Cell function and viability are dependent upon proper protein folding. To avoid abnormal and toxic deposition of proteins, the cells recruit the ubiquitin-proteasome system, by which ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) work together to assemble poly-ubiquitin chains on misfolded or misassembled proteins, to be marked for degradation by the 26S proteasome. With the maturation of the ubiquitin research field, a number of general principles have emerged about the enzymatic mechanisms of ubiquitin ligation to proteins. In particular, several high-resolution structures of different E2 and E3 enzymes have been determined, and these have yielded many insights into the mechanisms of ubiquitin-protein attachment. However, despite these advances, the fundamental mechanism(s) of ubiquitin-chain assembly remains unknown. The textbook version- which has not been proven incorrect- holds that ubiquitin molecules are added one at a time, first to the substrate protein and then to the distal end of the growing ubiquitin chain. Conversely, several recent publications have challenged this view by showing that polyubiquitin chains can pre-form on the catalytic cysteine of Ubc7, an E2 enzyme of the protein quality control ubiquitylation system, prior to transfer into a substrate. This led to our hypothesis that polyubiquitin-Ubc7 conjugates may represent an intermediate form, en route towards substrate ubiquitylation. Our specific aims are designed to provide a comprehensive evaluation of the enzymatic steps that precede substrate ubiquitylation in protein quality control pathways, with experimental emphasis on Ubc7-mediated ubiquitin chains catalysis. To this end, it is necessary to setup both cellular and cell-free ubiquitylation systems to provide the basic tools for our research. This will be accompanied by structure-function analyzes, to identify functional elements within Ubc7, and to evaluate their mode of action.
Field of science
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins/enzymes
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