Yes / The realisation that unregulated activation of the Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway is a key driver of a wide range of diseases has identified its components as targets for therapeutic intervention by small molecule inhibitors and biologicals. In this review, we discuss JAK-STAT signalling pathway inhibition by the inducible inhibitor “suppressor of cytokine signaling 3 (SOCS3), its role in diseases such as myeloproliferative disorders, and its function as part of a multi-subunit E3 ubiquitin ligase complex. In addition, we highlight potential applications of these insights into SOCS3-based therapeutic strategies for management of conditions such as vascular re-stenosis associated with acute vascular injury, where there is strong evidence that multiple processes involved in disease progression could be attenuated by localized potentiation of SOCS3 expression levels. / British Heart Foundation; Chief Scientist's Office; NHS Greater Glasgow and Clyde Research Endowment Fund; BBSRC

Ubiquilin (UBQL) is a member of type 2 ubiquitin-like (UBL) protein family. They structurally contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated (UBA) domain. Ubiquilin 2 (UBQL2) physically associates with poly ubiquitinated proteins and delivers them to the proteasome for degradation. This protein has been shown to play an important role in the regulation of aggregation and degradation of various neurodegenerative disease-associated proteins. In this study, we looked into the role of the ubiquilin-2 proteins in the AMPA receptor ubiquitination and proteasomal degradation pathway. Our results indicate that UBQL2 overexpression decreases AMPAR levels in neurons and also reduces GluA1 expression in HEK 293T cells. Moreover, by co-immunoprecipitation we found that UBQL2 interacts with ubiquitinated AMPARs. We, therefore propose that UBQL2 brings AMPARs to the proteasome for degradation. Consistent with this notion, expression of UBQL2 P497H, a mutant form incapable of interaction with proteasome, causes accumulation of AMPA receptors. These results indicate a role for UBQL2 in associating with and directing ubiquitinated AMPA receptors to the proteasome for degradation. / 2020-08-09T00:00:00Z

No description available.

The need for anabolic therapies to increase bone formation in difficult orthopaedic circumstances and to treat osteoporosis is an area of intense research focus. There is a current interest in the Wnt signalling pathway as a target for such treatment, with accumulating evidence for a role of this pathway in bone formation. Ectoderm Neural Cortex 1 (ENC1) is a Wnt target gene, not previously studied in bone, which was observed in our laboratory to be up-regulated in an anabolic surgical model of bone formation. The involvement of ENC1 in the differentiation of neuronal and adipocytic cells has previously been reported; therefore, this thesis investigates the expression of ENC1 in cells of the bone and the role of ENC1 during osteoblast differentiation. ENC1 transcript expression was localised to osteoblastic, chondrocytic and osteocytic cells in sections of healing fracture callus and normal mouse bone by in situ hybridisation. The expression of ENC1 was confirmed in differentiating primary osteoblasts and in osteoblastic and osteosarcoma cell lines by quantitative real time PCR and western blotting. ENC1 exists as two protein isoforms of 67 and 57kD in size, which are translated from alternatively spliced ENC1 transcripts. Both isoforms of the protein were detected in differentiating cultures of the pre-osteoblast cell line MC3T3-E1. To address the function of ENC1 in osteoblast differentiation, shRNA knockdown of the endogenous transcript was undertaken in MG63 osteosarcoma cells and in the MC3T3-E1 pre-osteoblastic differentiation model. Stable expression of shRNA targeted to both ENC1 spliceforms resulted in reduced accumulation of alkaline phosphatase positive nodules and alkaline phosphatase transcripts in MG63 cell culture. This reduction was not seen with targeted knockdown of 67kD ENC1 alone. Stable tetracycline-inducible shRNA knockdown targeted to both 57 and 67kD ENC1 isoforms in MC3T3-E1 cells resulted in a significant reduction of Alizarin Red S stained mineralised nodules. When expression of 67kD ENC1 alone was reduced, however, a significant increase in MC3T3-E1 nodule formation was observed. This knockdown had no effect on the expression of early genes involved in osteoblast differentiation Runx2 and osterix, but changes in expression of alkaline phosphatase and osteocalcin mRNA mirrored nodule formation. ENC1 is a member of the BTB-Kelch family of proteins. Some members of this family have recently been found to act as substrate adaptors for the E3 ubiquitin ligase, binding to the cullin 3 component of the complex. These adaptor proteins function to bring a substrate protein within the vicinity of the E2 ubiquitin-conjugating enzyme, thus targeting it for ubiquitination and subsequent proteasomal degradation. The ability of ENC1 to interact with cullin 3 was investigated as a possible mechanism by which it may affect a role in osteoblast differentiation. Full length ENC1 showed robust binding to cullin 3 and weak binding was seen between the N-terminally truncated 57kD isoform and cullin 3. ENC1, therefore, may act as a substrate adaptor protein for the cullin 3 based E3 ubiquitin ligase. These data present ENC1 as a novel candidate protein involved in osteoblast differentiation, and suggest the possible involvement of this protein in proteasomal degradation of a substrate involved in osteoblast differentiation. The ENC1 isoforms and the associated functional pathways thus are possible future therapeutic targets to treat bone loss and enhance or accelerate fracture healing.

