that UCH-L1 is required for maintaining LFA-1 in a low-avidity, non-adhesive state that favors the proliferation of malignant B-cells.
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