In the last two decades the eukaryotic cell cycle has gone from an enigma to a well articulated branch of cell biology. Two major gaps are apparent: integration of the cell cycle into aspects of cell physiology like growth and differentiation and establishing mechanistic understandings, especially around the complex processes of ubiquitylation and protein degradation. We address one aspect of the former and more broadly, the latter. Although ubiquitylation and phosphorylation are the major known regulatory mechanisms in the cell cycle, other modifications such as sumoylation and neddylation have been shown to have critical and specific roles. Using a recently developed protein array technology, we show that modification by FAT10, a ubiquitin-like protein that may serve as a degradation signal, is broadly regulated at the Metaphase/G1 transition. Building on our recent demonstration of the functional importance of Fat10ylation we plan to validate target proteins and use them to study the biochemical steps of FAT10ylation. We plan to examine whether FAT10 has a broad role in mitosis and to examine the biological role of particular substrates, focusing on those involved in metaphase arrest and apoptosis. The second project confronts the difficulty in resolving heterogeneous intermediates in the ubiquitin pathway and of identifying the logic of the pathway, key points of regulation and specificity. In particular the lack of mechanistic and dynamical understanding has stymied efforts to identify features that determine the timing and rate of degradation. Single molecule enzymatic studies have proven to be capable of resolving unappreciated mechanistic features of well studied reactions, but have not previously been used to analyze entire pathways. We have begun to develop single-molecule methods to probe ubiquitylation mediated by the anaphase promoting complex, including deubiquitinating enzymes, E2's and the proteasome. We describe initial experiments that have already shed light on previously impenetrable features. By combining these approaches with the production of substrates with chemically specified ubiquitin configurations we hope to learn how ubiquitin configuration controls the timing, specificity, and rate of degradation. Expanding these single molecule studies to the proteasome may reveal additional features of specificity. These studies offer real promise of predictive models for interpreting sequence features that serve as a code for protein degradation and may inform new pharmacological development.

Public Health Relevance

Among the most complex of the regulatory processes that govern protein abundance and activity is that of ubiquitin and ubiquitin-like protein conjugation;these play key roles in the cell cycle and cell physiology. Our goal is to extend our understanding of the diversity of this regulation beyond ubiquitin and understand the rules that select proteins in the right order for degradation in the cell cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM039023-27A1
Application #
8627814
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Hamlet, Michelle R
Project Start
1993-09-01
Project End
2018-03-31
Budget Start
2014-06-01
Budget End
2015-03-31
Support Year
27
Fiscal Year
2014
Total Cost
$522,574
Indirect Cost
$204,091
Name
Harvard University
Department
Biology
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Merbl, Yifat; Kirschner, Marc W (2014) Post-Translational Modification Profiling-a High-Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems. Curr Protoc Protein Sci 77:27.8.1-27.8.13
Wan, Lixin; Tan, Mingjia; Yang, Jie et al. (2014) APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction. Dev Cell 29:377-91
Wang, Weiping; Wu, Tao; Kirschner, Marc W (2014) The master cell cycle regulator APC-Cdc20 regulates ciliary length and disassembly of the primary cilium. Elife 3:e03083
Wang, Weiping; Kirschner, Marc W (2013) Emi1 preferentially inhibits ubiquitin chain elongation by the anaphase-promoting complex. Nat Cell Biol 15:797-806
Fukushima, Hidefumi; Ogura, Kohei; Wan, Lixin et al. (2013) SCF-mediated Cdh1 degradation defines a negative feedback system that coordinates cell-cycle progression. Cell Rep 4:803-16
Merbl, Yifat; Refour, Phillipe; Patel, Hevan et al. (2013) Profiling of ubiquitin-like modifications reveals features of mitotic control. Cell 152:1160-72
Ballabeni, Andrea; Park, In-Hyun; Zhao, Rui et al. (2011) Cell cycle adaptations of embryonic stem cells. Proc Natl Acad Sci U S A 108:19252-7
Ali, Fahad; Hindley, Chris; McDowell, Gary et al. (2011) Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis. Development 138:4267-77
Merbl, Yifat; Kirschner, Marc W (2011) Protein microarrays for genome-wide posttranslational modification analysis. Wiley Interdiscip Rev Syst Biol Med 3:347-56
Wu, Tao; Merbl, Yifat; Huo, Ying et al. (2010) UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex. Proc Natl Acad Sci U S A 107:1355-60

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