Regulating the human HECT E3 ligases

Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C terminus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibitory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruitment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein–protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways

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

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms

The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This singe amino-acid deletion is shared with apes and Old World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This hUBE3A-Iso2 isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a s

A novel UBE3A sequence variant identified in eight related individuals with neurodevelopmental delay, results in a phenotype which does not match the clinical criteria of Angelman syndrome

Background: Loss of functional UBE3A, an E3 protein ubiquitin ligase, causes Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, speech impairment, epilepsy, movement or balance disorder, and a characteristic behavioral pattern. We identified a novel UBE3A sequence variant in a large family with eight affected individuals, who did not meet the clinical AS criteria. Methods: Detailed clinical examination and genetic analysis was performed to establish the phenotypic diversity and the genetic cause. The function of the mutant UBE3A protein was assessed with respect to its subcellular localization, stability, and E3 ubiquitin ligase activity. Results: All eight affected individuals showed the presence of a novel maternally inherited UBE3A sequence variant (NM_130838.4(UBE3A):c.1018-1020del, p.(Asn340del), which is in line with a genetic AS diagnosis. Although they presented with moderate to severe intellectual disability, the phenotype did not match the clinical criteria for AS. In line with this, functional analysis of the UBE3A p.Asn340del mutant protein revealed no major deficits in UBE3A protein localization, stability, or E3 ubiquitin ligase activity. Conclusion: The p.(Asn340del) mutant protein behaves distinctly different from previously described AS-linked missense mutations in UBE3A, and causes a phenotype that is markedly different from AS. This study further extends the range of phenotypes that are associated with UBE3A loss, duplication, or mutation

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

Candidate CSPG4 mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10−5 and c.2702T > G [p.V901G], MAF 2.51 × 10−3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05–13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10−8), viability (P = 8.9 × 10−7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10−4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10−5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia

Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

In this study, we examine microbial communities of early developmental stages of the coral Porites astreoides by sequence analysis of cloned 16S rRNA genes, terminal restriction fragment length polymorphism (TRFLP), and fluorescence in situ hybridization (FISH) imaging. Bacteria are associated with the ectoderm layer in newly released planula larvae, in 4-day-old planulae, and on the newly forming mesenteries surrounding developing septa in juvenile polyps after settlement. Roseobacter clade-associated (RCA) bacteria and Marinobacter sp. are consistently detected in specimens of P. astreoides spanning three early developmental stages, two locations in the Caribbean and 3 years of collection. Multi-response permutation procedures analysis on the TRFLP results do not support significant variation in the bacterial communities associated with P. astreoides larvae across collection location, collection year or developmental stage. The results are the first evidence of vertical transmission (from parent to offspring) of bacteria in corals. The results also show that at least two groups of bacterial taxa, the RCA bacteria and Marinobacter, are consistently associated with juvenile P. astreoides against a complex background of microbial associations, indicating that some components of the microbial community are long-term associates of the corals and may impact host health and survival