Research

Our lab has two major interests: understanding the physiological function of poly(ADP-ribose) and the 17 PARPs which polymerize it, and understanding poly(ADP-ribose) and PARP function during cell stress.

Poly(ADP-ribose) and PARP function

Poly(ADP-ribose) is a poorly understood biopolymer and post-translational modification required for life in all multicellular organisms. While primarily known for its role in DNA damage repair, it functions in many essential cellular processes such as cell division and cell cycle progression as well as transcriptional and translational regulation. Poly(ADP-ribose) is polymerized onto acceptor proteins by a family of 17 PARPs using NAD+ as substrate. The majority of these are newly identified and uncharacterized. Several are up-regulated in cancers. Mis-regulation of poly(ADP-ribose) is lethal, and appears to be important in human diseases such as cancers, prompting pharma-ceutical companies to develop candidate therapeutics targeting the poly(ADP-ribose) polymerases (PARPs).

What makes poly(ADP-ribose) so interesting is that it acts as both a traditional protein modification, like phosphorylation or ubiquitination, and a macromolecule with chemical similarities to nucleic acids and carbohydrates. Like these other polymers, poly(ADP-ribose) binds specific proteins, however due to its rapid turnover dynamics, protein binding can be regulated in time and space making poly(ADP-ribose) an ideal mediator of dynamic protein localization.

To better understand poly(ADP-ribose) and PARPs, we are taking a systems-level approach by first determining where, when and how poly(ADP-ribose) and PARPs function in the cell. We have started by characterizing the localization and function of each of the 17 PARPs simultaneously using a combination of antibody staining, long-term imaging of GFP fusions, and RNAi. These preliminary experiments have identified several PARPs as essential for somatic cell function and localized the majority of the PARP proteins to the cytoplasm, membranes and vesicles during interphase, and to the mitotic spindle during mitosis. To better understand and identify the biological pathways of PARP function, and to identify functionally conserved mechanisms, we are identifying all PARP binding proteins, and poly(ADP-ribose) acceptor and binding proteins using biochemical approaches. Finally, to understand when PARPs are active, we are designing ways to monitor PARP activity in cells real time using fluorescence microscopy.

Poly(ADP-ribose) function during cell stress and disease

Some of the best understood poly(ADP-ribose) functions occur as a response to cell stresses such as DNA damage, and apoptosis. In collaboration with Phil Sharp's lab at MIT, we have identified a poly(ADP-ribose) and PARP requirement in new stress pathways, and have identified specific PARPs that function in these stress responses. Our lab is continuing to identify new stress pathways in which PARPs function, and, with our library of 17 PARP clones and siRNAs, have revisited functions for other PARPs in DNA damage and apoptosis. One long term goal of the lab is to identify mechanistic differences and similarities between physiological and stresss mediated PARP functions. With this information, we hope to determine how PARPs malfunction in human diseases.

 

 

Massachusetts Institute of Technology