No description available.

La protéine PCNA est un facteur d'échafaudage polyvalent pour plus de cinquante protéines impliquées dans le métabolisme d'ADN, notamment dans la réplication et la réparation. Comment les échanges entre les partenaires de PCNA sont régulés est actuellement mal compris. Parmi ses partenaires, CDT1, p21 et PR-Set7/Set8 possèdent un motif d'interaction avec PCNA particulier, nommé « PIP degron », qui favorise leur protéolyse d'une manière dépendante de l'E3 ubiquitine ligase CRL4Cdt2. Après irradiation aux UV-C, le facteur d'initiation de la réplication CDT1 est rapidement détruit d'une manière dépendante de son PIP degron, mais le rôle de cette dégradation est inconnu. Dans cette étude, j'ai analysé la fonction du PIP degron de CDT1 et fourni des évidences expérimentales qui montrent que l'inhibition de la dégradation de Cdt1 par CRL4Cdt2 dans les cellules de mammifères compromet la relocalisation de l'ADN polymérase translesionnelle eta en foyers nucléaires induits par les irradiations UV-C. En élargissant cette étude à d'autres partenaires de PCNA, nous avons constaté que seuls les protéines qui contiennent un PIP degron, et pas un PIP box canonique comme celui de FEN1 et p15 (PAF), interfèrent avec la formation de foyers de pol eta. La mutagenèse du PIP degron de CDT1 a révélé qu'un résidu de thréonine conservé parmi les PIP degrons est essentiel pour l'inhibition de la formation des foyers de pol eta. Les résultats obtenus suggèrent que l'élimination de protéines contenant des PIP degrons par la voie CRL4Cdt2 régule le recrutement de pol eta au niveau des sites de dommages induits par les UV-C. / The sliding clamp PCNA is a versatile scaffold for more than fifty proteins involved in DNA metabolism such as replication and repair. How the switch between PCNA partners is regulated is currently not fully understood. Among its partners, Cdt1, p21 and PR-Set7/Set8 contain a specialized PCNA-binding motif named « PIP degron » that promotes their proteolysis in a fashion dependent on the E3 ubiquitin ligase CRL4Cdt2. Upon UV-irradiation, the replication initiation factor Cdt1 is rapidly destroyed in a PIP degron-dependent manner but the role of this degradation is unknown. Here we have analyzed the function of Cdt1 PIP degron and we provide evidence that interference with CRL4Cdt2-mediated destruction of Cdt1 in mammalian cells compromises PCNA-dependent relocalisation of the DNA translesion polymerase eta into UV-induced nuclear foci. By extending this analysis to other PCNA partners, we found that only PIP degrons, as compared to canonical PCNA-binding motifs of Fen1 and p15(PAF), interfere with pol eta focus formation. Mutagenesis of Cdt1 PIP degron revealed that a threonine residue conserved in PIP degrons is critical for inhibition of pol eta focus formation. Our results suggest that removal of high-affinity PIP degron-containing proteins from PCNA by CRL4Cdt2 pathway regulates pol eta recruitment to sites of UV-damage.

