• Novel Signaling Mechanisms in the Regulation of Mitochondrial Dynamics

      Cherok, Edward Patrick, Jr.; Karbowski, Mariusz (2017)
      Mitochondria are dynamic organelles that constantly undergo fission and fusion events (referred to as mitochondrial dynamics) to form highly interconnected networks within cells. These networks allow mitochondria to share resources such as mitochondrial DNA and antioxidant molecules to maintain the health of the network. Because mitochondria are the main source of production of ATP through oxidative phosphorylation, and also regulate cell death through apoptosis, it is critically important to maintain homeostasis in these organelles. Indeed, dysfunction in mitochondrial dynamics has been linked to numerous diseases, including cancer, neurodegenerative, endocrine, and cardiovascular diseases. Therefore, understanding the mechanisms by which mitochondrial dynamics contributes to the overall health of this organelle is of great interest. The primary proteins involved in the regulation of mitochondrial fusion and fission, and the mechanisms by which they act, are generally understood. It is also well accepted that mitochondrial fusion and fission is balanced; however, how these two separate processes communicate and signal to each other is currently unknown. To better understand the crosstalk between mitochondrial fission and fusion, we studied a function of the outer mitochondrial membrane associated E3 ubiquitin ligase, MARCH5. We found that MARCH5 acts as a negative regulator of mitochondrial fission through the ubiquitin-dependent degradation of the fission factor, MiD49. Shedding light on a possible mechanism by which the activities of fission factors are coordinated, we found that the Drp1 receptor, Mff, promotes MiD49 stability by negatively regulating MARCH5 activity, thereby enhancing mitochondrial fission rates. Finally, supporting molecular crosstalk between fission and fusion, we found that Mff also regulates the stability of the outer mitochondrial membrane fusion factors, Mfn1 and Mfn2, and that loss of Mff expression/activity results in reduced mitochondrial fusion rates in those cells. Thus, the studies presented here display novel crosstalk and signaling mechanisms by which fission factors are able to fine-tune mitochondrial fission and fusion rates through modification of the ubiquitin-proteasome system.
    • The Role of the LDLR Family Member LRAD3 in the Brain

      Noyes, Nathaniel Christopher; Strickland, Dudley K. (2014)
      The LDLR family is a class of cell surface receptors important in a wide range of biological activities, including neuronal function and pathology. We discovered the highly conserved gene LDLRAD3 in a screen of the human EST database for novel LDLR-related genes expressed in the brain. The protein product for this gene, LRAD3, is highly conserved across species in amino acid sequence, specifically in functionally important protein domains and motifs. Therefore, we hypothesized that LRAD3 should have an important role in the brain. Indeed, we found LRAD3 to be highly expressed in the brain and specifically in neurons. We performed an unbiased screen searching for proteins that interact with LRAD3 in mouse brain and discovered several of the WW domain containing members of the Nedd4 family, including the E3 ligase Itch. This interaction results an increase in the ubiquitination activity of Itch that is dependent on the PPxY motifs in LRAD3. The increase in Itch activity leads to increased auto-ubiquitination and subsequent degradation of Itch. Finally, we demonstrate that the activation-induced degradation of Itch is also achieved by the protein Spartin and is therefore not unique to LRAD3. These results reveal that LRAD3 is a component of the ubiquitin proteasome system (UPS) similar to other Itch activating, PPxY containing proteins like NDFIP1 and Spartin. The UPS regulates proteins involved in Alzheimers Disease (AD) and the UPS is dysfunctional in AD. This led us to study the role of LRAD3 in the processing of the AD associated protein, APP, a protein regulated by a number of LDLR family members. We found that LRAD3 interacts with APP and this interaction is mediated through either their transmembrane domains or intracellular domains. The association of LRAD3 with APP increases the amyloidogenic processing of APP resulting in a decrease in sAPPα production and an increase in Aβ peptide production. Pulse-chase experiments revealed that LRAD3 expression significantly decreases the cellular half-life of mature APP. Our work indicates that LRAD3 may be an important component in AD, potentially through its regulation of the UPS.