The role of the N domain in substrate binding, oligomerization, and allosteric regulation of the AAA+ Lon protease

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2013."June 2013." Cataloged from PDF version of thesis.Includes bibliographical references.For cells and organisms to survive, they must maintain protein homeostasis in varied and often harsh environments. Cells utilize proteases and chaperones to maintain their proteomes. In bacteria, most cytosolic proteolysis is performed by self-compartmentalized AAA+ proteases, which convert the chemical energy of ATP binding and hydrolysis into mechanical work to unfold and translocate substrates into an internal degradation chamber. Substrates are targeted to AAA+ proteases by degradation tags (degrons). In E. coli, the Lon protease is responsible for the degradation of numerous regulatory proteins, including the cell-division inhibitor SulA, but also recognizes and degrades the majority of misfolded proteins. How Lon recognizes and prioritizes such a vast array of substrates is poorly understood. Active Lon is a homohexamer in which each subunit contains an N domain, a AAA+ module that mediates ATP binding and hydrolysis, and a peptidase domain. Degron binding allosterically regulates Lon activity and can shift Lon into conformations with higher or lower protease activity, but the mechanistic basis of this regulation is unknown. The low-protease conformation of Lon may serve as a chaperone. In Chapter 2, I describe the development and characterization of fluorescent model substrates that Lon degrades in vitro and in vivo. In Chapter 3, I describe collaborative experiments that show that Lon equilibrates between a hexamer and a dodecamer. Based on biochemical analysis and a low-resolution EM dodecamer structure, Lon appears to shift its substrate profile by changing oligomeric states and contacts between N domains appear to stabilize the dodecamer. In Chapters 4 and 5, 1 identify a binding site for the sul20 degron (isolated from SulA) in the Lon N domain and demonstrate that substrate binding to this site allosterically regulates protease and ATPase activity. I also show that the E240K mutation in the N domain alters Lon activity and stabilizes dodecamers. Finally, I provide evidence that E. coli Lon can act as a chaperone in vivo. These experiments demonstrate that the N domain integrates substrate binding, oligomerization, and regulation of the catalytic activities of Lon.by Matthew L. Wohlever.Ph.D

Altering the electron transfer mechanism of cytochrome P450 reductase through a single point mutation

Cytochrome P450 reductase (CPR) is an electron transporter enzyme that plays an essential role in xenobiotic transformations, including metabolism of carcinogens, environmental agents, and drugs. CPR, a membrane bound flavoprotein, contains two flavin cofactors--flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)--each bound to a separate protein domain. Both flavin cofactors utilize three distinct oxidation states: oxidized (OX), semiquinone (SQ) (one electron reduced), and hydroquinone (HQ) (two electron reduced). The multiple oxidation states of the flavin cofactors allow CPR to catalyze the essential transfer of electrons from the obligate two-electron donor NADPH to the one-electron acceptor heme-iron of cytochrome P450. A comparison of the FMN-binding domains in various related flavoproteins reveals that the FMN binding loops differ in their size, conformations, and primary structure yet each contains a conserved glycine residue. My research project was designed to evaluate the functional significance of this conserved glycine in rat CPR (Gly-141) through its replacement with threonine using site-directed mutagenesis. This replacement was made in both the intact reductase and the isolated FMN-binding domain (FBD). Based on previous research on the flavodoxin, we hypothesized that the larger, beta-branched side chain of threonine would disrupt the hydrogen bond between residue 141’s carbonyl group and the N5H of the reduced flavin, which should destabilize the functionally relevant semiquinone state. Using a standard steady-state turnover activity assay for cytochrome reduction and the physiological reductant NADPH, the G141T mutant was found to exhibit a specific activity that is 30% less than that of the wild type reductase, indicating that the conserved glycine residue helps modulate electron transfer. A distinctive characteristic of CPR is that the thermodynamically stable neutral FMN SQ serves as the primary electron donor to cytochrome P450. This phenomenon is the direct result of the substantial separation of the midpoint potentials for the OX/SQ and SQ/HQ redox couples (-43 mV and -280 mV, respectively). Reductive titrations of the G141T mutant revealed a significantly lower formation of the FMN SQ at thermodynamic equilibrium in both CPR and the FBD. The direct measurement of the midpoint potentials for this mutant indicated values for the OX/SQ and SQ/HQ couples of -250 mV and -218 mV, respectively. Thus, the midpoint potential for the OX/SQ couple has decreased substantially resulting in a significant loss in stability of the SQ state with the HQ state becoming the most stable thermodynamically. When wild-type CPR is mixed with an equimolar concentration of NADPH in a stopped-flow spectrophotometer, the disemiquinoid species is formed. However, when the experiment is repeated with G141T CPR, the FAD OX, FMN HQ species are preferentially formed. This data suggests that G141T CPR utilizes the FMN HQ as the primary electron donor to the cytochrome rather than the FMN SQ, but apparently in a less efficient manner. Thus the conserved glycine residue plays a critical role in stabilizing the reduced forms of the FMN cofactor, and in determining the mechanism of electron transfer in CPR. Advisor: Dr. Richard P. SwensonA one-year embargo was granted for this item

Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity

Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation.National Institutes of Health (U.S.) (Grant AI-16982)National Science Foundation (U.S.). Graduate Research Fellowship Progra

Collateral deletion of the mitochondrial AAA+ ATPase ATAD1 sensitizes cancer cells to proteasome dysfunction

The tumor suppressor gene PTEN is the second most commonly deleted gene in cancer. Such deletions often include portions of the chromosome 10q23 locus beyond the bounds of PTEN itself, which frequently disrupts adjacent genes. Coincidental loss of PTEN-adjacent genes might impose vulnerabilities that could either affect patient outcome basally or be exploited therapeutically. Here, we describe how the loss of ATAD1, which is adjacent to and frequently co-deleted with PTEN, predisposes cancer cells to apoptosis triggered by proteasome dysfunction and correlates with improved survival in cancer patients. ATAD1 directly and specifically extracts the pro-apoptotic protein BIM from mitochondria to inactivate it. Cultured cells and mouse xenografts lacking ATAD1 are hypersensitive to clinically used proteasome inhibitors, which activate BIM and trigger apoptosis. This work furthers our understanding of mitochondrial protein homeostasis and could lead to new therapeutic options for the hundreds of thousands of cancer patients who have tumors with chromosome 10q23 deletion