Neurogenin 2 (Ngn2) is a basic Helix-Loop-Helix (bHLH) transcription factor regulating differentiation and cell cycle exit in the developing brain. By transcriptional upregulation of a cascade of other bHLH factors, neural progenitor cells exit the cell cycle and differentiate towards a neuronal fate. Xenopus laevis Ngn2 (xNgn2) is a short-lived protein, targeted for degradation by the 26S proteasome. I have investigated the stability of Ngn2 mediated by post-translational modifications and structural disorder. Firstly I will describe work focused on ubiquitylation of xNgn2, targeting it for proteasomal degradation. xNgn2 is ubiquitylated on lysines, the recognized site of modification. I will discuss the role of lysines in ubiquitylation and stability of xNgn2. In addition to canonical ubiquitylation on lysines, I describe ubiquitylation of xNgn2 on non-canonical sites, namely its amino-terminal amino group, and cysteine, serine and threonine residues. I show that the ubiquitylation of cysteines in particular exhibits cell cycle dependence and is also observed in mammalian cell lines, resulting in cell cycle-dependent regulation of stability. I will then discuss whether phosphorylation, a regulator of xNgn2 activity, also affects xNgn2 stability. I will provide evidence of cell cycle-dependent phosphorylation of cyclin dependent kinase (cdk) consensus sites affecting the stability of xNgn2. Finally I describe studies on the folding properties of Ngn2 to assess their role in protein stability. xNgn2 associates with DNA and its heterodimeric binding partner xE12 and may interact directly with the cyclin-dependent kinase inhibitor Xic1. I will discuss the role of these interaction partners in xNgn2 stability. xNeuroD, a downstream target of xNgn2, is a related bHLH transcription factor which is stable. Here I describe domain swapping experiments between these two proteins highlighting regions conferring instability on the chimeric protein. Finally I will provide nuclear magnetic resonance (NMR) data looking at the effect of phosphorylation on protein structure in mouse Ngn2 (mNgn2).

Yes / The fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context. / RLI was supported by MRC New Investigator Research Grant: G0900936. RLI and SH are funded by BBSRC grants: BB/L006952/1 and BB/L006510/1 respectively. RLI is funded by BBSRC grant: BB/N006267/1. AT is funded by BBSRC grant: BB/J014567/1. ILT was the recipient of a Wellcome Trust Vacation Scholarship 2015. NMR experiments were performed at the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust

<p>Parkinson’s disease (PD) is a detrimental neurodegenerative disorder characterized by the presence of large protein aggregates in the brain called Lewy bodies, which are primarily composed of the protein α-synuclein (αSyn). Due to the dysregulation of αSyn levels in PD, controlling its levels through the manipulation of protein degradation pathways has been suggested as a therapeutic avenue for the treatment of PD and related diseases. Although αSyn is known to be degraded through the autophagy and proteasome pathways, it is one of only a few known substrates of the ubiquitin-independent proteasome pathway, which utilizes the 20S core particle of the proteasome (20S CP) to degrade proteins. We therefore hypothesize that small molecule stimulation of the 20S CP will enhance αSyn degradation and reduce αSyn pathology, providing a therapeutic benefit in PD models.</p><p>We began our studies by developing a fluorescence resonance energy transfer (FRET) reporter assay to monitor 20S CP activity and screen for small molecule stimulators. This assay provides a greater dynamic range to detect 20S CP stimulation compared to the most commonly used assay to monitor proteasome activity. Using the FRET assay, we were able to identify a number of novel 20S CP stimulators that differ in structure as well as potency and degree of stimulation. We next evaluated the ability of four small molecule stimulators to enhance protein degradation by the 20S CP in a biochemical assay using 15 different purified proteins. These 15 proteins include known substrates of the 20S CP and vary in size and degree of disorder. From this assay, we demonstrate that a 20S CP stimulator is likely to enhance the degradation of highly disordered proteins, such as αSyn, but the effect on other protein levels appears to be distinct for each stimulator. Two of our more potent stimulators, AM-404 and miconazole, were used with the proteasome inhibitor bortezomib for subsequent studies in HEK-293T cells in which we corroborated the results of our biochemical assay. While both AM-404 and miconazole were shown to impact highly disordered proteins, there was not much overlap between the proteins shown to be affected by each stimulator. Due to the distinct effect of each stimulator on protein regulation by the 20S CP, this study indicates the potential of tailoring a small molecule 20S CP stimulator to enhance the degradation of particular substrates.</p><p>Since AM-404 and miconazole were shown to impact 20S CP activity in different ways, we next evaluated whether either stimulator would be able to prevent the αSyn-induced inhibition of the 20S CP. High levels of αSyn have been shown to lead to proteasome impairment in biochemical and cell studies. We confirm 20S CP impairment in the presence of micromolar amounts of αSyn, and we demonstrate that miconazole, but not AM-404, is effective at maintaining 20S CP activity in the presence of increasing concentrations of αSyn. We also show that αSyn-overexpressing PC12 cells (PC12 C4 cells) display reduced proteasome activity compared to the parent cell line. Miconazole and AM-404 increased proteasome activity in PC12 C4 cells, which were more sensitive to 20S CP stimulation than non-transfected PC12 cells, but miconazole was shown to be more effective at modulating αSyn phosphorylated at Ser129 in PC12 C4 cells. </p><p>Our results reveal the dynamic nature of the 20S CP and the ways in which its activity can be modulated to affect protein levels. While AM-404 is effective at stimulating the 20S CP to enhance the degradation of some proteins, miconazole was shown to be more efficient at modulating αSyn levels and impacting αSyn pathology, as it relates to 20S CP impairment. While the results described here mark the beginning of an exciting area of study, we do demonstrate the therapeutic potential of 20S CP stimulation to combat PD. </p><div><br></div>

