This proposal is to 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 linkages 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 Ub-binding proteins that tie the polyUb signal to downstream events, the molecular basis of diversity in Ub-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 Ub's ability to serve as a versatile cellular signal. We will focus on the so-called non-canonical chains: linked via lysines other than Lys48 or Lys63 (e.g., Lys11), chains with heterogeneous linkages (e.g., Lys11, Lys48, Lys63, head-to-tail), both linear and branched, as well as heterologous chains composed of Ub and Ub-like proteins (Rub1/Nedd8). We will also analyze the canonical, Lys48-linked polyUb chain conjugated to a model substrate protein, in order to examine the effect of the conjugation on both the polyUb tag and the substrate. In addition, we will characterize (poly)Ub complexes with ubistatins, small molecule inhibitors that block polyUb signal recognition by the proteasome, in order to assist in designing the next generation of ubistatin derivatives. We will use modern NMR approaches to determine the three-dimensional structure of these chains in solution and characterize their binding properties and complexes with receptors. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from NOEs and paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. NMR data will be combined with small-angle X-ray and neutron scattering (SAXS, SANS) data, to better characterize the structure and the conformational ensemble of polyUb chains.
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 extend our understanding of the molecular mechanisms of recognition and regulation in ubiquitin-mediated signaling pathways and assist in the design of novel inhibitors that control ubiquitin-dependent protein degradation.
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