Demystifying the interaction of UGGT, the ER folding gatekeeper, with its substrates and co- chaperone Project Abstract Proteins must fold into specific 3-dimensional structures to attain their function, but this process can be error prone. Worse yet, accumulation of misfolded proteins can lead to diseases such as Alzheimer’s and Parkinson’s. To mitigate this risk, nature has evolved sophisticated mechanisms to assist protein folding and assess their folding status. One such example is the endoplasmic reticulum protein quality control (ERQC) cycle, where the enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) serves as the master regulator. UGGT can sense the folding status of its clients and selectively adds a glucose residue to the N-glycans of non-natively folded proteins. Substrates modified by UGGT are retained within the ERQC cycle for further attempts at productive folding. Prior studies have determined that UGGT prefers “near-native” substrates that present molten-globule conformations. Nonetheless, a structural description of the folding-sensing mechanism of UGGT remains unknown. UGGT can act alone, but also forms a complex with a co-chaperone, the 15-kDa selenoprotein (SEP15). This protein contains a redox-active selenocysteine residue and is thought to aid UGGT in QC of disulfide-rich substrates, but few structural and functional studies have been performed. This project will utilize a combination of cellular and biophysical experiments to investigate the interactions of UGGT with its substrates and co-chaperones. First, structural studies of UGGT and the UGGT/SEP15 complex are underway. These results will provide insights into the concerted action of UGGT and SEP15. In parallel, endogenous substrates of UGGT/Sep15 will be identified in mammalian cell culture using a glycoproteomics assay and their maturation characterized. Second, UGGT-substrate interactions will be investigated using the disease- causing null Hong Kong variant of ɑ1-antitrypsin as a model substrate. Specific UGGT residues involved in client recognition will be identified using photo-crosslinking mass spectrometry (XL-MS) experiments. This work will be performed under the guidance of two leaders in the field of protein homeostasis. Dr. Lila Gierasch is a leader in the field of protein chaperone biophysics, while Dr. Daniel Hebert is an expert in the cell biology of glycoprotein quality control. This project will leverage my background in analytical chemistry and provide critical training in biochemistry and cell biology. Additionally, I will become well- versed in the broader field of protein homeostasis (proteostasis). After completing the training described in this proposal, I will be ready to launch my independent academic career studying the structural biology of ER proteostasis.