Mitochondria are complex organelles that play a central role in metabolism. Dynamic membrane-associated processes regulate mitochondrial morphology and bioenergetics in response to cellular demand. In tumor cells, metabolic reprogramming requires active mitochondrial metabolism for providing key metabolites and building blocks for tumor growth and rapid proliferation. To counter this, the mitochondrial serine beta-lactamase-like protein (LACTB) alters mitochondrial lipid metabolism and potently inhibits the proliferation of a variety of tumor cells. Mammalian LACTB is localized in the mitochondrial intermembrane space (IMS), where it assembles into filaments to regulate the efficiency of essential metabolic processes. However, the structural basis of LACTB polymerization and regulation remains incompletely understood. Here, we describe how human LACTB self-assembles into micron-scale filaments that increase their catalytic activity. The electron cryo-microscopy (cryoEM) structure defines the mechanism of assembly and reveals how highly ordered filament bundles stabilize the active state of the enzyme. We identify and characterize residues that are located at the filament-forming interface and further show that mutations that disrupt filamentation reduce enzyme activity. Furthermore, our results provide evidence that LACTB filaments can bind lipid membranes. These data reveal the detailed molecular organization and polymerization-based regulation of human LACTB and provide new insights into the mechanism of mitochondrial membrane organization that modulates lipid metabolism.

Mitochondria are complex organelles that play a central role in metabolism. Dynamic membrane-associated processes regulate mitochondrial morphology and biogenesis in response to cellular demand. In tumor cells, metabolic reprogramming requires active mitochondrial metabolism for providing key metabolites and building blocks for tumor growth and rapid proliferation. To counter this, the mitochondrial serine beta-lactamase-like protein (LACTB) alters mitochondrial lipid metabolism and potently inhibits the proliferation of a variety of tumor cells. Mammalian LACTB is localized in the mitochondrial intermembrane space, where it assembles into filaments to regulate mitochondrial membrane organization and the efficiency of essential metabolic processes. However, the structural basis of LACTB polymerization and regulation remains incompletely understood. Here, we describe how human LACTB self-assembles into micron-scale filaments that increase their catalytic activity. The electron cryo-microscopy (cryoEM) structure defines the mechanism of assembly and reveals how highly ordered filament bundles stabilize the active state of the enzyme. We identify and characterize residues that are located at the filament-forming interface, and further show that mutations that disrupt filamentation reduce enzyme activity. Furthermore, our results provide evidence that LACTB filaments can bind lipid membranes. These data reveal the detailed molecular organization and polymerization-based regulation of human LACTB and provide new insights into the mechanism of mitochondrial membrane organization that modulates tumor suppression.

No abstract

Reactive Oxygen Species (ROS) at low levels can act as important signaling molecules; however, when there is a surplus of ROS this can lead to harmful and or lethal results for the cell. ROS are primarily generated in the mitochondria of cells. This development occurs in the electron transport chain (ETC) which is embedded in the inner membrane of the mitochondria. Production of the mitochondrial superoxide anion (a type of ROS), occurs at redox active prosthetic groups, or electron carriers, bound to ETC proteins where kinetic factors favor O2 to becoming the superoxide anion. These kinetic factors can include the inhibition of ETC by small molecules. If an electron interacts “early” with O2 then superoxide will form, which could ultimately result in peroxides or more toxic hydroxyl radicals (HO•). Both proteins and small molecules are used within cells to mediate ROS levels and maintain redox states within these mechanisms to ensure ROS levels do not reach damaging levels. The accumulation of too much ROS can prove to be harmful to the organism. Mutations and dysfunctional molecules can result from ROS reacting with both nuclear and mitochondrial DNA, proteins, and lipids; however, more recent research shows that low levels of ROS provide important signaling mechanisms. Our interest is in how metal cofactors are incorporated into the ETC protein complexes of yeast and how misincorporation or modulation of available metals, such as copper and iron, in yeast mitochondria leads to the production of ROS and how under these conditions ROS changes during yeast lifespan. This project is particularly interested in how ROS changes “early” in yeast lifespan, meaning timepoints before yeast cells complete stationary phase and begin to enter the death phase. To detect the ROS superoxide, yeast cultures were grown in rich media for one to three days and then stained with dyhydroethidum (DHE). DHE is a fluorescent indicator for cytosolic superoxide, and cells were assayed utilizing both a fluorescence plate reader and FACS. To enhance superoxide production cells were also treated with the ETC inhibitor Antimycin A (a Complex II inhibitor), and the control inhibitor Oligomycin A (an ATPase inhibitor). Our results indicate that we are able to detect cellular superoxide using the fluorescent dye DHE and that cells cultured in the presence of the inhibitor Antimycin A have higher DHE fluorescence values compared to cells cultured in the presence of Oligomycin A. Since Antimycin A inhibits Complex III in the ETC, these results are consistent with ROS being generated from this complex. Our current work is to culture cells in supplemental copper ranging from no treatment to 0.5 mM CuSO4, or bathocuproine disulphonate (BCS) a copper chelator. Ultimately, we intend to use DHE fluorescence to assess superoxide levels in multiple yeast strains to provide insight into how ROS changes during yeast lifespan.