TAOK1 is associated with neurodevelopmental disorder and essential for neuronal maturation and cortical development.

Thousand and one amino-acid kinase 1 (TAOK1) is a MAP3K protein kinase, regulating different mitogen-activated protein kinase pathways, thereby modulating a multitude of processes in the cell. Given the recent finding of TAOK1 involvement in neurodevelopmental disorders (NDDs), we investigated the role of TAOK1 in neuronal function and collected a cohort of 23 individuals with mostly de novo variants in TAOK1 to further define the associated NDD. Here, we provide evidence for an important role for TAOK1 in neuronal function, showing that altered TAOK1 expression levels in the embryonic mouse brain affect neural migration in vivo, as well as neuronal maturation in vitro. The molecular spectrum of the identified TAOK1 variants comprises largely truncating and nonsense variants, but also missense variants, for which we provide evidence that they can have a loss of function or dominant-negative effect on TAOK1, expanding the potential underlying causative mechanisms resulting in NDD. Taken together, our data indicate that TAOK1 activity needs to be properly controlled for normal neuronal function and that TAOK1 dysregulation leads to a neurodevelopmental disorder mainly comprising similar facial features, developmental delay/intellectual disability and/or variable learning or behavioral problems, muscular hypotonia, infant feeding difficulties, and growth problems.

LRSAM1-mediated ubiquitylation is disrupted in axonal Charcot-Marie-Tooth disease 2P.

Charcot-Marie-Tooth (CMT) disease type 2 is a genetically heterogeneous group of inherited neuropathies characterized by motor and sensory deficits as a result of peripheral axonal degeneration. We recently reported a frameshift (FS) mutation in the Really Interesting New Gene finger (RING) domain of LRSAM1 (c.2121_2122dup, p.Leu708Argfs) that encodes an E3 ubiquitin ligase, as the cause of axonal-type CMT (CMT2P). However, the frequency of LRSAM1 mutations in CMT2 and the functional basis for their association with disease remains unknown. In this study, we evaluated LRSAM1 mutations in two large Dutch cohorts. In the first cohort (n = 107), we sequenced the full LRSAM1 coding exons in an unbiased fashion, and, in the second cohort (n = 468), we specifically sequenced the last, RING-encoding exon in individuals where other CMT-associated genes had been ruled out. We identified a novel LRSAM1 missense mutation (c.2120C > T, p.Pro707Leu) mapping to the RING domain. Based on our genetic analysis, the occurrence of pathogenic LRSAM1 mutations is estimated to be rare. Functional characterization of the FS, the identified missense mutation, as well as of another recently reported pathogenic missense mutation (c.2081G > A, p.Cys694Tyr), revealed that in vitro ubiquitylation activity was largely abrogated. We demonstrate that loss of the E2-E3 interaction that is an essential prerequisite for supporting ubiquitylation of target substrates, underlies this reduced ubiquitylation capacity. In contrast, LRSAM1 dimerization and interaction with the bona fide target TSG101 were not disrupted. In conclusion, our study provides further support for the role of LRSAM1 in CMT and identifies LRSAM1-mediated ubiquitylation as a common determinant of disease-associated LRSAM1 mutations.

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

RATIONALE: 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. OBJECTIVE: To establish whether deubiquitylases counteract IDOL-mediated ubiquitylation and degradation of the LDLR. METHODS AND RESULTS: 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. CONCLUSIONS: Our findings identify USP2 as a novel regulator of lipoprotein clearance owing to its ability to control ubiquitylation-dependent degradation of the LDLR by IDOL.

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.

Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.

Mutations affecting the gene encoding the ubiquitin ligase UBE3A cause Angelman syndrome. Although most studies focus on the synaptic function of UBE3A, we show that UBE3A is highly enriched in the nucleus of mouse and human neurons. We found that the two major isoforms of UBE3A exhibit highly distinct nuclear versus cytoplasmic subcellular localization. Both isoforms undergo nuclear import through direct binding to PSMD4 (also known as S5A or RPN10), but the amino terminus of the cytoplasmic isoform prevents nuclear retention. Mice lacking the nuclear UBE3A isoform recapitulate the behavioral and electrophysiological phenotypes of Ube3am-/p+ mice, whereas mice harboring a targeted deletion of the cytosolic isoform are unaffected. Finally, we identified Angelman syndrome-associated UBE3A missense mutations that interfere with either nuclear targeting or nuclear retention of UBE3A. Taken together, our findings elucidate the mechanisms underlying the subcellular localization of UBE3A, and indicate that the nuclear UBE3A isoform is the most critical for the pathophysiology of Angelman syndrome.

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.