A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex posttranslational modifications in a cellular setting

Mono-ubiquitination of Rabphilin 3A by UBE3A serves a non-degradative function

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function

A matter of perspective:The multifaceted role of UBE3A in Angelman syndrome development

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the UBE3A gene, which encodes an E3 ubiquitin protein ligase. E3 ligases are essential components of the UPS (ubiquitin proteasome system). The UPS is a finely tuned machinery that has an essential role in cellular homeostasis, and any perturbation of the UPS may lead to irreparable cellular damage. In this thesis we have employed molecular and biochemical techniques to unravel how the loss-of-function (deletion or mutations) of the E3 ubiquitin protein ligase UBE3A results in AS development, with emphasis on the identification and characterization of brain-specific targets of UBE3A. Using protein-protein interaction techniques we have identified several bona fide UBE3A interacting proteins, among which a pre-synaptic protein. Detailed biochemical and mutational analyses of these proteins have provided the first insights into the intracellular pathways where UBE3A exerts its function. By studying the localization of UBE3A isoforms we uncovered that UBE3A is predominantly localized in the nucleus and that AS-linked mutations can disturb nuclear localization. These observations suggest that UBE3A has an important role in the nucleus, providing further direction for future studies on the relation between UBE3A subcellular localization and the pathophysiology of AS

A matter of perspective: The multifaceted role of UBE3A in Angelman syndrome development

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the UBE3A gene, which encodes an E3 ubiquitin protein ligase. E3 ligases are essential components of the UPS (ubiquitin proteasome system). The UPS is a finely tuned machinery that has an essential role in cellular homeostasis, and any perturbation of the UPS may lead to irreparable cellular damage. In this thesis we have employed molecular and biochemical techniques to unravel how the loss-of-function (deletion or mutations) of the E3 ubiquitin protein ligase UBE3A results in AS development, with emphasis on the identification and characterization of brain-specific targets of UBE3A. Using protein-protein interaction techniques we have identified several bona fide UBE3A interacting proteins, among which a pre-synaptic protein. Detailed biochemical and mutational analyses of these proteins have provided the first insights into the intracellular pathways where UBE3A exerts its function. By studying the localization of UBE3A isoforms we uncovered that UBE3A is predominantly localized in the nucleus and that AS-linked mutations can disturb nuclear localization. These observations suggest that UBE3A has an important role in the nucleus, providing further direction for future studies on the relation between UBE3A subcellular localization and the pathophysiology of AS

A matter of perspective: The multifaceted role of UBE3A in Angelman syndrome development

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutation or deletion of the UBE3A gene, which encodes an E3 ubiquitin protein ligase. E3 ligases are essential components of the UPS (ubiquitin proteasome system). The UPS is a finely tuned machinery that has an essential role in cellular homeostasis, and any perturbation of the UPS may lead to irreparable cellular damage. In this thesis we have employed molecular and biochemical techniques to unravel how the loss-of-function (deletion or mutations) of the E3 ubiquitin protein ligase UBE3A results in AS development, with emphasis on the identification and characterization of brain-specific targets of UBE3A. Using protein-protein interaction techniques we have identified several bona fide UBE3A interacting proteins, among which a pre-synaptic protein. Detailed biochemical and mutational analyses of these proteins have provided the first insights into the intracellular pathways where UBE3A exerts its function. By studying the localization of UBE3A isoforms we uncovered that UBE3A is predominantly localized in the nucleus and that AS-linked mutations can disturb nuclear localization. These observations suggest that UBE3A has an important role in the nucleus, providing further direction for future studies on the relation between UBE3A subcellular localization and the pathophysiology of AS

A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex post-translational modifications in a cellular setting

A versatile plasmid system for reconstitution and analysis of mammalian ubiquitination cascades in yeast

Ubiquitination is a posttranslational protein modification that regulates most aspects of cellular life. The sheer number of ubiquitination enzymes that are present in a mammalian cell, over 700 in total, has thus far hampered the analysis of distinct protein ubiquitination cascades in a cellular context. To overcome this complexity we have developed a versatile vector system that allows the reconstitution of specific ubiquitination cascades in the model eukaryote Saccharomyces cerevisae (baker’s yeast). The vector system consists of 32 modular yeast shuttle plasmids allowing inducible or constitutive expression of up to four proteins of interest in a single cell. To demonstrate the validity of the system, we show that co-expression in yeast of the mammalian HECT type E3 ubiquitin ligase E6AP (E6-Associated Protein) and a model substrate faithfully recapitulates E6AP-dependent substrate ubiquitination and degradation. In addition, we show that the endogenous sumoylation pathway of S. cerevisiae can specifically sumoylate mouse PML (Promyelocytic leukemia protein). In conclusion, the yeast vector system described in this paper provides a versatile tool to study complex posttranslational modifications in a cellular setting

The Deubiquitylase USP2 Regulates the LDLR Pathway by Counteracting the E3-Ubiquitin Ligase IDOL

The low-density lipoprotein (LDL) receptor (LDLR) is a central determinant of circulating LDL-cholesterol and as such subject to tight regulation. Recent studies and genetic evidence implicate the inducible degrader of the LDLR (IDOL) as a regulator of LDLR abundance and of circulating levels of LDL-cholesterol in humans. Acting as an E3-ubiquitin ligase, IDOL promotes ubiquitylation and subsequent lysosomal degradation of the LDLR. Consequently, inhibition of IDOL-mediated degradation of the LDLR represents a potential strategy to increase hepatic LDL-cholesterol clearance. To establish whether deubiquitylases counteract IDOL-mediated ubiquitylation and degradation of the LDLR. Using a genetic screening approach, we identify the ubiquitin-specific protease 2 (USP2) as a post-transcriptional regulator of IDOL-mediated LDLR degradation. We demonstrate that both USP2 isoforms, USP2-69 and USP2-45, interact with IDOL and promote its deubiquitylation. IDOL deubiquitylation requires USP2 enzymatic activity and leads to a marked stabilization of IDOL protein. Paradoxically, this also markedly attenuates IDOL-mediated degradation of the LDLR and the ability of IDOL to limit LDL uptake into cells. Conversely, loss of USP2 reduces LDLR protein in an IDOL-dependent manner and limits LDL uptake. We identify a tri-partite complex encompassing IDOL, USP2, and LDLR and demonstrate that in this context USP2 promotes deubiquitylation of the LDLR and prevents its degradation. Our findings identify USP2 as a novel regulator of lipoprotein clearance owing to its ability to control ubiquitylation-dependent degradation of the LDLR by IDO

The Deubiquitylase USP2 Regulates the LDLR Pathway by Counteracting the E3-Ubiquitin Ligase IDOL

10.1161/CIRCRESAHA.115.307298CIRCULATION RESEARCH1183410-41