159 research outputs found
MOLECULAR RECOGNITION MECHANISM OF p63 BY ITCH-E3 LIGASE: ADVANCES AND EFFECTS OF A p63 MUTATION RELATED TO ECTODERMAL DYSPLASIAS.
No full textRecently, it has been shown that Itch mediates the degradation of TAp63 and ΔNp63 proteins1. Itch E3–ligase contains four WW domains important in the recognized process. Several signalling complexes, that these domains mediate, have been implicated in human diseases (Muscular Dystrophy, Alzheimer's Disease, Huntington Disease etc.). WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. Based on their ligand-binding specificity they have been categorized into four groups. WW domains fold into stable three-stranded antiparallel b-sheet structures, and their primary sequence share two conserved tryptophan residues spaced 20-22 amino acids apart. The four WW domains of Itch are considered belonging to the Group I, which binds polypeptides with a PY motif characterized by a PPXY consensus sequence, where X can be any residue. It is likely that the Itch-p63 interaction results from a direct interaction of Itch-WW2 domain with the PY motif of p63. Here, we present a structural characterization of the interaction by fluorescence, CD and NMR spectroscopy of the Itch-WW2 domain. Interaction studies in vitro between Itch-WW2 domain and pep63, which correspond to the fragment of the p63 protein including the PY motif, were performed. Moreover, the effects of a site specific mutation of p63, that has been reported in both Hay–Wells syndrome and Rapp–Hodgkin syndrome, was also evaluated both on the conformation of pep63 and on the WW-pep63 interaction.
1Rossi M., Aqeilan I., Neale M., Candi E., Salomoni P., Knight R.A., Croce C.M., Melino G. PNAS (2006) 103: 12753-5
Progress for dengue virus diseases: Towards the NS2B-NS3pro inhibition for a therapeutic-based approach
No full textTransmitted by the Aedes aegypti mosquito, the dengue virus is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, and, as such, is a significant factor in the high death rate found in most tropical and subtropical areas of the world. Dengue diseases are not only a health burden to developing countries, but pose an emerging problem worldwide. The immunopathological mechanisms appear to include a complex series of immune responses. A rapid increase in the levels of cytokines and chemical mediators during dengue disease plays a key role in inducing plasma leakage, shock and hemorrhagic manifestations. Currently, there are no vaccines available against dengue virus, although several tetravalent live-attenuated dengue vaccines are in clinical phases I or II, and prevention through vaccination has become a major priority on the agendas of the World Health Organization and of national ministries of health and military organizations. An alternative to vaccines is found in therapeutic-based approaches. Understanding the molecular mechanisms of viral replication has led to the development of potential drugs, and new molecular viral targets for therapy are emerging. The NS3 protease domain of the NS3 protein is responsible for processing the viral polyprotein and its inhibition is one of the principal aims of pharmacological therapy. This review is an overview of the progress made against dengue virus; in particular, it examines the unique properties - structural and functional - of the NS3 protease for the treatment of dengue virus infections by the inhibition of viral polyprotein processing. © 2007 The Authors
Recognition of p63 by the E3 ligase ITCH: Effect of an ectodermal dysplasia mutant
No full textThe E3 ubiquitin ligase Itch mediates the degradation of the p63 protein. Itch contains four WW domains which are pivotal for the substrate recognition process. Indeed, this domain is implicated in several signalling complexes crucially involved in human diseases including Muscular Dystrophy, Alzheimer's Disease and Huntington Disease. WW domains are highly compact protein-protein binding modules that interact with short proline-rich sequences. The four WW domains present in Itch belong to the Group I type, which binds polypeptides with a PY motif characterized by a PP xY consensus sequence, where x can be any residue. Accordingly, the Itch-p63 interaction results from a direct binding of Itch-WW2 domain with the PY motif of p63. Here, we report a structural analysis of the Itch-p63 interaction by fluorescence, CD and NMR spectroscopy. Indeed, we studied the in vitro interaction between Itch-WW2 domain and p63(534-551), an 18-mer peptide encompassing a fragment of the p63 protein including the PY motif. In addition, we evaluated the conformation and the interaction with Itch-WW2 of a site specific mutant of p63, I549T, that has been reported in both Hay-Wells syndrome and Rapp-Hodgkin syndrome. Based on our results, we propose an extended PP xY motif for the Itch recognition motif (P-P-P-Y-x(4)-[ST]-[ILV]), which includes these C-terminal residues to the PP xY motif
p63 threonine phosphorylation signals the interaction with the WW domain of the E3 ligase Itch
No full textBoth in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the four WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition
Recognition mechanism of p63 by the E3 ligase Itch Novel strategy in the study and inhibition of this interaction.
