Liu, Fen2012-01-232012-01-232011-12https://hdl.handle.net/11299/119964University of Minnesota Ph.D. dissertation. April 2009. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Dr. Kylie J. Walters. 1 computer file (PDF); x, 119 pages.Misfolded proteins are routinely produced in cells due to faulty protein synthesis or folding, or to environmental damage such as oxidative stress. Maintaining protein structural integrity is critical to cell viability and human health, as misfolded proteins often aggregate and disrupt vital cellular processes. Protein aggregation and the resulting cytotoxity are hallmarks of neurodegenerative diseases. To provide mechanistic insights into how aberrant cytoplasmic proteins are recognized and eliminated in mammalian cells, the proteolysis of naturally occurring human variants of arylamine N-acetyltransferase 1 (NAT1) and parkin were studied. NAT and parkin variants are associated with cancer and parkinsonism, respectively. Initially, biophysical and biochemical characterization was done to unravel the unique properties that lead to the ubiquitination of NAT1 R64W variant. Data from multiple techniques including 2-dimensional nuclear magnetic resonance (NMR) spectroscopy, circular dichorism spectroscopy, dynamic light scattering, sedimentation velocity experiments, and enzyme kinetics analysis indicated that the arginine to tryptophan mutation does not disrupt NAT1 protein structure, thermal stability or enzymatic activity, but rather makes it aggregation prone. Indirect immunofluorescence indicated this NAT1 protein variant to form microaggegrates in cells that co-localize with ubiquitin. Altogether, these findings suggest an interplay between NAT1 R64W aggregation and its constitutive ubiquitination. Studies were expanded to define the cellular pathway for the ubiquitination and degradation of misfolded cytosolic proteins. Human NAT1 R64W and parkin R42P were used as model substrates and studied via a variety of techniques, including confocal microscopy, cell fractionation, co-immunoprecipitation, mass spectrometry, protease protection, and immunogold electron microscopy. A novel pathway of protein quality control was revealed that involves the routing of aberrant cytosolic proteins through the endoplasmic reticulum (ER). ER-associated protein degradation (ERAD) is typically used for the quality control of ER proteins. However, the data presented in this dissertation suggest that two unrelated structurally compromised cytosolic protein variants, human NAT1 R64W and parkin R42P, are recognized by the molecular chaperone Hsc70 in the cytosol, trafficked to the ER, where they are translocated into the lumen and later retro-translocated out of the ER for ubiquitination. Knockdown of a key ERAD component, p97, by RNAi results in inefficient degradation of ubiquitinated NAT1 R64W and the activation of autophagy and degradation by the lysosome. These observations indicate that the ER plays a broader role in protein quality control than previously anticipated and this study is expected to have significant impact in areas including protein quality control and ER transportation.en-USEndoplasmic reticulumMisfolded cytosolic proteinsN-acetyltransferase 1Protein aggregatesProtein quality controlUbiquitinationBiochemistry, Molecular Bio, and BiophysicsThesis or Dissertation