Novel Phosphorylation Sites on Cdt1 Regulate Its Activity

Origin licensing is a process responsible for preparing the genome for replication during the cell cycle. The overexpression of chromatin licensing and DNA replication factor 1 (Cdt1), an origin licensing protein, is toxic to cells because it induces re-replication of DNA. Protein activity is often regulated through the mechanism of phosphorylation. However, the phospho-regulation of Cdt1 during the cell cycle is not fully understood.Bachelor of Scienc

HECT E3 Ubiquitin Ligase Itch Functions as a Novel Nega

The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases

Locating the post-national activist:Migration rights, civil society and the practice of post-nationalism

Theorists of post-nationalism examine the (re)configuration of national identity, membership and rights. Yet while normative scholarship has conceptualized post-nationalism as an ongoing practice of discursive contestation over the role of national group membership in liberal democratic societies, more empirical studies have tended to overlook these features to predominantly focus instead on top-down legal and political institution-building as evidence of post-nationalism. In this article I argue in favour of an empirical conceptualization of post-nationalism which more effectively captures micro-level practices of discursive contestation. Specifically I posit that post-national activists, or actors engaging in post-national practices of contestation from within the state, are a key focus of analysis for scholars of post-nationalism. I develop this claim through the analysis of data collected with individuals working on civil society campaigns for migration rights in Europe, Australia and the USA who–I demonstrate–embody many of the characteristics of the post-national activist

Frequency of pathogenic germline variants in cancer susceptibility genes in 1336 renal cell carcinoma cases

Background Renal cell carcinoma (RCC) occurs in a number of cancer predisposition syndromes but the genetic architecture of susceptibility to RCC is not well defined. We investigated the frequency of pathogenic germline variants in cancer susceptibility genes (CSGs) within a large series of unselected RCC participants. Methods Whole genome sequencing data on 1336 RCC participants and 5834 controls recruited to the UK 100000 Genomes Project, a nationwide multicentre study, was analysed to identify rare pathogenic or likely pathogenic (P/LP) short variants (SNVs and INDELs) and structural variants in 121 CSGs. Results Among 1336 RCC participants (mean 61.3 years [±12SD], range 13–88 years; 64% male), 85 participants (6.4%; 95% CI [5.1, 7.8]) had one or more P/LP germline variant in a wider range of CSGs than previously recognised. A further 64 intragenic variants in CSGs previously associated with RCC were classified as a variant of uncertain significance (VUS) (24 ‘hot VUSs’) and were considered to be of potential clinical relevance as further evaluation might result in their reclassification. Most patients with pathogenic variants in well-established RCC-CSGs were aged < 50 years. Burden test analysis for filtered variants in CSGs demonstrated a significant excess of CHEK2 variants RCC European participants compared to the healthy European controls (P = 0.0019). Conclusions Approximately 6% of patients with RCC unselected for family history have a germline variant requiring additional follow-up analysis. To improve diagnostic yield we suggest expanding the panel of RCC-CSGs tested to include CHEK2 and all SDHx subunits and raising the eligibility criteria for age-based testing

HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity

<div><p>The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a <i>PPxY</i> motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the <i>PPxY</i> motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous <i>Ins2</i> expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases.</p></div

Itch, Smurf2, and NEDD4 polyubiquitinate Glis3.

<p>A-B. HEK293T cells were transfected with CMV-HA-Ubiquitin, FLAG-Glis3 or FLAG-Glis3-ΔC480 or their respective <i>PY</i>^<i>461</i> mutants, and Myc-Itch, Smurf2, NEDD4, or empty vector as indicated. Cells were treated with 10 μM MG132 for 6 h prior to harvest. Co-immunoprecipitation was performed using an anti-M2 FLAG antibody and immunoprecipitated proteins were analysed by Western blot using a high affinity anti-HA, anti-M2 FLAG-HRP, anti-Myc or goat anti-mouse-HRP antibodies. C. HEK293T cells were transfected with FLAG Glis3, CMV-HA-Ubiquitin, and Myc-Itch or its catalytically inactive mutant as indicated. Co-IP was performed as described in A-B. D. HEK293T cells were transfected with FLAG-Glis3, Myc Itch, and HA-Ubiquitin or the K48R or K63R ubiquitin mutants as indicated. Co-IP was performed as described in A-B.</p

Glis3 associates with Itch, Smurf2, and NEDD4.

<p>A-C. HEK293T cells were transfected with FLAG-Glis3 or the FLAG-Glis3-<i>PY</i>^<i>461</i> mutant and Myc empty vector, Myc-Itch-C832G, Myc-Smurf2-C716G, or Myc-NEDD4-C867G as indicated. Co-immunoprecipitation was performed using a mouse anti-Myc antibody and immunoprecipitated proteins were examined by Western blot analysis using anti-M2 FLAG-HRP or anti-Myc and goat anti-mouse-HRP antibodies. D. HEK293T cells were transfected with FLAG-Glis3-ΔC480 or its respective <i>PY</i>^<i>461</i> mutant and Myc empty vector, Myc-Itch-C832G, Myc Smurf2-C716G, or Myc-NEDD4-C867G and co-IPs performed as described for A-C. E. HEK293T cells were transfected with FLAG-Glis3-ΔN496 and Myc empty vector or Myc-Itch-C832G and co-IPs performed as described in A-C. F. HEK293T cells were transfected with FLAG empty vector, FLAG-Glis3 or the FLAG-Glis3-<i>PY</i>^<i>461</i> mutant as indicated. After 48 h co-immunoprecipitation was performed using a mouse anti-M2 FLAG antibody and immunoprecipitated proteins were examined by Western blot analysis using mouse anti-ITCH primary and goat anti-mouse-HRP antibodies.</p

Itch targets Glis3 for degradation through the 26s proteasome.

<p>A. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch or empty vector. Cells were treated with DMSO, 10 μM MG132, 10 mM ammonium chloride, 200 μM chloroquine, or 100 μM leupeptin as indicated for 7 h. Proteins were analysed by Western blotting using anti-M2 FLAG-HRP antibody or anti-Myc and goat anti-mouse-HRP antibodies. GAPDH is shown as a loading control. B. HEK293T cells were transfected with FLAG-Glis3 and Myc-Itch, catalytically inactive Myc-Itch C832G, or empty vector. Cells were treated with 10 μM MG132 or DMSO for 6 h as indicated. Proteins were analysed by western blotting using an anti-M2 FLAG-HRP antibody.</p

Itch inhibits Glis3-mediated transactivation of target genes.

<p>A. INS1 832/13 cells were transfected with pGL4.27 or p3xGlisBS-Luc, FLAG-Glis3, the <i>PY</i>^<i>461</i> mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, cells were assayed for luciferase and β-galactosidase activity and the normalized relative luciferase activity (nRLU) was calculated and plotted. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. B. HEK293T cells were transfected with p-mIP-696-Luc, FLAG-Glis3, the <i>PY</i>^<i>461</i> mutant, or empty vector, and Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h cells were assayed as described for A. Each bar represents the mean +/- SEM. * Indicates statistically different value from corresponding Myc empty vector control p < 0.02. C. INS1 832/13 cells were transfected with Myc-Itch, the C832G mutant, or empty vector as indicated. After 48 h, RNA was collected and rIns2 mRNA was measured by qRT-PCR analysis. Each bar represents relative <i>Ins2</i> mRNA normalized to 18s rRNA +/- SEM. * Indicates statistically different value compared to empty vector control p < 0.02.</p