No full textThe HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73,
involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer’s or Huntington’s diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly 13C-15N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534–551 residue of p63, encompassing the PP xY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PP xY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex.
Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase
Thiosulfate-Cyanide Sulfurtransferase a Mitochondrial Essential Enzyme: From Cell Metabolism to the Biotechnological Applications
No full textThiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron–sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme
Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities.
No full textThe ubiquitin proteasome system (UPS) plays a role in the regulation of most cellular pathways, and its deregulation has been implicated in a wide range of human pathologies that include cancer, neurodegenerative and immunological disorders and viral infections. Targeting the UPS by small molecular regulators thus provides an opportunity for the development of therapeutics for the treatment of several diseases. The proteasome inhibitor Bortezomib was approved for treatment of hematologic malignancies by the FDA in 2003, becoming the first drug targeting the ubiquitin proteasome system in the clinic. Development of drugs targeting specific components of the ubiquitin proteasome system, however, has lagged behind, mainly due to the complexity of the ubiquitination reaction and its outcomes. However, significant advances have been made in recent years in understanding the molecular nature of the ubiquitination system and the vast variety of cellular signals that it produces. Additionally, improvement of screening methods, both in vitro and in silico, have led to the discovery of a number of compounds targeting components of the ubiquitin proteasome system, and some of these have now entered clinical trials. Here, we discuss the current state of drug discovery targeting E3 ligases and the opportunities and challenges that it provides
Thiosulfate sulfurtransferase: a model of essential enzyme with potential applications in medicine and biotechnology
No full textSlow H2S-Releasing Donors and 3D Printable Arrays Cellular Models in Osteo-Differentiation of Mesenchymal Stem Cells for Personalized Therapies
No full textThe effects of the hydrogen sulfide (H2S) slow-releasing donor, named GSGa, a glutathione-conjugate water-soluble garlic extract, on human mesenchymal stem cells (hMSCs) in both bidimensional (2D) and three-dimensional (3D) cultures were investigated, demonstrating increased expression of the antioxidant enzyme HO-1 and decreased expression of the pro-inflammatory cytokine interleukin-6 (IL-6). The administration of the H2S donor can therefore increase the expression of antioxidant enzymes, which may have potential therapeutic applications in osteoarthritis (OA). Moreover, GSGa was able to promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), but not of cardiac mesenchymal stem cells (cMSCs) in a 2D culture system. This result highlights the varying sensitivity of hMSCs to the H2S donor GSGa, suggesting that the induction of osteogenic differentiation in stem cells by chemical factors is dependent on the tissue of origin. Additionally, a 3D-printable mesenchymal stem cells–bone matrix array (MSCBM), designed to closely mimic the stiffness of bone tissue, was developed to serve as a versatile tool for evaluating the effects of drugs and stem cells on bone repair in chronic diseases, such as OA. We demonstrated that the osteogenic differentiation process in cMSCs can be induced just by simulating bone stiffness in a 3D system. The expression of osteocalcin, RUNX2, and antioxidant enzymes was also assessed after treating MSCs with GSGa and/or increasing the stiffness of the culture environment. The printability of the array may enable better customization of the cavities, enabling an accurate replication of real bone defects. This could optimize the BM array to mimic bone defects not only in terms of stiffness, but also in terms of shape. This culture system may enable a rapid screening of antioxidant and anti-inflammatory compounds, facilitating a more personalized approach to regenerative therapy
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