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.
