No description available.

A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.

No description available.

Shigella flexneri is a highly adapted pathogen that invades the host cytosol and causes bacillary dysentery. Shigella has evolved powerful countermeasures to disarm host defense mechanisms; amongst them a family of twelve bacterial E3 ubiquitin ligases (IpaH) that are structurally unrelated to eukaryotic enzymes. IpaH ligases are injected into the host cytosol via the bacterial type III secretion system (T3SS) to manipulate the host cell and counteract anti-bacterial defense pathways. My work demonstrated that IFN-induced guanylate-binding proteins (GBPs) are novel targets for IpaH9.8. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria unless they are ubiquitylated by IpaH9.8 and consequently degraded by the proteasome. IpaH9.8 targets GBP1, GBP2, and GBP4, thereby causing a transient poly-ubiquitin coat comprising K48 and K27-linked chains around S. flexneri, which leads to the proteasome-dependent destruction of existing GBP coats and the re-establishment of bacterial motility and cell-to-cell spread. So far, ubiquitylation of bacteria has mostly been associated with anti-bacterial autophagy or immune signaling. However, the ubiquitin coat assembled around intracellular Shigella by IpaH effectors, in particular IpaH9.8, serves a pro-bacterial function, the first observed so far. In addition, I characterized IpaH1.4 and IpaH2.5 for their ability to prevent NF-κB activation by targeting LUBAC. I found that IpaH1.4 specifically binds the LUBAC component HOIP and mediates its proteasomal degradation, thus abolishing linear ubiquitylation of bacteria and consecutive NF-κB activation via NEMO and autophagy induction via optineurin. Lastly, I identified novel potential ubiquitylation targets for IpaH effectors in human cells using a mass spectrometry-based approach. The resulting IpaH interactome presents the groundwork for further investigations and will help to identify potentially unknown cellular defense mechanisms that are antagonized by Shigella flexneri.

Bone morphogenetic protein 4 (Bmp4) is a well known growth factor that regulates gene expression through the SMAD signaling pathway. Bmp4 is involved in many developmental processes and has been identified as an important factor in several cancers, including melanoma, ovarian cancer, and colon cancer. Madoz-Gurpide et al. recently observed Bmp4 in the nuclei of a minor percentage of cells in colon cancer tissues. In addition, our lab has recently discovered a nuclear variant of Bmp2 (nBmp2), the TGF-β family member most closely related to Bmp4. These observations led us to hypothesize that a nuclear variant of Bmp4 (nBmp4) also exists. The results of chapter one report the existence of a nuclear variant of Bmp4. nBmp4 is translated from an alternative start codon downstream of the signal peptide sequence which allows a bipartite nuclear localization signal to direct translocation of nBmp4 to the nucleus. Chapter 2 and 3 further report that nBmp4 interacts with several subunits in the SCF E3 ubiquitin ligase, namely two Regulator of Cullins (ROC) proteins, five Cullin proteins, and two F-box proteins. Due to the known role of the SCF E3 ubiquitin ligase in regulating the cell cycle, the effect of nBmp4 on cell cycle progression was analyzed and the results show that nBmp4 affects the cell cycle by causing cells to accumulate in G0/G1. The association of nBmp4 and the SCF E3 ubiquitin ligase components and the affect that nBmp4 has on the cell cycle suggest that nBmp4 functions in the nucleus by inhibiting the SCF E3 ubiquitin ligase from ubiquitinating target proteins that are involved in regulating cell cycle progression. Finally, the initial stages in the generation of an nBmp4 over-expression mouse are described. The results of this research clearly change the traditional paradigm that Bmp4 performs all of its functions via extracellular signaling and introduce the existence of a nuclear variant that is involved in cell cycle regulation.

