Angelman syndrome is a complex neurodevelopmental disorder caused by the lack of function in the brain of a single gene, UBE3A. The E3 ligase coded by this gene is known to build K48-linked ubiquitin chains, a modification historically considered to target substrates for degradation by the proteasome. However, a change in protein abundance is not proof that a candidate UBE3A substrate is indeed ubiquitinated by UBE3A. We have here used an unbiased ubiquitin proteomics approach, the bioUb strategy, to identify 79 proteins that appear more ubiquitinated in the Drosophila photoreceptor cells when Ube3a is over-expressed. We found a significantly high number of those proteins to be proteasomal subunits or proteasome-interacting proteins, suggesting a wide proteasomal perturbation in the brain of Angelman patients. We focused on validating the ubiquitination by Ube3a of Rngo, a proteasomal component conserved from yeast (Ddi1) to humans (DDI1 and DDI2), but yet scarcely characterized. Ube3a-mediated Rngo ubiquitination in fly neurons was confirmed by immunoblotting. Using human neuroblastoma SH-SY5Y cells in culture, we also observed that human DDI1 is ubiquitinated by UBE3A, without being targeted for degradation. The novel observation that DDI1 is expressed in the developing mice brain, with a significant peak at E16.5, strongly suggests that DDI1 has biological functions not yet described that could be of relevance for Angelman syndrome clinical research.

• MeSH
• Angelmanův syndrom genetika   patofyziologie   MeSH
• aspartátové proteasy genetika   MeSH
• Drosophila MeSH
• fotoreceptory metabolismus   patologie   MeSH
• lidé MeSH
• myši MeSH
• neurony metabolismus   patologie   MeSH
• proteiny Drosophily genetika   MeSH
• proteomika MeSH
• regulace genové exprese genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• ubikvitinace genetika   MeSH
• ubikvitinligasy genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové proteasy MeSH
• DDI1 protein, S cerevisiae MeSH Prohlížeč
• DDI2 protein, human MeSH Prohlížeč
• proteiny Drosophily MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• Ube3a protein, Drosophila MeSH Prohlížeč
• UBE3A protein, human MeSH Prohlížeč
• ubikvitinligasy MeSH

The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains.

• MeSH
• interakční proteinové domény a motivy * MeSH
• katalytická doména MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multigenová rodina MeSH
• poškození DNA * MeSH
• proteomika metody   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• substrátová specifita MeSH
• ubikvitin metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDI1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• ubikvitin MeSH

Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization.

• MeSH
• aminokyselinové motivy MeSH
• aspartátové proteasy chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• konzervovaná sekvence MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• maloúhlový rozptyl MeSH
• mapování interakce mezi proteiny MeSH
• molekulární evoluce MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• polyubikvitin metabolismus   MeSH
• proteinové domény MeSH
• proteolýza MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza proteinů MeSH
• strukturní homologie proteinů * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aspartátové proteasy MeSH
• DDI2 protein, human MeSH Prohlížeč
• polyubikvitin MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny MeSH