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Faculty and Areas of Research Paul Chang, 2007
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Our lab has two primary areas of interest:
1) Understanding Poly(ADP-ribose) function
Poly(ADP-ribose) is a ubiquitous and poorly understood protein modification essential for life in multicellular organisms. It is involved in cell division, DNA damage repair, cell cycle progression, transcriptional and translational regulation and appears to be important in the development of human diseases such as cancer.
What makes poly(ADP-ribose) so interesting is that it acts as both a protein modification and a macromolecule in its own right, binding its own set of proteins (Fig. 1). The polymer’s chemical structure is unique, but has similarities to nucleic acids and carbohydrates (Fig. 2). Like these other polymers, poly(ADP-ribose) binds specific proteins but, since it is highly structurally dynamic, this binding can be regulated in time and space making poly(ADP-ribose) an ideal mediator of dynamic protein localization.
Poly(ADP-ribose) is polymerized onto acceptor proteins by a family of 17 PARPs. The majority of these are newly identified and uncharacterized. The end product of this polymerization is a branched polymer containing two distinct chemical motifs, linear poly(ADP-ribose) and branched poly(ADP-ribose). The factors that regulate polymer length and branching are not well understood, but the ratio of length to branching likely dictates which proteins bind to poly(ADP-ribose).
The unique chemistry of poly(ADP-ribose) has made studying the molecule difficult in the past, requiring development of new techniques and technologies. We are therefore using a combined chemistry and biology approach to study the function of the polymer in mitosis, where we first became interested in poly(ADP-ribose), and elsewhere in the cell. We will eventually extend these studies to whole organisms.
A global approach to poly(ADP-ribose) function: We are taking a global approach to understanding the cellular mechanism of poly(ADP-ribose) function. This approach initially focuses on understanding the PARP family as a whole, and identifying poly(ADP-ribose) acceptor and binding proteins through mass spectrometry. To start, we have characterized the localization and function of each of the 17 PARPs. These preliminary experiments identified several PARPs as essential for somatic cell function and localized the majority of the PARP proteins to the cytoplasm, and to membranes and vesicles during interphase, suggesting functions at these locations.
To identify poly(ADP-ribose) binding proteins we have begun by showing that poly(ADP-ribose) binds distinct sets of proteins during mitosis and interphase, and that protein binding to poly(ADP-ribose) is PARP-dependent suggesting that each PARP polymerizes polymer of different structure. To determine which chemical structures within poly(ADP-ribose) mediate binding of proteins, we (with collaborators Tim Mitchison and Dan Kahne) have chemically synthesized the branched portion of poly(ADP-ribose) and showed that it binds a subset of proteins bound to full poly(ADP-ribose) polymer. We hope to synthesize linear poly(ADP-ribose) for similar analyses. Importantly, the branch molecule serves as a PARP substrate, resulting in polymerization of poly(ADP-ribose) allowing us to study poly(ADP-ribose) polymerization dynamics, and identify poly(ADP-ribose) binding proteins independently of polymerizing PARP. Using this compound we hope to determine how polymerization is regulated at the chemical level, and how changes in polymerization dynamics affect protein binding and thus regulate function.
Poly(ADP-ribose) function in the mitotic spindle: The mitotic spindle is a highly dynamic structure composed primarily of microtubule polymers and associated proteins which serve to segregate the chromosomes during cell division. Poly(ADP-ribose) is enriched in the spindle (Fig. 3), and is required for spindle assembly and function. We have identified PARP-5a, a spindle pole protein, as the PARP which polymerizes the majority of poly(ADP-ribose) bound to the spindle and the PARP required for mitosis. Many proteins are modified by poly(ADP-ribose) in mitosis, with NuMA, a spindle pole protein required for mitosis, and PARP5a itself, being the major acceptors. Importantly, we have recently shown that the poly(ADP-ribose) bound to PARP5a recruits the direct binding of other spindle pole proteins suggesting that the scaffold mechanism of poly(ADP-ribose) function is important in spindle function.
2) Identifying novel molecules required for mitotic spindle assembly
We have developed a method to assemble mitotic spindles in human somatic cell extracts using purified components. This method allows us to integrate techniques from biochemistry such as fractionation, protein depletion and addition, with somatic cell culture genetic techniques such as RNAi and exogenous gene expression. Using this technique we hope to identify novel molecules and organelles required for spindle function, and determine when and where specific protein modifications are required for spindle function. We also hope to determine if genotoxic or cytotoxic damage to specific organelles such as DNA and centrosomes affects spindle function or assembly.
Chang P, Coughlin M, Mitchison TJ. Tankyrase-1 polymerization of poly(ADP-ribose) is required for spindle structure and function. Nature Cell Biology 2005 Nov;7(11):1133-9.
Chang P, Jacobson MK, Mitchison TJ. Poly(ADP-ribose) is required for spindle assembly and structure. Nature 2004 Dec 2;432(7017):645-9.
Louie RK, Bahmanyar S, Siemers KA, Votin V, Chang P, Stearns T, Nelson WJ, Barth AI. Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes. J Cell Science 2004 Mar 1;117(Pt 7):1117-28.
Chang P, Giddings TH, Winey M, Stearns T. Epsilon-tubulin is Required for Centriole Duplication and Microtubule Organization. Nature Cell Biology 2003 Jan;5(1):71-6.
Chang P, Stearns T. Delta-tubulin and epsilon-tubulin: two new human centrosomal tubulins reveal new aspects of centrosome structure and function. Nature Cell Biology 2000 Jan;2(1):30-5.
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