This work will characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, which function as signaling molecules in the regulation of a host of cellular processes, ranging from progression through the cell cycle, to transcriptional activation, antigen processing and vesicular trafficking of proteins. Conjugation of substrates to polyUb chains of different linkage types commits the target protein to distinct fates in the cell. In particular, Lys48-linked polyUb acts as a universal signal in the ubiquitin- proteasome proteolytic pathway, the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Despite an increasing wealth of information on the cellular processes regulated by polyubiquitination and the identification of numerous ubiquitin-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in ubiquitin-mediated signaling remains unclear. The mechanisms underlying the ability of different polyUb chains to signal for distinct outcomes remain to be elucidated before the origin of specificity in polyUb signaling is understood. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. The objective of the proposed work is to characterize the structure and recognition properties of various types of polyUb chains, in order to understand at a molecular level the structural basis for ubiquitin's ability to serve as a versatile yet specific cellular signal. These studies will focus on the so-called non-canonical ubiquitin chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys6, Lys27) and chains containing heterogeneous linkages (e.g., Lys11 & Lys48), branched and unbranched. We will also determine the conformations of the canonical, Lys48-linked tetraUb chains in solution in order to gain insights into the mechanisms of their recognition by cellular receptors. Finally, these studies will characterize the interactions and determine the structures of polyUb complexes with the recently designed cyclic peptides that bind polyUb with high affinity and specificity in order to facilitate further development and optimization of this entirely novel class of modulators and potential therapeutics for ubiquitin-mediated signaling pathways. We will use modern NMR approaches in combination with small-angle X-ray and neutron scattering (SAXS, SANS) to determine the three-dimensional structures and conformational ensembles of polyUb chains in solution and to characterize their binding preferences and complexes with receptors.
Ubiquitin-mediated signaling is involved in the regulation of a wide variety of biological processes including cell development and division, the immune response, and signal transduction. The ubiquitin-proteasome proteolytic pathway, the principal mechanism for turnover of short-lived proteins, has been the target for extensive drug design efforts, as defects in this pathway are associated with a variety of diseases, including cancers, neurodegenerative disorders, developmental disorders, immune and inflammatory disorders, and muscle wasting. This research will enhance our understanding of the molecular mechanisms underlying recognition and regulation in ubiquitin-mediated signaling pathways and will guide the design of novel type of inhibitors targeting ubiquitin-dependent protein degradation and other pathways.
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