Sna3p and Sna4p are small proteins of unknown function possessing two transmembrane domains and belong to a small family of conserved proteins present in plant and fungi. The budding yeast has four SNA proteins (Sna1–4) that have different localizations in the cell. Sna3p is targeted to the vacuolar lumen by the multivesicular body pathway. Two observations marked Sna3p as a multivesicular body cargo that is sorted in an ubiquitin-independent manner. First, Sna3p-GFP is still correctly transported to internal multivesicular body vesicles under conditions of ubiquitin depletion, which impairs multivesicular body sorting of certain other cargoes. Second, a mutant form of Sna3p-GFP lacking the only potential positions for ubiquitylation is still correctly targeted to the vacuolar lumen. It has thus been postulated that ubiquitylation marks, but not all, membrane proteins for sorting into the interior of the vacuole. In this study we present a further characterization of the Golgi to vacuole trafficking of Sna3p together with its ubiquitylation status. We observed that Sna3p physically interacts with the E3 ligase Rsp5p and that this interaction is essential for sorting of Sna3p to the endosomal pathway. Sna3p is ubiquitylated on its Lys125 residue by Rsp5p and modified by Lys 63-linked ubiquitin chains. In contrast to the conclusions from prior reports, we demonstrated that, as noticed for most other multivesicular body cargoes, Sna3p ubiquitylation is required for its multivesicular body sorting. Sna4p is localized to the vacuolar membrane and interior. Sna4p contains an acidic di-leucine motif, that could be a sorting signal specific for AP-3 dependent pathway directing Sna4p to the vacuolar membrane. In apm3∆ cells, where µ subunit of the AP-3 complex is deleted, Sna4p is missorted to the vacuolar interior. Strikingly, this localization is different from localization of markers of AP-3 dependent pathway. This dissimilarity indicates that Sna4p possesses an additional characteristic, absent in other AP-3 cargoes, driving it to the vacuolar interior. In this study we have shown that the acidic di-leucine motif is indeed the sorting signal of Sna4p to the vacuolar membrane through the AP-3 dependent pathway, and that a part of Sna4p is targeted to the vacuole lumen via the multivesicular body pathway. The ability to enter multivesicular bodies is linked to the c-terminal PPPY sequence of Sna4p. Sna4p interacts with Rsp5p via this PY motif, resulting in Sna4p ubiquitylation on its lysine 128 and incorporation into the multivesicular bodies. Thus, Sna4p possesses two functional sorting signals which allow it to use two different pathways directing the protein to the vacuole.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

Development requires the acquisition of different cell fates. A major conserved pathway required for cell fate determination is the Notch signalling pathway. Neuralized is a key regulator of the Notch pathway and is essential for embryonic development in Drosophila melanogaster. I have been studying the role of Neuralized during Drosophila development, focusing on the regulation of this protein. Neuralized is an E3 ubiquitin ligase that targets Notch ligands for ubiquitination and endocytosis in the signal sending cell. This endocytic event is required for signal transduction, and cells lacking Neuralized fail to signal through Notch. I have identified a conserved interaction between Neuralized and phosphoinositides that is essential for the ability of Neuralized to promote ligand endocytosis and Notch signalling. Interactions between Neuralized and phosphoinositides are not required for ligand ubiquitination, identifying a role for Neuralized in downstream aspects of ligand trafficking. I have also determined that Neuralized is dynamically regulated through a combination of tissue specific expression, subcellular trafficking, protein interactions and posttranslational modification. Neuralized contains two related protein domains of unknown function called Neuralized homology repeats (NHR). To gain insight into the function of the NHR domain, I characterized another NHR containing protein, CG3894. CG3894 is required for development and preliminary data indicate that NHR domains dimerize, suggesting a possible interaction between Neuralized and CG3894. The study of Neuralized in Drosophila has contributed to our understanding of this essential protein both at a developmental and cellular level, and has provided a means through which to ask questions about regulation of Notch signalling in a relatively simple context. Given the importance of Notch signalling to development, and contributions that aberrations in signalling make to cancer and diseases of the nervous system, expanding our understanding of the regulation of Notch signalling is essential to understanding how this important pathway functions.

Tumor susceptibility gene 101¡]TSG101¡^encodes an inactive ubiquitin conjugating E2 enzyme implicated in regulation of protein sorting, vesicular trafficking, transcription activation of nuclear receptor, cell growth and differentiation. Previous studies showed that TSG101 can be mono- or poly- ubiquitinated, which is relevant to its functional status. There are seven Lysine (K) sites, K6, K11, K27, K29, K33, K48 and K63, on ubiquitin (Ub). Polyubiquitination using different Ub K sites confers differential function for protein degradation, DNA damage repair, endocytosis and protein sorting. AIP4 E3 ubiquitin ligase modifies its substrates involved in erythroid and lymphoid lineage differentiation and the associated immune responses. Mutation in AIP4 gene resolves in multisystemic autoimmune disease. TSG101 was recently shown to be a molecular checkpoint for T cell receptor downregulation. Here we investigate the ubiqutination status of TSG101. The ubiquitin-conjugated protein in lysate of cells co-transfected with pHA-TSG101 and His-tagged wild type Ub or each K site mutant ubiquitin expression plasmids was purified on nickel beads and then subjected to western blotting using antibodies against HA-TSG101 or His-tag. The results showed that K series mutant had differential effect on the steady-state of HA-TSG101. Proteasome inhibitor could alleviate its degradation especially in the K63 ubiquitin expression group, implying K63 ubiquitination E3 ligase is critical in maintaining HA-TSG101 level. Our coimmunoprecipitation result demonstrated the interaction between AIP4 and HA-TSG101, implying that TSG101 might be a substrate for AIP4. The ectopic overexpression of AIP4 increased the amount of HA-TSG101 in an E3 ligase activity depended manner. Taken together, these results indicated that AIP4 activity mediating Ub K63 modification might be critical for regulating cellular TSG101 protein level. Further experiment should clarify this issue.