Browsing by Subject "Adenosine Triphosphatases"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Neuronal Maintenance via a Neuron-Specific Degradation Pathway(2015-01-26) Schmidt, Taylor; Jin, Eugene Jennifer; Ozel, Mehmet Neset; Epstein, Daniel; Marchant, Corey; Hiesinger, RobinBACKGROUND: Neurons can survive for decades via cell maintenance and protein degradation. This process includes the general protein endolysosomal degradation pathway, an integral part of which is the Rab GTPase proteins. Recently, components of a neuron-specific protein degradation pathway were discovered, which include the neuronal vesicle ATPase component V100 and the synaptic vesicle protein neuronal Synaptobrevin (n-Syb). While this neuron-specific degradation pathway has been shown as necessary for neuronal maintenance in adult Drosophila melanogaster fruit flies, it is not known what this neuron-specific degradation pathway does, nor how it interacts with the general protein degradation pathway. Our research aimed to fill this gap in knowledge. Such research may be salient because the misregulation of protein degradation in neurons leads to neurodegenerative diseases like dementia. OBJECTIVE: We hypothesized that neurons either have an increased or a specialized need for protein degradation in comparison to other cells. METHODS: 1. The lab chose a myristoylated protein (myr) to represent general proteins found in every cell, and Synaptotagmin1 (Syt1) to represent neuron-specific proteins. The acidification-sensitive tag mCherry-pHluorin, which changes color with a decrease in pH, was placed on Syt1 and myr to visualize acidification and degradation of the two proteins. 2. The lab generated Drosophila lines to compare acidification and degradation of Syt1 and myr in wild-type versus the following three mutants: rab7 mutants to disrupt general protein degradation, v100 to disrupt the neuron-specific protein degradation, and synaptobrevin also to disrupt neuron-specific degradation. 3. We performed live imaging to visualize acidification and protein degradation at synaptic terminals. Brains of Drosophila pupae from each cross were dissected, mounted onto Petri dishes, and surrounded with a culture medium to be kept alive. A resonant confocal microscope was used to observe the brain's lamina, a layer of neurons between the eye and the brain. At the lamina, we recorded 30-minute videos showing changes in fluorescence representing protein degradation. RESULTS AND CONCLUSION: Preliminary data show that nsyb and v100 mutations may cause defects in the degradation of neuron-specific cargo. Such evidence suggests that the neuron-specific endolysosomal degradation pathway specifically degrades the synaptic vesicle protein Synaptotagmin1. Also, the experiments indicate that disruption of either the neuron-specific or the general endolysosomal degradation pathway has no effect on the acidification of the myristoylated protein. Such evidence implies that the general pathway of protein degradation occurs at synapses, but has no specificity for protein cargo. A greater sample size is needed for future experiments, as well as quantitative analysis.Item Sequential Actions of VCP/p97 and the Proteasome 19S Regulatory Particle in Sterol-Accelerated, ER-Associated Degradation of HMG CoA Reductase(2014-05-28) Morris, Lindsey LaChelle; Goodman, Joel M.; DeBose-Boyd, Russell A.; Lehrman, Mark A.; De Martino, GeorgeAccelerated endoplasmic reticulum (ER)-associated degradation (ERAD) of the cholesterol biosynthetic enzyme HMG CoA reductase results from its sterol-induced binding to ER membrane proteins called Insig-1 and Insig-2. This binding allows for subsequent ubiquitination of reductase by Insig-associated ubiquitin ligases. Once ubiquitinated, reductase becomes dislocated from ER membranes into the cytosol for degradation by 26S proteasomes through poorly defined reactions mediated by the AAA-ATPase VCP/p97 and augmented by the nonsterol isoprenoid geranylgeraniol. Here, we report that the oxysterol 25-hydroxycholesterol and geranylgeraniol combine to trigger extraction of reductase across ER membranes prior to its cytosolic release. This conclusion was drawn from studies utilizing a novel assay that measures membrane extraction of reductase by determining susceptibility of a lumenal epitope in the enzyme to in vitro protease digestion. Susceptibility of the lumenal epitope to protease digestion, and thus membrane extraction of reductase, was tightly regulated by 25-hydroxycholesterol and geranylgeraniol. The reaction was inhibited by RNA interference mediated knockdown of either Insigs or VCP/p97. In contrast, reductase continued to become membrane extracted, but not cytosolically dislocated, in cells deficient for AAA-ATPases of the proteasome 19S regulatory particle. These findings establish sequential roles for VCP/p97 and the 19S regulatory particle in the sterol-accelerated ERAD of reductase that may be applicable to the ERAD of other substrates.