The cardiac rapidly activating delayed rectifier potassium channel (IKr) is encoded by the human ether-a-go-go related gene (hERG), which is important for repolarization of the cardiac action potential. Reduction in hERG expression levels due to genetic mutations or drugs causes Long QT Syndrome (LQTS). Recently, we demonstrated that ubiquitination of hERG channels is involved in low K+ induced hERG endocytic degradation. Since homeostatic degradation is an important pathway in maintaining hERG membrane expression levels, we investigated the molecular mechanisms for hERG degradation by focusing on the role and consequence of overexpressing the ubiquitin (Ub) ligase, Nedd4-2 (Neural Precursor Cell- Expressed Developmentally Downregulated Gene 4- 2) (Yang & Kumar, 2010). Previous work in the lab demonstrated that Ub plays a role in the internalization of cell-surface hERG channels, and I hypothesized that ubiquitination of hERG channels is facilitated through Nedd4-2. To study the effects of Nedd4-2 on hERG channels, I overexpressed Nedd4-2 in human embryonic kidney (HEK) 293 cells that stably express the hERG channels. Electrophysiological recordings, Western blot, co-immunoprecipitation analysis, and confocal microscopy were performed to identify Nedd4-2’s role in hERG expression. The data from whole-cell patch clamp recordings demonstrated that, among hEAG, Kv1.5 and hERG, Nedd4-2 specifically eliminates the hERG channel current. Western blot and confocal imaging analyses showed that Nedd4-2 overexpression led to a significant reduction in mature hERG channels in the plasma membrane. Data obtained using co-immunoprecipitation indicated that Nedd4-2 significantly increases ubiquitinated hERG channels. These data indicate that Nedd4-2 may play a role in hERG homeostatic degradation. / Thesis (Master, Physiology) -- Queen's University, 2011-08-15 18:17:00.452

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

3BP2 has been previously described as the protein mutated in the osteoporotic disorder, Cherubism. The gain of function mutation that characterizes Cherubism is the result of an uncoupling of its interaction with Tankyrase 2, which has been reported to stimulate 3BP2 ubiquitination. Here we describe an attempt at identifying the E3 ligase responsible for mediating this ubiquitination using four candidate members from the Nedd4 family. Based on their respective abilities to bind and ubiquitinate 3BP2, as well as their sensitivity to the presence of Tankyrase 2 and to 3BP2 mutations (including Cherubism mutations and mutations within the 3BP2 PPxY motif thought to confer binding to the Nedd4 proteins), we have determined that Smurf1 best fits our model. Further supporting these findings, we have seen an elevation in 3BP2 protein levels in macrophages derived from Smurf1-/-/Smurf2+/- mice. This work supports a role for the Nedd4 family member, Smurf1, in mediating 3BP2 ubiquitination.

The HECT E3 ubiquitin ligase Nedd4-1 was previously shown to regulate diverse processes such as cell and animal growth, insulin signaling, and lysosomal trafficking. To further elucidate the cellular functions of Nedd4-1, Nedd4-1 knockout mouse embryonic fibroblasts were characterized relative to their wild type counterparts. Immunofluorescence experiments revealed an altered lysosomal distribution in the knockout cells, although their lysosomal proteolytic function appeared normal. Transmission Electron Microscopy revealed striking morphological differences, especially regarding the lysosome and endoplasmic reticulum of the knockout cells. Another aspect of my studies examined the interaction between Nedd4-1 and Angiomotin (p130-AMOT), which involves the same motifs required to sequester transcriptional co-activators YAP and TAZ in the cytoplasm. To test either a competitive or non-competitive mode of binding, co-immunoprecipitation experiments involving p130-AMOT, the Nedd4 proteins, and YAP or TAZ were performed, with results not supporting a competitive mode of interaction. Overall, my results demonstrate new Nedd4-1 cellular functions.

Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.

Drosophila Nedd4 (DNedd4), an E3 ubiquitin ligase, is known to be involved in neuromuscular (NM) synaptogenesis during embryogenesis. To further elucidate its mechanism and function in this process, two major splice isoforms, dNedd4 short (dNedd4S) and dNedd4 long (dNedd4L), were studied. My work shows that while dNedd4S positively regulates NM synaptogenesis, dNedd4L plays a negative role in this process. Unique regions in dNedd4L, including the N-terminal 66 amino acid-long sequence (but not the putative dAkt phosphorylation site) and the middle 159 amino acid-long sequence, as well as the catalytic site, are required for its negative function. I proposed one possible mechanism of dNedd4L acting as a negative regulator of dNedd4S. Results from my studies of the putative effect of dNedd4L on the catalytic activity of dNedd4S in vitro, as well as on the function of dNedd4S towards Comm in Drosophila S2 cells, did not support this mechanism.