Das Neuroblastom ist der am weitesten verbreitete solide, extrakranielle Tumor im Kindesalter. Trotz intensiver Forschung sind die Überlebensraten von Patienten mit fortgeschrittenem Tumorwachstum nach wie vor schlecht. Die Idee, eine zelluläre, langanhaltende Immunantwort im Körper zu induzieren, vermittelt durch zytotoxische CD8+T-Zellen, die sich gegen den Tumor richten, scheint dabei besonders attraktiv. Als tumorassoziiertes Antigen (TAA) wurde zu diesem Zweck für diese Arbeit die murine Tyrosinhydroxylase (mTH), das Schrittmacherenzym der Katecholaminbiosynthese, gewählt, da sie in der Mehrzahl der Neuroblastome stark überexprimiert ist. Für die Impfexperimente wurden sog. DNA-Minigen-Vakzine, die für Peptide aus der mTH-Sequenz kodieren, konstruiert. Die Auswahl Minigen-Peptide erfolgte mit dem MHC-Klasse-I-Liganden-Vorhersageprogramm syfpeithi, welches drei vorhergesagte starke H2-Kk-Liganden lieferte (mTH3k). Außerdem wurden zwei weitere Vakzine hergestellt: als Negativkontrolle das Vakzin mTHlowest, dessen mTH-Peptide laut syfpeithi schlechte MHC-Klasse-I-Liganden darstellen und das Vakzin Ersatzepis, dessen Peptide auf der Oberfläche von murinen NXS2-Neuroblastomzellen aus MHC-Klasse-I-Komplexen isoliert werden konnten. Sowohl in prophylaktischen als auch therapeutischen Impfversuchen in Mäusen konnte das Tumorwachstum und die spontane Metastasierung in sekundäre Organe wie die Leber signifikant verhindert werden. Außerdem konnte gezeigt werden, daß der Antitumoreffekt auf der Induktion mTH-spezifischer, zytotoxischer CD8+T Zellen (CTLs) beruht. Zusätzlich und insbesondere interessant für eine eventuelle klinische Anwendung eines auf der TH basierenden DNA-Vakzins verursachte das mTH-Minigen-Vakzin zumindest in Mäusen keine Aktivierung selbst-reaktiver CD8+T-Zellen. Alles in allem lassen die in dieser Arbeit erhaltenen Ergebnisse den Schluß zu, daß sich die Tyrosinhydroxylase als TAA in Form eines DNA-Vakzins zur adjuvanten Therapie des Neuroblastoms eignet. / Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we demonstrate for the first time effective therapeutic vaccination followed by eradication of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I H2-Kk according to prediction program syfpeithi and computer modeling of epitopes into MHC class I binding groove. Subsequently, a DNA minigene vaccine based on pCMV-F3Ub encoding for mutated ubiquitin (G76 to A76) and mTH3 was generated. Prophylactic and therapeutic efficacy of this vaccine was established following oral delivery using attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependant on ubiquitin and high affinity of the mTH epitopes to MHC class I. Specifically, we demonstrated a crucial role for minigene expression as a stable ubiquitin-Ala76 fusion peptide for vaccine efficacy. Interestingly, the unstable wild type ubiquitin-Gly76 vaccine was completely ineffective. The immune response following mTH3 DNA minigene vaccination was mediated by CD8+ T-cells as indicated by infiltration of primary tumors and TH specific cytolytic activity in vitro. Importantly, no infiltration was detectable in TH expressing adrenal medulla, indicating the absence of auto immunity. In summary, we demonstrate effective therapeutic vaccination against neuroblastoma with a novel rationally designed tyrosine hydroxylase minigene vaccine without induction of autoimmunity providing an important base line for clinical application of this strategy.