Modulation of the deubiquitnating system during lymphoid cell activation and viral infection

Abstract

The family of ubiquitin-specific proteases (USPs) removes ubiquitin from its conjugates and regulates the production and recycling of ubiquitin. USPs are thereby critically involved in the control of important functions such as cell growth, differentiation and apoptosis. Increasing evidence implicates USPs deregulation in malignant transformation.

The human genome contains many putative USP-encoding genes but little is known about USP tissue distribution, pattern of expression, activity and substrate specificity. We have used a chemistry-based functional proteomics approach to identify active USPs 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 HPV E6/E7 immortalized keratinocytes. Both tumor specific and tissue specific patterns could be identified when profiling USP activity in a panel of tumor cell lines of different tissue origin. Only few USPs were active in primary cells of hematopoietic origin.

The activity of specific USPs, 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 several tumor cell lines of epithelial and hematopoietic cell origin but was not detected in freshly isolated and mitogen activated cells. Upregulation of this LISP was a late event in the establishment of EBV immortalized lymphoblastoid cell lines and correlated with enhanced proliferation suggesting a possible role in growth transformation.

To further investigate the role of USPs in tumor development, the activity profiles of human papillomavirus (HPV) carrying cervical carcinomas and the adjacent normal tissue were compared. The activity of UCH-L3 and UCH37 was upregulated in the majority of tumor tissues as compared to the adjacent normal tissue. UCH-L1 activity appeared to be lower in a significant proportion of the tumors, to a less extent in more advanced stages of the tumor. In order to assess the contribution of HPV proteins, LISP activity was also monitored in a panel of HPV positive and negative cervical carcinoma cell lines and in HPV E6/E7 immortalized human keratinocytes.

In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines while seven out of eight cell lines expressed significant levels of USP7 that was only occasionally detected in tumor biopsies. UCHL1, UCH-L3, USP7 and USP9X were upregulated in HPV E6/E7 immortalized keratinocytes suggesting that changes in the pattern of activity of these enzymes may play a role in growth transformation.