The human ether-à-go-go-related gene (hERG) encodes the pore-forming α-subunits of the Kv11.1 channel that is responsible for the cardiac rapidly activating delayed rectifier K+ current (IKr), which plays a critical role in cardiac repolarization. Dysfunction of hERG causes long QT syndrome (LQTS), a cardiac electrical disorder that can lead to severe cardiac arrhythmias and sudden death (Mitcheson et al., 2000a; Roden, 2004; Maier et al., 2006; Misner et al., 2012). The overall function of hERG channels is dependent on the channel density at the plasma membrane as well as proper channel gating. Previous work from our lab demonstrated that degradation of hERG protein in the lysosome is regulated by ubiquitin ligase Nedd4-2-mediated monoubiquitination (Sun et al., 2011; Guo et al., 2012). However, whether the internalized hERG proteins can be recycled back to the plasma membrane remains to be determined. In the present study, we investigated the regulatory effects of various Rabs on hERG channels using Western blot analysis, co-immunoprecipitation (Co-IP), whole-cell patch clamp and immunofluorescence microscopy. The data revealed that, among hERG, human Kv1.5 (cardiac ultra-rapidly activating delayed rectifier K+ channel), and human EAG (ether-à-go-go gene) potassium channels, Rab4 selectively decreased the mature hERG protein expression on the plasma membrane. Mechanistically, Rab4 did not directly target the internalized hERG protein for recycling. Instead, Rab4 increased the expression level of the E3 ubiquitin ligase Nedd4-2 (Neural Precursor Cell-expressed Developmentally Downregulated Protein 4-2), which has been shown to mediate hERG ubiquitination and degradation (Guo et al., 2012). Nedd4-2 binding site mutations ∆1073 (binding site is removed) and Y1078A (binding site is modified) in hERG completely abolished the effect of Rab4. It has been shown that Nedd4-2 undergoes self-ubiquitination after targeting substrates (Bruce et al., 2008). My data further demonstrated that Rab4 decreased the degradation rate of Nedd4-2 and increased the rate of recycling. The increased Nedd4-2 then decreases hERG expression at the plasma membrane by targeting the PY-motif in the C-terminus of hERG channels. In summary, the present study showed that Rab4 decreases the expression and function of hERG potassium channels on the plasma membrane through enhancing the recycling of the ubiquitin ligase Nedd4-2. / Thesis (Master, Physiology) -- Queen's University, 2013-08-09 12:11:27.938

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.

No description available.

The lysosomal-associated protein transmembrane 5 (LAPTM5) is a protein preferentially expressed in the immune cells. LAPTM5 was isolated in our laboratory as an interacting partner of the ubiquitin ligase, Nedd4. The intracellular domains of LAPTM5 contain three PY (L/PPxY) motifs, which bind the WW domains of Nedd4, as well as a ubiquitin-interacting motif (UIM). Here, I show that sorting of LAPTM5 from the Golgi to the lysosomes requires its association with Nedd4 and the clathrin adaptor GGA3. Although the Nedd4-LAPTM5 interaction leads to the ubiquitination of LAPTM5, this event is not necessary for LAPTM5 sorting. Rather, the Nedd4-LAPTM5 complex recruits ubiquitinated GGA3, which binds the UIM of LAPTM5. Hence, I propose a novel mechanism by which the ubiquitin ligase Nedd4, via interactions with GGA3 and cargo (LAPTM5), regulates cargo trafficking to the lysosome without requiring cargo ubiquitination. Because nothing was known about the biological function of LAPTM5, at the beginning of my Ph.D. training, I set out to determine the role of LAPTM5 in the innate immune cells. I demonstrate that LAPTM5 interacts with kinesin, a motor protein previously implicated in the anterograde movement of the late endosomal/lysosomal compartments. In dendritic cells, I show that upon maturation LAPTM5 is present within endolysosomal tubules formed by class II MHC molecules. Although I find that LAPTM5 is dispensable for the translocation of peptide-loaded MHC II molecules to the cell surface, this study extends our knowledge of the repertoire of proteins present within tubules formed by the MHC II compartments in activated dendritic cells. In macrophages, I demonstrate that LAPTM5 acts as a positive regulator of NFκB and MAPK signaling cascades, and promotes efficient proinflammatory cytokine production in response to several inducers of macrophage activation. During TNFα stimulation, LAPTM5 is required for proper initiation of NFκB signaling by acting at the receptor-proximate level. Thus, my findings indicate that LAPTM5 is an important component of inflammatory signaling cascades in macrophages and highlight a role for the endosomal/lysosomal system in regulating these cascades. Collectively, the work presented in this thesis broadens our understanding of lysosomal membrane protein sorting and function.